Microsoft Word - 476hernandez.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 43, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Chief Editors: Sauro Pierucci, Jiří J. Klemeš Copyright © 2015, AIDIC Servizi S.r.l., ISBN 978-88-95608-34-1; ISSN 2283-9216 Evolution of Polyphenols, Volatile Aroma Compounds and Natural Yeast Flora of Coda di Volpe White Grape Floriana Boscaino*a, Gerry Cutrib, Maria Grazia Volpea, Giuseppe Blaiottac, Alida Sorrentinoa a Istituto di Scienze dell’Alimentazione, CNR, Via Roma 64, 83100 Avellino, Italia b ITC-Via A. De Gasperi, 10 - 83035 Grottaminarda, Avellino, Italia c Dipartimento di Agraria, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, NA, Italy fboscaino@isa.cnr.it Wine is one of the main product of the agro-food products in Italy especially for its economic importance to local and regional level. The Campania region is excellent for the production of wine especially those of value, thanks to the presence of wide known varieties of native grapes. The aim of this work is been to characterize the chemical, biochemical and microbiological properties of Coda di Volpe grapes, cultivated in two areas, the hills and flat lands of Benevento. The name Coda di Volpe means tail of the fox, which was given in reference to the varieties of long, pendulous bunches of grapes, which resembles a fox's bushy tail. The polyphenols, pH, total acidity and °Brix were determined at different stages of maturation in order to identify the optimal time for the harvest. In particular, the total polyphenols content was evaluated with Folin-Ciocalteu method, while the specific polyphenols were determined by RP-HPLC. The grapes were harvested in sterile bag, sealed, pressed and incubated at 20°C for 1 month. During the fermentation it was isolated epiphytic microflora and characterized in order to analyse the physiological, oenological and technological properties. Three different yeast strains were assayed in their oenolegical ability, these were used in experimental fermentation with Coda di Volpe must. During alcholic fermentation have been evaluated yeast population, alcoholic grade and the volatile profile by the SPME-GC/MS technique. The data obtained have highlighted their ability to conduct the fermentation process, dominate the indigenous microflora and contribute to the definition of the flavour profile. 1. Introduction Coda di Volpe is a golden-yellow grapes variety, used to produce wines in and around the region of Campania in southern Italy. The wine produced from this cultivar is often described as medium to medium-full bodied, fruity (citrus, peach, pineapple) and spicy (nutmeg, cinnamon) and flavors of grapefruit, lemon, and almond. Since the 1980’s, wineries in Campania have begun making single-varietal wines from Coda di Volpe, and now used in the DOC wines of Irpinia and Sannio area. Industrial wine fermentations are currently conducted by starters of special wine yeast strains of Saccharomyces cerevisiae in contrast to traditional spontaneous fermentations conducted by the flora present on the grapes and in the winery. Despite the advantages of using pure cultures of S. cerevisiae with regard to the easy control and homogeneity of fermentations, wine produced with pure yeast monocultures lacks the complexity of flavour, stylistic distinction and vintage variability caused by indigenous yeasts (Swiegers et al., 2005). In recent years the inclusion of non-Saccharomyces wine yeast species as part of mixed starters together with S. cerevisiae to improve wine quality has been suggested as a way of taking advantage of spontaneous fermentations without running the risks of stuck fermentations or wine spoilage (Romano et al., 2003; Ciani et al., 2006). Although non-Saccharomyces wine yeast species have traditionally been associated with high volatile acidity, ethyl acetate production, off-flavours and wine spoilage (Sponholz, 1993; Ciani and Maccarelli, 1997), the potential positive role they play in the organoleptic characteristics of wine has been emphasized in numerous studies DOI: 10.3303/CET1543002 Please cite this article as: Boscaino F., Cutri G., Volpe M.G., Blaiotta G., Sorrentino A., 2015, Evolution of polyphenols, volatile aroma compounds and natural yeast flora of coda di volpe white grape, Chemical Engineering Transactions, 43, 7-12 DOI: 10.3303/CET1543002 7 (Fleet, 2003). Metabolic interactions between non-Saccharomyces and S. cerevisiae wine yeasts during fermentation could positively or negatively interfere with the growth and fermentation behaviour of yeast species, particularly S. cerevisiae. It has also been noted that the presence/absence of S. cerevisiae differs according to each plant and grape cluster (Pretorius et al., 1999). For this reason it is not always possible to obtain the same product from spontaneous wine fermentation. This problem is being solved at present by the use of commercial strains in the fermentation process, to the detriment of the wine’s autochthonous character. On the other hand, the widespread use of LSA has led to a leveling of the aromatic characteristics of wines produced with different cultivars. Today is increasing the demand for selected native yeasts starter that could enhance the sensory characteristics peculiar of the wine. The purpose of this work was the identification of cultures of native yeast to be used in starter mix for use on a large scale 2. Materials and Methods 2.1 Chemicals The culture media for the isolation, the reactivation and the growth of yeast strains were purchased from Oxoid (Hampshire, UK). Chemical reagents and solvents are all of analytical grade and were from Sigma- Aldrich (St.Louis, MO, USA). The SPME fibers PDMS- 100μm (polydimethylsiloxane), was from Supelco (Bellefonte, PA, USA). 2.2 Sampling In order to evaluate the change in total polyphenols and antioxidant activity, the Coda di Volpe grapes were harvested in two different stages of maturation, in the last week of September and in the second week of October respectively. Figure 1 reported the experimental protocol diagram. 2.3 Analysis of total polyphenols Total polyphenol content was determined using the Folin-Ciocalteu reagent (Waterman and Mole,1994) following a micro scale as described by Arnous et al. (2002). The absorbance at 750 nm was recorded, and the total polyphenol concentration was calculated from a calibration curve using gallic acid as standard and expressed as mg gallic acid equivalents/L (GAME). 2.4 HPLC analysis of phenolic compounds Phenolic compounds analysis was performed by HPLC/UV with a C18 column Hypersil (ThermoElectron Corporation, Bellefonte, PA, North America). The injected sample volume was 20 μL. Peak identifications were confirmed from retention times and direct comparison to pure standards. The solvent flow rate was 0.9 mL/min and the mobile phase was a four-step linear solvent gradient system(0–5 min, 10 % B; 5-40 min, 45 % B; 40– 45 min, 100 % B; 45-50 min, 100 % B; 50-55 min, 10 % B)using 2 % acetic acid in water as solvent A and 0.5 % acetic acid in 50 % acetonitrile as solvent B. UV detection was carried out at 280 nm (Katalinić et al., 2010). 2.5 Antioxidant Activity of grape skin extracts Antioxidant properties of grape skin extracts were determined as free radical-scavenging ability (DPPH method), reducing power (FRAP method), Fe2+-chelating ability of plant extracts, and ability to prevent oxidation of linoleic acid (Katalinić et al., 2010). 2.6 Isolation of yeasts To isolate the different yeast populations, serial dilutions of Coda di Volpe grape from sealed bag were prepared. 1 mL of must was used to prepare Ringer solution serial dilutions and appropriate dilutions were plated on two different solid agar media: WL-nutrient and Lysine agar. On WL medium, yeast species have been distinguished by different colony morphologies and colours. The isolation of non- Saccharomyces species has been carried out on Lysine medium, that does not allow the growth Saccharomyces spp (Cavazza et al 1992). After 4 days of incubation at 25 °C, the colonies were enumerated to determine the order of population. Five colonies were isolated randomly from the plates and stored at 4°C for physiological and technological characterization. Figure 1: experimental protocol diagram 8 The culture yeasts were identified according to their morphological aspect (elliptical and lemon shaped) and biochemical test API 20C Aux (bioMerieux, France). 2.7 Determination of the yeast fermentative power The must (100mL) after pasteurization (100 °C for 30 min) was inoculated with a 1 % (v/v) microbial biomass suspension (24h growth). The must fermentation tests were carried out in shake-flasks incubated at 28 °C. The flasks weight decrease was measured every day, until a constant weight has been reached and maintained for three days (Zambonelli, 1998). 2.8 Yeasts selection for the resistance to sulphur dioxide The tests were carried out on Coda di Volpe must which was pasteurized and inoculated with a 1 % microbial biomass suspension (24h growth). Potassium metabisulfite (MBK) was used as the source of sulphur dioxide and the yeasts resistance at concentrations of 100 and 250 mg/L was checked. The tests were performed at 28 °C for 7 days (Costanti et al., 1998). 2.9 Inoculated fermentations in laboratory-scale Three yeasts (YOCMCVWL4, YTECMCVWL4, YTECCVWL7), selected on the base of technological traits were inoculated in must in single and different combination. Inoculated fermentation assay was performed in 1L Erlenmeyer flasks filled with 500 mL of Coda di Volpe grape must with 10% MBK. Grape must characteristics were: 21,5 °Brix, pH 3,2. Each strain was inoculated in grape must at a concentration of 106 cells/mL. The fermentation was performed at 20°C and the fermentative course was monitored by measuring weight loss, determined by carbon dioxide evolution during the process. At the end of the process, the wine samples, immediately, were analyzed by SPME-GC/MS to evaluate the volatile compounds. 2.10 Analysis of volatile compounds by HS-SPME-GC/MS The SPME fiber (PDMS-100μm, polydimethylsiloxane) was conditioned according to the manufacturer’s recommendations prior to its first use (Pawliszyn 2009). To a 20 mL Headspace vial was added 5 mL of wine samples, together to 3g of NaCl and octan-3-ol, in hydro-alcoholic solution (1/1, v/v) at 100 μg/L, as Internal Standard. The solution was homogenized with a vortex shaker and then loaded onto a Gerstel autosampling device. The program consisted of swirling the vial at 250 rpm for 5 min at 40 °C, then inserting the fiber into the headspace for 30 min at 40 °C as the solution was swirled again, then transferring the fiber to the injector for desorption at 240 °C for 30 min. 2.11 GasCromatography-MassSpectrometry Gas chromatography analysis were carried out using a 7890 Agilent GC system coupled to an Agilent 5975 inert quadrupole mass spectrometer equipped with a Gerstel MPS2 autosampler. The capillary column employed was a HP-Innowax (Agilent technologies) (30 m x 0,25 mm id. 0,50 μm film thickness) and the carrier gas was Helium. Splitless injections were used. The initial oven temperature was set to 40 °C for 1 min. The temperature was increased in four steps: 40–60 °C at 2 °C/min; 60–150 °C at 3 °C/min, 150–200 °C at 10 °C/min and 200-240 °C at 25 °C/min; the final temperature was maintened for 7 min. The injector, the quadrupole, the source and the transfer line temperature were maintained at 240 °C, 150 °C, 230 °C and 200 °C, respectively. Electron ionization mass spectra in full-scan mode were recorded at 70eV electron energy in the range 40–300 amu. Peaks were identified using both the NIST 98 and Wiley libraries. Quantification was performed by using the relative concentration in μg/L of the Internal Standard, calculated as the ratio between each compound area and the internal standard area. The samples were analyzed in triplicate and blank runs were made by using an empty vial every two analysis (Sorrentino et al., 2013). 3. Results and Discussion 3.1 Total polyphenols and antioxidant activity The data reported in Fig.2 show the content of total polyphenols and antioxidant activity in Coda di Volpe berry grapes. They show that the amounts was highest in the skin respect to pulp berry. These results were confirmed in all samples collected to the different times (last Figure 2: total phenols and antioxidant activity relative to skin and pulp grape berry in two different time sampling 9 week of September and the second week of October). In addition, experimental data have shown that polyphenols were higher in the skin of the grape berry harvested in the first sampling while the antioxidant activity was higher in the berry in second. The HPLC analysis on grape skin (data not shown), confirmed the presence of catechins and epicatechins peaks in both sampling, this data was in according to literature (Katalinić et al., 2010). 3.2 Monitoring of yeast populations on Coda di Volpe grapes The results of microbiological analyses on the grapes showed that in the first sampling the epiphytic microflora was around 3*103 cfu/g on WL agar and 2*104 cfu/g on Lysine medium, while on the second sampling the load of yeast population was respectively 5*105 cfu/g and 6*105 cfu/g. After enumeration, from the plates contained from 15 to 300 colonies were isolated randomly five colonies. These were stored at 4°C for identification and technological characterization. After physiological screening, were selected yeasts with elliptical and lemon shaped (Martini et al., 1996). The yeasts selected were S.cerevisae YTECCVWL7 (a) and YOCMCVWL4 (c), non-Saccharomyces YTECMCVWL4 (b), they were used in technological tests and to performe microfermentations. 3.3 Technological characterization On yeast selected, was carried out the technologic test in order to ascertain resistance of the indigenous yeasts to sulphur dioxide. Sulphur dioxide is the most widely antiseptic agent used in wine cellars to control the growth of spontaneous microflora during fermentation. For our selection, we checked the resistance at two different concentrations (100 mg/L and 250 mg/L). The results demonstrated that most of the yeasts were resistant to SO2 concentration 100 mg/L. Our data, revealed that many indigenous yeast belonged to non-Saccharomyces genera showed resistance to SO2, to despite the literature data (Romano and Suzzi, 1993; Henick-Kling et al., 1998) that suggest the lack of such resistance in many yeast non-Saccharomyces genera. The resistance to sulphur dioxide could be caused by a “selective pressure” of sulphur levels used on grapevines throughout the years. Moreover, a significant number of indigenous yeasts non-Saccharomyces can grow after the addition of the usual dose of SO2 until they die of ethanol intoxication after three or four days from the beginning of fermentation (Fleet, 2003). Based on the results, we chosen three yeasts that showed best performance. These yeasts were used to perform fermentative power test along and in mix in Coda di Volpe must. The results (Fig.3) showed that all yeast as along as mix, were able to start and complete the wine fermentation. Moreover, the abc mix showed the best fermentative power as demonstrated by higher CO2 production. 3.4 Volatile compounds The results obtained from SPME-GC/MS analysis on the product of microvinification in batch, were reported in Table 1. The Esters are considered important to the sensory properties of wines, contributing with positive aroma, essentially with fruity notes (Pereira et al., 2014). The results (Fig.4) showed that esters were one of the most abundant groups (about 55%) as they are secondary aromas. While, the higher alcohols may be present Figure 3: fermentative power of single and mix yeast selected, evaluated by loss in CO2 Figure 4: principal VOC classes on Coda di Volpe wine, determined by SPME-GC/MS, reported in percentage. a: YTECCVWL7; b: YTECMCVWL4; c: YOCMCVWL4; ab: YTECCVWL7+ YTECMCVWL4; ac: YTECCVWL7+ YOCMCVWL4; bc: YTECMCVWL4+ YOCMCVWL4; abc: YTECCVWL7+ YTECMCVWL4+ YOCMCVWL4 10 in healthy grapes, but seldom occur in significant amount, they are essentially formed either from sugar catabolism or from amino acid decarboxylation and deamination and they have high perception threshold. They commonly account for about 50% of the aromatic constituents of wine, excluding ethanol. Moreover, the terpenes are varietal compounds present in grapes, especially in skins, o arise from glycosil precursors and they have low perception threshold. In conclusion, we selected the mix abc, to apply in future winemaking process on large scale, because it showed the best fermentative power and a balance in the amount of esters and alcohols products, and a higher content of terpenes (2%), being these volatile compounds classes responsible to modulation of wine aroma and flavor. References Arnous A., Makris D., Kefalas P., 2002, Correlation of pigment and flavanol content with antioxidant properties in selected aged regional wines from Greece, Journal of Food Composition and Analysis, 15, 655-665. Cavazza A., Grando M.S., Zini C.,1992, Rilevazione della flora microbica di mosti e vini, Vignevini 9, 17–20. Ciani M., Maccarelli F., 1997, Oenological properties of non-Saccharomyces yeasts associated with wine- making. World Journal of Microbiology & Biotechnology 14, 199-203. Ciani M., Beco L., Comitini F., 2006, Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations. International Journal of Food Microbiology, 108, 239–245. Ciani M., Comitini F., 2015, Yeast interactions in multi-starter wine fermentation. Current Opinion in Food Science, 1, 1–6. Table 1: Volatile compounds identified in microvinification with single and different combination of selected yeasts. Each value is expressed in µg/L and is the mean of three replicates ± sd (standard deviation) a b c ab ac bc abc odor a µg/L sd µg/L sd µg/L sd µg/L sd µg/L sd µg/L sd µg/L sd Esters (14) ethyl acetate 3166,6 ± 241, 1134,6 ± 118,2 871,4 ± 20,3 3110,3 ± 44,9 1840, ± 4,1 2066,1 ± 153, 2704,3 ± 54,4 fruity isobutyl acetate 7,7 ± 0,6 19,8 ± 2,1 nd 19,4 ± 0,2 9,6 ± 0,02 nd 8,90 ± 0,2 fruity Ethyl butanoate 121,1 ± 9,2 90,4 ± 9,4 92,2 ± 2,1 178,0 ± 2,5 117,2 ± 0,2 126,5 ± 9,4 123,3 ± 2,4 floral, ethyl i l 9,27 ± 0,7 10,54 ± 1,1 8,26 ± 0,1 16,50 ± 0,2 11,75 ± 0,03 15,76 ± 1,1 9,34 ± 0,1 sweet fruit Ethyl h 1153,3 ± 87,9 1284,8 ± 133,9 760,1 ± 17,7 1693,1 ± 24,4 1112, ± 2,5 1100,3 ± 81,9 1034,6 ± 20,8 fruity Hexyl acetate 8,5 ± 0,6 63,6 ± 6,6 6,7 ± 0,1 26,1 ± 0,3 40,2 ± 0,01 7,1 ± 0,5 9,2 ± 0,1 herbaceou ethyl lactate nd nd 7,8 ± 0,2 nd 112,0 ± 0,3 54,2 ± 4,1 nd Milk,butter Ethyl octanoate 6493,1 ± 495, 9094,5 ± 947,9 6364, ± 148, 8058,5 ± 116, 7898, ± 17,8 6422,6 ± 478, 5638,2 ± 113, sweet fruit isoamyl acetate 783,2 ± 59,7 1332,8 ± 138,3 539,5 ± 12,6 1322,2 ± 19,1 1013, ± 2,3 940,6 ± 70,0 803,8 ± 16,1 banana Ethyl d 3612,17 ± 275, 4112,4 ± 301,3 3172, ± 74,1 4270,2 ± 61,7 2767, ± 100, 3400,6 ± 250, 2560,7 ± 51,5 oily, fruity diethyl i 13,7 ± 1,1 16,3 ± 1,7 18,6 ± 0,4 17,6 ± 0,2 38,1 ± 0,1 45,3 ± 3,3 29,8 ± 0,6 fruity ethyl 9- d 848,8 ± 64,7 2496,1 ± 260,1 2795, ± 65,3 1720,8 ± 24,7 3298, ± 70,4 1267,5 ± 94,7 1075,9 ± 21,5 - Phenethylaceta 351,4 ± 26,8 306,4 ± 10,1 328,8 ± 7,6 443,5 ± 9,1 491,5 ± 20,3 349,7 ± 25,5 403,5 ± 8,2 flowery ethyl laurate 345,5 ± 26,3 410,6 ± 42,8 269,2 ± 6,2 482,2 ± 6,9 188,4 ± 10,2 337,8 ± 30,3 271,5 ± 5,7 sweet Alcohols (8) Isobutanol 733,7 ± 55,9 962,7 ± 100,3 303,3 ± 7,1 1111,5 ± 16,1 790,3 ± 1,7 736,4 ± 54,8 857,5 ± 17,6 fusel isoamyl alcohol 10629,7 ± 810, 10340, ± 1077, 8088, ± 188, 13202, ± 190, 9897, ± 22,4 11728, ± 873, 10553, ± 212, fusel 4-methyl-1- l nd 10,5 ± 1,1 5,5 ± 0,1 nd nd 6,4 ± 0,4 3,9 ± 0,1 almond 3-methyl 1- nd 15,3 ± 1,6 17,6 ± 0,4 13,5 ± 0,2 11,9 ± 0,03 16,9 ± 1,6 10,7 ± 0,2 vinous 1-hexanol 53,1 ± 4,05 6,8 ± 0,7 38,7 ± 0,9 52,5 ± 0,7 9,1 ± 0,02 nd 45,8 ± 0,2 herbaceou 2-ethyl hexanol nd nd 4,3 ± 0,1 nd nd nd 8,7 ± 0,2 mushroom 2,3-butandiol 33,6 ± 2,5 nd nd 18,3 ± 0,2 nd 17,6 ± 1,3 9,4 ± 0,2 butter phenethyl l h l 3122,59 ± 238, 1084,6 ± 113,1 1621, ± 37,8 2838,3 ± 41,1 1878, ± 4,2 2147,2 ± 159, 2256,5 ± 45,1 rose Acids (3) acetic acid 232,9 ± 17,7 31,0 ± 3,2 24,8 ± 0,6 255,4 ± 3,6 69,1 ± 0,1 62,9 ± 4,6 237,9 ± 4,9 vinegar octanoic acid 391,6 ± 29,8 65,3 ± 6,8 386,4 ± 9,1 12,2 ± 0,1 869,5 ± 1,9 475,1 ± 35,3 512,6 ± 10, 2 fatty decanoic acid 352,1 ± 26,8 69,3 ± 7,2 486,6 ± 11,4 128,2 ± 1,8 894,6 ± 2,1 533,3 ± 39,7 504,4 ± 10,1 fatty carbonyl comp (2) acetoino 29,5 ± 2,2 38,9 ± 4,1 nd 61,0 ± 0,8 42,5 ± 0,1 nd nd butter acetaldehyde 76,3 ± 5,8 14,0 ± 1,4 30,4 ± 0,7 61,3 ± 0,8 47,2 ± 0,1 25,6 ± 1,9 93,9 ± 1,8 ripe apple Terpenes (6) beta farnesene 11,1 ± 0,8 16,3 ± 1,7 17,6 ± 0,4 16,6 ± 0,2 27,5 ± 0,1 20,8 ± 1,5 22,4 ± 0,4 citrus alfa-farnesene 5,2 ± 0,4 12,9 ± 1,3 11,4 ± 0,2 11,9 ± 0,1 17,6 ± 0,11 19,7 ± 1,4 14,6 ± 0,3 wood nerolidol 93,7 ± 7,1 186,6 ± 19,3 175,2 ± 5,1 180,1 ± 2,1 129,6 ± 0,3 151,3 ± 11,2 173,6 ± 3,4 wood 2,3- dih d f l 60,7 ± 4,6 97,7 ± 5,6 92,5 ± 3,7 80,0 ± 0,4 96,9 ± 0,3 75,3 ± 6,5 143,5 ± 4,1 swee farnesol 85,3 ± 9,3 98,2 ± 4,6 94,1 ± 3,8 80,2 ± 0,6 78,1 ± 0,3 80,3 ± 9,7 112,7 ± 3,2 flower beta citronellol nd nd 14,5 ± 0,3 nd 9,3 ± 0,02 10,6 ± 0,7 39,2 ± 0,7 citrus a Based on flavornet (www.flavornet.org) and pherobase (www.pherobase.com) online databases nd: not detected 11 Constanti M., Reguant C., Poblet M.,Zamora F., Mas A., Guillamòn J.M., 1998, Molecular analysis of yeast population dynamics: effect of dioxide and inoculums on must fermentation, International Journal of Food Microbiology, 41, 169-175. 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