283 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XIII, Issue 4- 2014, pag. 283 - 289 EFFECT OF TREATMENT PARAMETERS ON THE CAROTENOID EXTRACTION FROM TOMATO PEELS OF BULGARIAN INDUSTRIAL VARIETIES Milena NIKOLOVA1, *Tsvetko PROKOPOV 1, Daniela GANEVA 2, Galina PEVICHAROVA 2 1Department of Environmental Engineering, University of Food Technologies, Plovdiv, Bulgaria 2Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgaria tsvetko_prokopov@abv.bg *Corresponding author Received October 15th 2014, accepted December 29th 2014 Abstract: Tomato by-products are an attractive source of natural carotenoids. This study was carried out to investigate the extractability of carotenoids from tomato peels of two Bulgarian industrial varieties, named “Stela” and “Karobeta”, and to assess the effects of the extraction parameters (time, extraction steps, temperature, solid/liquid ratio and solvent type) on the yield of carotenoids. Individual carotenoid identification in dried tomato peels was carried out by using a HPLC system. The content of total carotenoids, lycopene and -carotene in the obtained extracts was measured spectrophotometrically. Carotenoid recovery was significantly (p < 0.05) affected by the investigated extraction parameters. The maximum amounts of total carotenoids (111.58±2.11 and 76.59±2.13 mg/100g), -carotene (54.69±2.15 and 64.70±1.42 mg/100g) and lycopene (49.07±0.19 and 6.51±0.57 mg/100g) extracted from dried tomato peels of “Stela” and “Karobeta” varieties, respectively, were obtained by using acetone as solvent, for three successive extraction steps of 30 min, at 40oC and solid/liquid ratio of 1:30. It was experimentally established that tomato peels of “Stela” variety are suitable for lycopene and -carotene recovery, while those of “Karobeta”variety for the recovery of -carotene only. Keywords: vegetable, processing, waste, by-products, lycopene, -carotene, extractability 1. Introduction The food processing industry produced large quantity of waste by-products. One- tird of the used product in fruit and vegetable processing was reported that being discarded which creates a significant environmental problem [1]. Today, food wastes including fruit and vegetable by-products are considered as a cheap source of valuable components since the existent technologies allow the recovery of target compounds and their recycling inside food chain as functional additives in different products [2]. For example, tomato is a food product containing large amounts of high added- value compounds such as carotenoids, mainly in the form of lycopene and -carotene. At present, large quantities of tomato skin and outer pericarp tissue are discarded as a waste product from the peeling operation. About 10-40 % of the total tomato processed for tomato products are as skins and seeds. The quantity of wastes generated during tomato production and processing, combined with Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 284 the beneficial characteristics of components of these wastes justifies the great interest of researchers and manufacturers in extracting carotenoids from tomato wastes [3]. By-products of tomato processing are attractive source of valuable bioactive components and colour pigments. These products can be used as functional foods, dietary supplements and can also be applied to cosmetic and pharmaceutical products. Extraction with organic solvents is a well-established method in the food industry. As the main tomato carotenoids are lipid-soluble, common organic solvents such as acetone, hexane, ethanol, ethyl acetate, methanol, petroleum ether and solvent mixtures in different ratios (e.g. 50:50 hexane-ethyl acetate, hexane- acetone, 50:25:25 hexane-acetone-ethanol) have been tested for carotenoids extraction. Although carotenoids extraction yields obtained from tomato by-products and comparison of efficiency among different solvents are presented in the literature [3- 8] there are limited data about the effect of treatment parameters on the carotenoids extraction from tomato by-products including the influence of tomato varieties. There are needs for further research concerning optimization of tomato carotenoids extraction and factors affecting carotenoids recovery from tomato by- products which could be applied on a commercial scale. This study was carried out to investigate the extractability of carotenoids from peels of two Bulgarian industrial tomato varieties and to assess the effects of extraction parameters, such as time, successive extraction steps, temperature, solid/liquid ratio and type of organic solvent on the yield of total carotenoids, lycopene and -carotene. 2. Materials and methods 2.1. Raw materials Two Bulgarian industrial tomato varieties named “Stela” and “Karobeta” were used in present study. Raw tomatoes were grown under field conditions within the Maritsa Vegetable Crops Research Institute, Plovdiv, Bulgaria. Tomatoes were harvested from the selected plants in technological maturity for each variety and were transported to the laboratory within 24 h. 2.2. Preparation of tomato peels Raw tomatoes were blanched at 95oC for 2 min, cooled on tap water and hand peeled. The obtained tomato peels were subsequently air dried at 25±1oC, ground in a laboratory mill (Bosh MKM 6003, Germany) and sieved through a 1.00 mm sieve. Moisture content of ground dry tomato peels was determined by gravimetric method at 105oC and was 4.61±0.21 % and 5.30±0.32 % for “Stela” and “Karobeta” variety, respectively. The obtained dry ground material was kept in glass jars closed with aluminium caps and wrapped with aluminium foil at -20oC until conduction of experiments. 2.3. Chemicals Acetone, n-hexane, ethanol, methanol, tetraclormethane, acetonitrile and methyl tert-butyl ether (MTBE) in analytical grade were purchased from Sigma (Germany). Used standards of lutein, lycopene and -carotene were purchased from Extrasynthese (France). 2.4. Carotenoids extraction The extraction of carotenoids was performed into 250 mL conical glass flask wrapped with aluminium foil. The flask was plased in a temperature-controlled (±1oC) water bath and continuously agitated with magnetic stirrer (VELP Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 285 Scientifica Aluminium Hot Plate Stirrer- ARE, Italy) at 400 rpm. Ground dry tomato peels (1.00 g) were plased in the extraction flask and stirred with extraction solvent at different extraction conditions, such as time (5, 10, 15, 30 and 40 min), temperature (20, 30, 40 and 50oC) and tomato peels/solvent ratio (1:5, 1:10, 1:20, 1:30 and 1:50). The obtained extract was vacuum filtered through filter paper MN640de and analysed for carotenoids content. The effect of extraction steps was examined as follows: Successive extractions with acetone were conducted under the same conditions as above for 30 min each at 20oC and peels/solvent ratio 1:10. At the end of the first extraction step, the mixture was vacuum filtered, the residue was collected, put again in the flask and re-extracted with another quantity of acetone. The whole procedure was repeated four times to complete the successive extraction steps. The carotenoids content of the extract in each successive extraction were determined. When the effect of type of organic solvent was studied the obtained extracts were vacuum filtered, the liquid fraction was vacuume dried (t < 40oC), redisolved in acetone and analysed for carotenoids content. 2.5. Determination of carotenoids content Total carotenoids, lycopene and - carotene contents in the extracts were measured spectrophotometrically (UV- VIS Helios Omega Spectrophotometer, Thermo Fisher Scientific, Madison, WI, USA) at max 448 and 472 nm against acetone as blank, according to Manuelyan [9]. Carotenoids content was expressed as mg/100g dry weight. 2.6. Carotenoids analysis For the identification of individual carotenoids dryed tomato peels were analysed by HPLC system (Waters, Milford, USA) composed of a UV-VIS deterctor (Waters 2487 Dual ), a Waters 1525 binary pump and thermostat (LCO 102), according to the method proposed by [10]. The HPLC system was equipped with a (Supelco Discovery HS) C18 column (25 cm x 4.6 mm, 5 m particle). A mobile phases of methanol:acetonitrile in ratio 8:2 (A) and MTBE (B) with following gradient elution were used: 95 % (A) and 5 % (B) initially, 95 % (A) and 5 % (B) in 3 min, 80 % (A) and 20 % (B) in 4.5 min, 65 % (A) and 35 % (B) in 10 min, 95 % (A) and 5 % (B) in 15 min. The flow rate was maintained at 1 mL.min-1, the column temperature at 30oC and detection was carried out at 270 nm and 290 nm. The analysis of the chromatographic data was carried out on a Breeze 3.30 (Waters, Milford, USA) software. The identification of major carotenoids in tomato dry peels was carried out by comparing the retention times and absorption spectra with reference standards as described by [10]. The calibration curves were linear from 5 to 50 g/mL (r2 > 0.99). The percentage of HPLC identified carotenoids in dried tomato peels was calculated as the ratio of the concentration of each carotenoid (based on the respective standard curve) to the sum of all identified carotenoids in the chromatogram, multiplied by 100 [4]. 2.7. Statistical analysis All experiments were run in triplicate. The data were analysed and presented as mean values with standard deviation. Statistical analysis was conducted by using of Statgraphics Centurion XVI Version 16.2.04 software (Statpoint Technologies Inc., USA). The analysis of variance technique, incl. Lavene’s test, ANOVA, and Duncan’s Multiple Range Test were Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 286 used to determine significant differences at 95 % confidence (p < 0.05) level. 3. Results and discussion 3.1. Carotenoids identification HPLC analysis of dried tomato peels of the two investigated varieties indicated that the main carotenoid was -carotene, while lycopene and lutein are also presented (Table 1). This is contrary to the results obtained of [3], who reported lycopene as the main carotenoid of processing tomato waste composed of skins and seeds. Tomato peels of “Stela” variety were riched in -carotene (61.8 %) and lycopene (35.3 %), while these of “Karobeta” variety were riched in -carotene (86.7 %). Table 1 Carotenoids concentration (%) in dried tomato peels Tomato varieties Carotenoids concentration (%) Lycopene -carotene Lutein Stela 35.34 61.78 2.86 Karobeta 8.09 86.72 5.17 3.2. Effect of extraction time The effect of time on carotenoids extraction from dried tomato peels was investigated by conducting experiments during the extraction with acetone in solid:liquid ratio 1:10 at 20oC. 0 10 20 30 40 50 60 70 80 5 10 15 30 40 Time, min C ar ot en oi ds c on te nt , m g/ 10 0g Total Caroten oids Beta-carotene Lycopene Fig.1. Effect of time on carotenoids extraction (peels/acetone 1:10, t = 20oC) from dried tomato peels of “Stela” variety The obtained results are shown in Figure 1 and Figure 2 for “Stela” and “Karobeta” variety, respectively. As it was observed, carotenoids content of the extracts depended on extraction time, showing a high intial rate of extraction that decreased with time until an almost equilibrium was riched. The experimental results indicated the suitable extraction time of 30 min for total carotenoids, lycopene and -carotene extraction from peels of the two tomato varieties. This is in accordance with the results obtained by another research [3]. 0 10 20 30 40 50 60 70 5 10 15 30 40 Time, min C ar ot en oi ds c on te nt , m g/ 10 0g Total Carotenoids Beta-carotene Lycopene Fig.2. Effect of time on carotenoids extraction (peels/acetone 1:10, t = 20oC) from dried tomato peels of “Karobeta” variety 3.3. Effect of extraction steps The effect of successive extraction steps on carotenoid extraction is illustrated in Table 2. It is clearly indicated that the total carotenoids, lycopene and -carotene contents in the obtained extracts were significantly (p < 0.05) affected by the number of extractions. As it was observed, no significant differences (p < 0.05) or in some cases decreasing was obtained for the carotenoids contents between third and fourth extraction steps. Triple extraction was found as adequate for all experiments. 3.4. Effect of temperature The carotenoids content obtained by three successive extraction of 30 min each with acetone in solid/liquid ratio 1:10 at Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 287 temperatures ranging from 20 to 50oC is presented in Table 3. Limiting factor for the choise of extraction temperature was the boiling point of acetone (56oC) and the need to avoid isomerisation and/or oxidation of carotenoids. The increase in extraction temperature generally increased the extractability of carotenoids. The contents of total carotenoids, lycopene and -carotene in the obtained extracts were significantly (p < 0.05) higher at 40oC than at 20oC. However, when the extraction temperature rose to 50oC, an important extractability of carotenoids was not noticed. Finally, at 50oC decreasing of the carotenoids content was oserved, excluding the content of -carotene in the extract from tomato peels of “Karobeta” variety. Following the obtained results, a suitable extraction temperature of 40oC was chosen for adequate extraction of carotenoids. 3.5. Effect of solid/liquid ratio Solid/liquid ratio is another factor which affects the extraction of carotenoids. An equilibrium between the use of high and low solid/liquid ratios, involving a balance between high costs and solvent wastes, on the one side, and avoidance of insufficient mixing and saturation effects, on the other side, has to be found to optimized value [5]. Table 2 Carotenoids content (mg/100g) in extracts from dried tomato peels (peels/acetone 1:10, 30 min extraction, t = 20oC) depending of successive extraction steps Parameter Tomato variety Extraction steps 1 2 3 4 Total carotenoids, mg/100g Stela 31.84±0.09a 44.45±1.05b 54.17±1.00c 54.58±0.82c Karobeta 12.05± 0.07a 14.78±0.43b 23.92±1.78c 20.38±1.26d Lycopene, mg/100g Stela 6.03±0.34a 10.59±0.30b 12.62±0.44c 12.71±0.35c Karobeta 0.61±0.06a 0.66±0.03a 1.62±0.07b 1.44±0.21b β-carotene, mg/100g Stela 24.07±0.81a 34.16±1.02b 41.44±0.57c 42.11±1.52c Karobeta 10.80±0.23a 13.96±0.27b 22.27±1.71c 18.94±1.05d The values are mean of three replicates  SD. Values bearing different lowercase letters (a, b, c, d) in the same row differ significantly (p < 0.05). Table 3 Carotenoids content (mg/100g) in extracts from dried tomato peels (peels/acetone 1:10, triple extraction of 30 min) depending of temperature Parameter Tomato variety Temperature of extraction, oC 20 30 40 50 Total carotenoids, mg/100g Stela 10.17±1.38a 23.62±1.65b 31.16±1.21c 26.92±1.10d Karobeta 19.28±0.28a 21.45±0.25b 23.35±0.55c 23.59±0.32c Lycopene, mg/100g Stela 1.36±0.31a 3.54±0.37 b 5.29±1.08c 3.36±0.28b Karobeta 1.59±0.07a 2.79±0.36b 3.61±0.03c 1.33±0.03a β-carotene, mg/100g Stela 8.08±0.97a 18.41±1.17b 27.4±1.10c 21.66±0.75b Karobeta 16.34±0.19a 17.15±0.12b 18.21±0.43c 20.39±0.53d The values are mean of three replicates  SD. Values bearing different lowercase letters (a, b, c, d) in the same row differ significantly (p < 0.05). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 288 Table 4 presents the effect of solid/liquid ratio on carotenoids extractability. The increased organic solvent volume increased the carotenoids extractability up to solid/liquid ratio of 1:30, than at ratio 1:50 it decreased. The solid/liquid ratio of 1:30 produced significantly (p < 0.05) higher contents of total carotenoids, -carotene and lycopene in all samples in comparison with the other ratios. Table 4 Carotenoids content (mg/100g) in extracts from dried tomato peels (triple extraction of 30 min with acetone, t = 20oC) depending of solid/liquid ratio Parameter Tomato variety Solid/liquid ratio 1:5 1:10 1:20 1:30 1:50 Total carotenoids, mg/100g Stela 42.13±0.25a 59.92±0.21b 84.41±0.19c 98.26±0.67d 58.16±5.54b Karobeta 26.40±0.46a 27.02±0.18a 29.26±0.30b 64.08±0.25c 27.17±0.32a Lycopene, mg/100g Stela 6.92±0.18a 10.75±0.55b 13.65±0.64c 16.94±1.01d 12.50±1.19c Karobeta 0.17±0.04a 1.63±0.26b 2.01±0.12c 3.9±0.12d 1.09±0.16 e β-carotene, mg/100g Stela 32.30±0.79a 41.65±2.57b 64.25±0.29c 73.98±1.89d 44.87±0.6 b Karobeta 24.81±0.18a 23.63±0.34b 25.21±0.22a 56.06±1.40c 23.88±0.98b The values are mean of three replicates  SD. Values bearing different lowercase letters (a, b, c, d, e) in the same row differ significantly (p < 0.05). 3.6. Effect of extraction solvent Solvent selection is usually considered as the most important factor [5]. The contents of total carotenoids, lycopene and - carotene in the extracts obtained by three successive extractions of 30 min with two different solvents and one solvents mixture at 40oC and solid/liquid ratio of 1:30 are presented in Table 5. The extraction efficiency was affected by the solvent type and its polarity. As indicated in Table 5, acetone presented significant (p < 0.05) higher content of carotenoids extracted from tomato peels of “Karobeta” variety, compared to the hexane and hexane/acetone/ethanol mixture, possibly due to better penetration of the acetone to plant cells where carotenoids are enclosed. Concerning “Stela” variety, no significant differences (p > 0.05) was observed for carotenoids content extracted from tomato peels using acetone and hexane, respectively. Table 5 Carotenoids content (mg/100g) in extracts from dried tomato peels (solid/liquid ratio 1:30, triple extraction of 30 min, t = 40oC) depending of used organic solvent Parameter Tomato variety Type of solvent Acetone Hexane Hexane/Acetone/ Ethanol 50/25/25 Total carotenoids, mg/100g Stela 111.58±2.11a 108.42±1.06a 96.83±2.11b Karobeta 76.59±2.13a 69.14±1.07b 65.95±0.01c Lycopene, mg/100g Stela 49.07±0.19a 49.05±0.21a 41.18±2.35b Karobeta 6.51±0.57a 4.66±0.25b 5.45±2.46a b β-carotene, mg/100g Stela 54.69±2.15a 51.78±0.77a 48.86±0.39b Karobeta 64.70±1.42a 59.63±0.75b 55.39±1.98c The values are mean of three replicates  SD. Values bearing different lowercase letters (a, b, c) in the same row differ significantly (p < 0.05). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIII, Issue 4 – 2014 Milena NIKOLOVA, Tsvetko PROKOPOV, Daniela GANEVA, Galina PEVICHAROVA, Effect of treatment parameters on the carotenoids extraction from tomato peels of Bulgarian industrial varieties, Food and Environment Safety, Volume XIII, Issue 4 – 2014, pag. 283 – 289 289 From the experimental results presented in Table 5 can be concluded that extraction of total carotenoids, lycopene and -carotene from tomato peels of “Stela” and “Karobeta” varieties with acetone is adequate. 4. Conclusion The extractability of carotenoids from dried peels of two Bulgarian industrial tomato varieties, named “Stela” and “Karobeta”, was investigated. The effects of extraction time, extraction steps, temperature, solid/liquid ratio, two organic solvents and one solvents mixture on the contents of total carotenoids, lycopene and -carotene in the obtained extracts were established. The maximum amounts of total carotenoids, lycopene and -carotene extracted from tomato peels of the investigated tomato varieties were obtained in our experiments using acetone for three successive extractions of 30 min, at 40oC and solid/liquid ratio of 1:30. Carotenoids contents in the extracts obtained at these optimal extraction conditions, for “Stela” and “Karobeta” varieties, respectively were as follow: total carotenoids of 111.58±2.11 and 76.59±2.13 mg/100g, -carotene of 54.69±2.15 and 64.70±1.42 mg/100g and lycopene of 49.07±0.19 and 6.51±0.57 mg/100g. Experimentally was established that tomato peels of “Stela” variety are suitable for lycopene and -carotene recovery, whiles these of “Karobeta”variety for the recovery only of -carotene. 5. References [1]. PROKOPOV Ts., Utilization of by- products from fruit and vegetable processing: a review, Journal of Food and Packaging Science, Technique and technologies, 3. 49-54, (2014) [2]. 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