LYCOPENE AND [Beta]-CAROTENE RECOVERY FROM FERMENTED TOMATO WASTE AND THEIR ANTIOXIDANT ACTIVITY

Abstract

Waste generated from tomato processing poses disposal challenge even though it is a potential source of bioactive compounds. Acetone/ethanol/hexane mixture was used to recover lycopene and β-carotene from tomato pomace obtained from tomato must fermentation (pH of 4.11, 3.40, 3.20; temperature of 15, 20°C). Tomato must pH and temperature influenced (P<0.05) both lycopene and β-carotene recovery from the pomace. The highest total antioxidant activity and reducing power values were obtained from the tomato pomace of must pH 3.20, and fermented at 20°C. Tomato pomace from winemaking is a rich source of the bioactive compounds, lycopene and β-carotene, and possess substantial levels of antioxidant activity.

Headnote

Waste generated from tomato processing poses disposal challenge even though it is a potential source of bioactive compounds. Acetone/ethanol/hexane mixture was used to recover lycopene and β-carotene from tomato pomace obtained from tomato must fermentation (pH of 4.11, 3.40, 3.20; temperature of 15, 20°C). Tomato must pH and temperature influenced (P<0.05) both lycopene and β-carotene recovery from the pomace. The highest total antioxidant activity and reducing power values were obtained from the tomato pomace of must pH 3.20, and fermented at 20°C. Tomato pomace from winemaking is a rich source of the bioactive compounds, lycopene and β-carotene, and possess substantial levels of antioxidant activity.

Keywords: fermented tomato waste, fermentation temperature, bioactive compounds, lycopene, β-carotene

Introduction

Bioactive compounds have recently been a major subject of research. The interest in bioactive compounds probably stems from their ability to scavenge free radicals, to act as antibacterial and antiviral agents, immune system stimulators, and cell proliferation and apoptosis regulators (Kris-Etherton et al., 2002). Fruits and vegetables are a major source of bioactive compounds (Steinmetz and Potter, 1991). During processing of fruits and vegetables, wastes are generated, and their disposal poses a major challenge to the environment. As a result, bioactive compound recovery from food waste have been studied (Nagarajaiah and Prakash, 2011; Xu et al., 2012; Farcas et al., 2013).

Tomato has many bioactive compounds, including lycopene, β-carotene, vitamins E and C, phenolics and flavonoids (Kaur et al., 2002; Periago and Garcia-Alonso, 2009). Lycopene is known to protect humans against prostate and other cancers (Kris-Etherton et al., 2002). Significant correlation between the lycopene content of tomato and antioxidant activity was reported (Martínez-Valverde et al., 2002). Tomato processing generates waste, mainly in the form of seeds and peels. The skin and the seeds are collectively known as pomace. When juice is extracted from tomato the waste generated is about 20-30% of the total tomato fruit (Haddadin et al., 2001). Studies on tomato fruits have indicated that the principal source of its bioactive compounds is the peel (George et al., 2004; Chandra and Ramalingam, 2011).

Bioactive compound recovery from grape pomace (Hogan et al., 2010; Ghafoor et al., 2011), apple pomace (Grigoras et al., 2012), industrial tomato waste (Riggi and Avola, 2008; Naviglio et al., 2008) and mango peels (Tunchaiyaphum et al., 2013) have been studied. Since tomato waste poses disposal challenges, and is a source of bioactive compounds, the objective of this research was to evaluate the amount of lycopene and β-carotene recovered from the waste generated from tomato after winemaking at different fermentation temperatures and different tomato must pH levels, and, in addition, to evaluate their antioxidant activity.

Materials and methods

Source of material and material preparation

Tomato (Lycopersicon esculentum var. Hong Xia Hybrid No.2) fruits were purchased from the Zhenjiang local market in China. Spoiled tomatoes were removed from the lot. They were thoroughly washed several times with tap water, sterilized with 2% potassium metabisulphite, rinsed several times with distilled water, and the water dried with napkin paper. They were then cut into smaller pieces with a sterilized knife and blended with a sterilized Kenwood blender (Philips HR 2006, China) to obtain tomato must.

Fermentation

The initial pH of the tomato must was 4.11, and this was changed to two other levels, 3.40, and 3.20 using tartaric acid. The experiments conducted with the musts with pH level 4.11 were designated as the Control. Triplicate tomato musts were fermented at temperatures 15°C and 20°C, using the yeast culture (Saccharomyces bayanus, BV 818) of concentration 0.03 g/100 mL prepared as described by Owusu et al. (2014). Tomato must of volume 4.5 L was inoculated with 0.18 L of inoculum. During fermentation, which lasted for 12 and 15 days at 20°C and 15°C respectively (Results not shown), the must was in contact with the wine produced. After fermentation, the tomato wine was separated from the pomace, and stored at -20°C until needed for further experiment.

Determination of moisture content of tomato pomace

The moisture content of the tomato pomace was determined using AOAC (2000) and the value obtained ranged from 78.02 ±2.43 to 79.43 ±2.43%.

Determination of pH, titratable acidity and ethanol content of tomato wine

The pH of the tomato wine was measured with a pH Meter (PHS-2C Precision pH/mV meter, China). Briefly, the pH Meter was calibrated with buffer solutions of pH 7 and 4. After this, the electrode of the pH Meter was placed in the wine and the pH value was recorded. Titratable acidity (TA) was determined as described b y Sadler and Mulphy (2004), and the ethanol content was measured by the method described by Caputi et al. (1968). All measurements were triplicated.

Lycopene and beta-carotene determination

Total lycopene and ß-carotene content of the wines/tomato pomace were extracted following the method described by Fish et al. (2002). Triplicate samples (0.5 g) were weighed into vials. A mixture of hexane: acetone: ethanol [10: 5: 5 (v/v/v)] was used for the extraction. Five milliliters of acetone containing 0.05% (w/v) butylated hydroxytoluene (BHT), 5 mL of 95% ethanol, 10 mL hexane and 0.1 g CaCO3 were added to the sample. The mixture obtained was centrifuged at 5000 rpm for 10 min and afterwards combined with 3 mL distilled water. In the following procedure, the mixture was shaken for phase separation using an incubator shaker (QYC 211 Incubator Shaker, Shanghai Test Equipment Co. Ltd) at a speed of 160 rpm for 15 min. After shaking, the hexane layer (non-polar) containing carotenoids was transferred into a separate test tube and shortly thereafter measured spectrophotometrically at absorbances 505, 453 and 663 nm blanked with hexane and calculated as described by Nagata and Yamashita (1992):

Lycopene (mg/100 gFW) = -0.0458 A663 + 0.372 A505 - 0.0806 A453

ß-carotene (mg/100 gFW) = 0.216 A663 - 0.304 A505 + 0.452 A453,

where FW means fresh weight. The values obtained were then expressed as mg/100 g DW (dry weight).

Determination of total antioxidant activity

The total antioxidant activity (TAA) was determined by the phosphomolybdate assay (Prieto et al., 1999). Briefly, an aliquot of diluted sample (0.3 mL), obtained as in the lycopene and ß-carotene determination, was combined in a vial with 3 mL of molybdate reagent solution (0.6 M sulphuric acid, 28 Mm sodium phosphate and 4 Mm ammonium molybdate). The vials were capped and incubated in a water bath at 95°C for 90 min. The sample mixture was cooled to room temperature, and the absorbance was measured at 695 nm against a blank. Ascorbic acid (diluted in 13% ethanol v/v) was used to prepare a standard curve in the range 20-100 mg/L, and the total antioxidant activity was expressed in terms of milligram per liter ascorbic acid equivalent (AAE mg/L). All measurements were triplicated.

Reducing power

The reducing power (RP) of the sample was determined according to the method of Oyaizu (1986). Diluted sample of volume 0.6 mL (obtained as in the lycopene and ß-carotene determination) was mixed with 1.5 mL phosphate buffer (0.2 M, pH 6.6) and 1.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min and 1.5 mL of 10% trichloracetic acid was added to the mixture to terminate the reaction. It was then centrifuged at 5000 rpm for 10 min and 1.5 mL of the supernatant solution was mixed with 1.5 mL of distilled water. Freshly prepared 0.1% ferric chloride solution of volume 0.3 mL was added and the absorbance was read at 700 nm. Increased absorbance of the reaction mixture indicates elevated reducing power. Reducing power was expressed as ascorbic acid equivalents in mg/L (AAE mg/L). Determinations were made in triplicates.

Statistical analysis

Experimental results were reported as mean ± standard deviation. The data was analyzed using ANOVA, and differences in means were determined by using the Duncan's Multiple Range Test. Differences were deemed to be significant at P<0.01.

Results and discussion

Compositional attributes of tomato wine

The tomato wines recorded compositional attribute shown in Figures 1, 2, and 3. Must pH had effect (P<0.01) on the pH of the wines produced.

With the exception of the Control, the effect of fermentation temperature on the wine pH was also significant. The TA values of the tomato wines were higher (P<0.01) at the higher fermentation temperature. This may be due to the higher production of organic acids by higher yeast population at 20°C than at 15°C (Jackson, 2008). The effect of must pH and the fermentation temperature on ethanol content of the wine was not significant (P>0.01) even though there were some slight variations. The lycopene and β-carotene contents of the tomato wines are shown in Figure 3. The effect of both must pH and the fermentation temperature on the contents of both lycopene and β-carotene of the wines was significant (P<0.01).

The effect of pH on the concentration of lycopene and β-carotene in the tomato wines is similar to the results reported for phenolic compounds extracted from mango peels (Tunchaiyaphum et al., 2013). There was a general reduction in the contents of both bioactive compounds when fermentation temperature increased from 15°C to 20°C.

This may be due to the higher adsorption of bioactive compounds b y the yeast at the higher temperature which produced higher yeast population (Balík, 2006).

Lycopene and β-carotene recovery from tomato pomace

Lycopene and β-carotene were extracted from the tomato pomace using the solvent extraction method. The solvent mixture (hexane: acetone: ethanol) used in this study was in the ratio [10: 5: 5 (v/v/v)]. Extraction of lycopene with the same solvent mixture as the one used in this study was found to be more efficient than the one where either hexane or ethyl acetate alone was used (Kumar et al., 2013).

Lycopene and β-carotene recovered from the tomato pomace are shown in Table 1. The lycopene concentration of the tomato pomace ranged from 2.48-3.06 mg/100 g DW (representing 2.25-2.78% of the tomato pomace, Table 2), and this is comparable to the results reported earlier (Naviglio et al., 2008), but lower than the result of Chun et al. (2009). This may be due to the fact that the method used in the previous work was supercritical fluid extraction, which is different from the one used in the present study. The β-carotene concentration of the tomato pomace, 0.78-0.93 mg/100 g DW (representing 0.71-0.85%, Table 2) is lower than the results reported for tomato waste (Riggi and Avola, 2008), and this may be due to the differences in the source of waste and solvent ratios used for the two experiments. The results are similar to what was reported previously where the lycopene content of tomato was higher than β-carotene (Chérif et al., 2010). The recovery of both bioactive compounds from the pomace were significantl y influenced by both must pH and fermentation temperature.

The lycopene recovery was higher (P<0.01) in the pomace obtained from the fermentation conducted at 15°C than that at 20°C for all levels of pH. Higher yeast population at higher fermentation temperature (Results not shown) might have led to higher adsorption of bioactive compounds by the yeast (Balík, 2006). The pomace obtained from must pH 3.20 recorded the highest content (P<0.01) of lycopene at both fermentation temperatures. In a previous work on extraction of carotenoids from tomato waste, pulsed electric field treatment (5 kV/cm) was reported to have enhanced the permeability and extraction of carotenoids (Luengo et al., 2014).

In this study, low pH could enhance the porosity of the tomato pomace and diffusivity of lycopene from the pomace, and hence lead to increased recovery. β-carotene also showed the same trend as lycopene, however, the exception was the content obtained from the fermentation conducted with must pH 3.20. Similar results on the effect of pH on the extraction yield of phenolics from mango peels were reported (Tunchai yaphum et al., 2013). The combined effect of must pH and fermentation temperature on the amount of lycopene and β-carotene recovered from the pomace was also highly significant (P<0.01). The amount of dependent variable variance accounted for by the corrected model was measured by the R2 value. The variance accounted for by the two main effects, must pH and fermentation temperature as well as their interaction was 100 and 98.8% for lycopene and β-carotene contents of the tomato pomace respectively (Tables 3 and 4). This means that must pH, fermentation temperature as well as their interaction accounted for 100 and 98.8% of the variation in lycopene and β-carotene contents, respectively, of the tomato pomace. During fermentation, the pomace was in contact with the wine, and this was separated after the fermentation. Thus, the different pH and temperature conditions in the fermenting media may influence the composition of bioactive compounds in the pomace significantly. Ethanol can influence the porosity of the pomace to enhance the diffusivity and extractability of the carotenoids. However, the content of ethanol was not significantly different, so the ethanol content of the wines may not have any significant effect on the contents of the lycopene and carotene extracted.

The lycopene content of the tomato waste was expressed as a percentage of lycopene in tomato wine (Table 5), and the values obtained ranged from 1.70-6.67%. This indicates that higher concentration of lycopene was found in the tomato pomace than the tomato wine (George et al., 2004; Chandra and Ramalingam, 2011). In terms of the percentages, the values obtained for fermentation temperature 20°C were higher than those for 15°C. β-carotene in the tomato pomace was also expressed as a percentage of that in the tomato wine (Table 5), and the values obtained were in the range 1.71-17.68%.

Higher concentrations of β-carotene were also obtained from the pomace than in the wine (George et al., 2004; Chandra and Ramalingam, 2011).

Antioxidant properties of the extract from the tomato pomace

The antioxidant properties of the extract from the tomato pomace were determined using total antioxidant activity (TAA) and ferric reducing power (RP) methods.

The TAA of the wines was determined using the phosphomolybdenum assay in which the antioxidant reduces Mo (VI) to Mo (V) leading to the formation of green phosphate/Mo (V) complex under acidic conditions, which has a maximum absorbance at 695 nm (Shajiselvin and Muthu, 2011). A compound's RP is linked to its ability to transfer electrons and hence an indicator of its potential antioxidant activity. The results on TAA and RP are shown in Fig. 4. The different values of the RP of the extracts indicate that they may have different abilities to react with free radicals to stabilize and terminate radical chain reaction (Shimada et al., 1992).

The TAA and the RP of the tomato pomace extract were influenced by both must pH and fermentation temperature (P<0.05). The results are similar to those of the bioactive compounds, lycopene and β-carotene. The acidity level and the fermentation temperature may influence the porosity of the pomace, increase the permeability of the extracting solvent, and hence diffusivity of the lycopene and β-carotene. In a previous work on extraction of carotenoids from tomato waste, pulsed electric field treatment (5 kV/cm) was reported to have enhanced the permeability and extraction of carotenoids (Luengo et al., 2014). The highest TAA and RP were obtained from the tomato pomace which had must pH 3.20 and fermentation temperature 20°C. Higher temperature may encourage the extraction of the bioactive compounds more than the lower one. The highest TAA and RP for the tomato pomace obtained from the must of pH and fermentation temperature 20°C may be due to higher lycopene and β-carotene contents than the others. This agrees with the results of a previous study where higher carotenoid content of tomato waste was found to have the highest antioxidant capacity (Luengo et al., 2014). The fermented pomace exhibited substantial levels of antioxidant activity.

Conclusion

The recovery of lycopene and β-carotene from tomato pomace obtained after fermentation was studied. Both must pH and fermentation temperature influenced the concentrations of lycopene and β-carotene recovered from the pomace. The tomato pomace was found to be a rich source of lycopene and β-carotene. The best fermentation conditions which gave the highest TAA and RP were must pH 3.20, and fermentation temperature 20°C. Fermented tomato pomace exhibited substantial level of antioxidant activity.

Acknowledgements

The funding for this project was provided by Open Fund of Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products (JAPP2010-6), the Jiangsu Province Science and Technology Support Project (BE2011382).

Conflict of interest

None of the authors has any conflict of interest.

Ethical statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

References

References

AOAC. 2000. Official Methods of Analysis of the Association of Official Analytical Chemists. 17th ed. AOAC International, Maryland.

Balík, J. 2006. Dynamics of changes in total anthocyanins during the fermentative maceration of grapes. Horticultural Science (Prague), 33 (3), 103-107.

Caputi, A.J.R., Masao, U., and Brown, T. 1968. Spectrophotometric determination of ethanol in wine. American Journal of Enology and Viticulture, 19, 160-165.

Chandra, H. M., and Ramalingam, S. 2011. Antioxidant potentials of skin, pulp, and seed fractions of commercially important tomato cultivars. Food Science and Biotechnology, 20, 15-21.

Chérif, J. K., Jémai, S., Rahal, N. B., Jrad, A., and Trabelsi-Ayadi, M. 2010. Study of antioxidant and antiradical capacity of fresh and industrial waste of Tunisia tomato. Valorization of Tunisian tomato in bioactive molecules. Tunisian Journal of Medicinal Plants and Natural Products, 4, 116-125.

Chun, Y., John, S., Sophia, J. X., Yueming, J., Dong, L. 2009. Effects of supercritical fluid extraction parameters on lycopene yield and antioxidant activity. Food Chemistry, 113 (4), 1088-1094.

Grigoras, C.G., Destandau, E., Lazar, G., Elfakir, C. 2012. Bioactive compounds extraction from pomace of four apple varieties. Journal of Engineering Studies and Research, 18 (1), 96-103.

Farcas, A., Tofana, M., Socaci, S., Scrob, S., Salanta, L., Borsa, A. 2013. Preliminary study on antioxidant activity and polyphenols content in discharged waste from beer production. Journal of Agroalimentary Processes and Technology, 19 (3), 319-324.

Fish, W.W., Perkins-Veazie, P., Collins, J.K. 2002. A quantitative assay for lycopene that utilizes reduced volumes of organic solvents. Journal of Food Composition and Analysis, 15, 309-317.

George, B., Kaur, C., Khurdiya, D. S., Kapoor, H. C. 2004. Antioxidants in tomato (Lycopersium esculentum) as a function of genotype. Food Chemistry, 84, 45-51.

Ghafoor, K., Al-Juhaimi, F., Choi, Y. H. 2011. Effects of grape (Vitis labrusca B.) peel and seed extracts on phenolics, antioxidants and anthocyanins in grape juice. Pakistan Journal of Botany, 43 (3), 1581-1586.

Haddadin, M. S. Y., Abu-Reesh, I. M., Haddadin, F. A. S., Robinson, R. K. 2001. Utilization of tomato pomace as a substrate for the production of vitamin B12-a preliminary appraisal. Bioresource Technology, 78, 225-230.

Hogan, S., Zhan, L., Li, J., Sun, S., Canning, C., Zhou, K. 2010. Antioxidant rich grape pomace extract suppresses postprandial hyperglycemia in diabetic mice by specifically inhibiting alpha-glucosidase. Nutrition and Metabolism, 7 (71), 1-9

Jackson, R. S. 2008. Wine Science: Principles and Applications, 3rd ed. Elsevier Inc., USA.

Kaur, R., Savage, G. P., Dutta, P. C. 2002. Antioxidant vitamins in four commercially grown tomato cultivars. Proceedings of the Nutrition Society of New Zealand, 27, 69-74.

Kris-Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E., Hilpert, K. F., Griel, A. E., Etherton, T. D. 2002. Bioactive compound in foods: their role in prevention of cardiovascular disease and cancer. American Journal of Medicine, 113, 71-88.

Kumar, S. R., Sundar, S. S., Afandi, A., and Rahman, S. (2013). Optimization of lycopene from Malaysian all season coloured fleshy fruits. International Journal of Biology, Pharmacy and Allied Sciences, 2 (7), 1455-1467.

Luengo, E., Álvarez, I., and Raso, J. (2014). Improving carotenoid extraction from tomato waste by pulsed electric fields. Frontiers in Nutrition, 12 (1), 1-10.

Martínez-Valverde, I., Periago, M. J., Provan, G., Chesson, A. 2002. Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato (Lycopersicon esculentum). Journal of the Science of Food and Agriculture, 82, 323-330.

Nagarajaiah, S. B., Prakash, J. 2011. Chemical composition and antioxidant potential of peels from three varieties of banana. Asian Journal of Food and Agro-Industry, 4 (01), 31-46.

Nagata, M.,Yamashita, I. 1992. Simple method for simultaneous determination of chlorophyll and carotenoids in tomato fruit. Nippon Shakuhin Kogyo Gakkaish, 39, 925-928.

Naviglio, D., Pizzolongo, F., Ferrara, L., Naviglio, B., Aragòn, A., Santini, A. 2008. Extraction of lycopene from industrial tomato waste in water using the extractor Naviglio®. African Journal of Food Science, 2 (2), 037-044.

Owusu, J., Ma, H., Wang, Z., Afoakwa, N. A., Amissah, A. 2014. Volatile profiles of tomato wine before and after ageing. Maejo International Journal of Science and Technology, 8 (02), 129-142.

Oyaizu, M., 1986. Studies on products of browning reaction: Antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition, 44, 307-315.

Periago, M. J., Garcia-Alonso, J. 2009. Changes in bioactive compounds, folates and antioxidant properties of tomatoes (Lycopersicon esculentum) during vine ripening. International Journal of Food Science and Nutrition, 60 (8), 694-708.

Prieto, P., Pineda, M., Anguilar, M. 1999. Spectrophotometric quantification of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Analytical Biochemistry, 269, 337-341.

Riggi, E,., Avola, G. 2008. Fresh tomato packing houses waste as high added-value biosource. Resources, Conservation and Recycling, 53, 96-106.

Sadler, G.D., Murphy, P.A. 2010. pH and Titratable Acidity. In: Nielsen, S. S. (Ed.), Food Anal ysis. Springer Science + Business Media, LLC, New York. p. 219-238.

Shimada, K., Fujikawa, K., Yahara, K., and Nakamura, T. 1992. Antioxidative properties of xanthan on the antioxidation of soybean oil in cyclodextrin emulsion. Journal of Agriculture and Food Chemistry, 40, 945-948.

Steinmetz, K. A., Potter, J. D. 1991. Cancer causes control. American Journal of Medicine, 2 (6), 427-442.

Tunchaiyaphum, S., Eshtiaghi, M. N., Yoswathana, N. 2013. Extraction of bioactive compounds from mango peels using green technology. International Journal of Chemical Engineering Application, 4 (4), 194-198.

Xu, P., Bao, J., Gao, J., Zhou, T., Wang, Y. 2012. Phenolic antioxidants from tea fruit peel. BioResources, 7 (2), 2431-2443.