Enzymatic Browning and Its Prevention - American Chemical Society

Chapter 16

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on August 24, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch016

Antioxidant Characteristics of Melanin-Related Products from Enzymatic Browning Reaction of Catechin in a Model System 1

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Hong-Sik Cheigh , Soo-Hyoun Um , and Chang Y. Lee 1

Department of Food Science and Nutrition, Pusan National University, Pusan 609-735, Korea Department of Food Science and Technology, Cornell University, Geneva, NY 14456 2

Antioxidant activity of melanin related products obtained from enzymatic oxidation of catechin was studied in a model system. Melanin related products were obtained from the catechinpolyphenol oxidase reaction at pH 6.5 and 25 °C at various time intervals. All catechin-enzyme reaction products (CERPs) were brown in varied intensities with increased absorption at 210-220, 380-390 nm, and 420-430 nm. CERPs obtained at the early stage of the reaction showed a higher antioxidant activity than those from the later stage. Antioxidant activity of CERPs may be explained by their abilities of hydrogen atom donation, free radical scavenging, and lipoxygenase inhibition. However, the exact mechanism of the antioxidant activity of CERPs cannot be explained until their chemical structures are elucidated. Enzymatic browning of fruit and vegetable products is mainly caused by the oxidative reactions of various polyphenol compounds catalyzed by polyphenol oxidase (PPO) in the tissues. The enzyme catalyzes the oxidation of diphenols to 0-quinones and further reaction leads to melanin, brown pigments. Some of these reaction products have shown specific chemical characteristics depending on the particular polyphenol compounds (1-3). The antioxidant activity from nonenzymatic browning reaction products in processed foods has been reported (4-7). There have been reports on the antioxidant activity of the enzymatic browning reaction products in apples (8-10). However, there has been no report on the antioxidant activity of the enzymatic browning reaction products from specific polyphenol compounds. Catechin, one of the major phenolic compounds found in many fruits, has shown various antioxidant activities such as tocopherol as a free radical scavenger in the lipid oxidation systems (11-13). On the other hand, catechin itself is the major cause of enzymatic browning acting as a good substrate for polyphenol oxidase in many food products, such as tea and fruits (14, 15). The objective of this study was to obtain information on the antioxidant activity of the polyphenol oxidase reaction products in a model system using catechin. 0097-6156/95/0600-0200$12.00/0 © 1995 American Chemical Society In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Materials and Methods Reaction Products and Their Absorption Spectra. Melanin related reaction products were prepared by the following method. A sample of 19.5 mL of catechin solution (2 mM) in Mc'Ilvaine's citric acid-phosphate buffer (0.02 M, pH 6.5) was mixed with 0.5 mL polyphenol oxidase (EC 1.14.18.1, tyrosinase; 1000 unit/mL) solution in the buffer and kept at 25 °C in a water bath while stirring. Samples of the catechin-enzyme reaction products (CERPs) were taken at 0, 1, 3, 6, 15, 30, 60, and 120 min intervals. Some samples were measured directly for antioxidant activity while other samples were freeze-dried for later use. The absorption spectra (190-700 nm) of CERPs were measured using a spectrophotometer (Shimadzu UV 2100, Japan). The catechin content during enzymatic browning was monitored by HPLC (16) and the rate of browning was measured spectrophotometrically at 420 nm. Inhibitory Effect of CERPs on Peroxide Formation. Antioxidant activity of CERPs was determined by measuring peroxide produced from the linoleic acid oxidation system. A 2.5 mL of CERPs was mixed with linoleic acid solution (15 mg in 2.5 mL ethanol) and kept at a constant temperature of 37 °C for 2-4 days for autooxidation. The formed peroxides were then measured using a ferric thiocyanate method (17,18). Measurement of Free Radicals. Antioxidant activity of CERPs in terms of free radical scavenging property was measured using a,a'-diphenyl-P-picrylhydrazyl (DPPH) (19, 20). DPPH (16 mg) was dissolved in 100 mL ethanol and diluted to 200 mL with H20 and then filtered using Whatman filter paper No. 2. A 5 mL DPPH solution was mixed with 1 mL of CERPs and the absorbance decrease at 528 nm was recorded. Measurement of Conjugated Dienoic Acid. Antioxidant activity of CERPs was also measured by their inhibitory effects on the formation of conjugated dienoic acid from linoleic acid in the presence of lipoxygenase. A 0.1 mL of CERPs was mixed with 0.9 mL of the enzyme solution, kept at 25 °C for 10 min, and to which 2 mL of the linoleic acid solution was added. The conjugated dienoic acid formed in the linoleic acid-lipoxygenase system was measured by the increase in absorbance at 238 nm using a spectrophotometer (Cecil CE 599, England). The enzyme solution was prepared by dissolving lipoxygenase (EC 1. 13. 11.12, 52,000 unit/mg) in 0.17 M boric acid buffer at a weight ratio of 1:30 to linoleic acid. Linoleic acid was dissolved in ethanol at ratio of 1.7 mL to 1 mL (21, 22). Enzymes used in this experiment were from Sigma Chemical Co. (St. Louis, MO). Other chemicals were acquired from Fluka Chemical Corp. (Ronkonkoma, NY). Results and Discussion Changes in Catechin and the Browning Rate During Catechin Oxidation Catalyzed by Tyrosinase. Catechin was oxidized rapidly during the first 15 min. More than 50% of the catechin was converted to CERPs within the first 12 min of the reaction time (Figure 1). The reaction rate slowed at 30 min when more than 80% of catechin was lost and remained at the low level thereafter. At the same time, the rate of browning (absorbance at 420 nm) was fast at the beginning, up to the 30 min reaction time, and then increased slowly at the steady rate reaching a maximum absorption (7.9) at 120 min reaction time. Practically 90% of the original catechin was converted to CERPs at the end of the experiment (at 120 min) with only a small amount of residual catechin remaining.

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Absorption Spectra of CERPs. Figure 2 represents the absorption spectra of catechin and CERPs taken at 15, 30, and 120 min. Catechin (control) showed a maximum absorption at 200-219 nm with an increased absorption at 290 nm. CERPs obtained at the early stages of the reaction showed a similar spectral pattern but the reaction products obtained later (after 15 min) showed increased absorbance at 380-420 nm. A rapid visual color change was observed at 15 min which coincided with the increased absorption at 210 nm, 390 and 420-440 nm. This increase in absorbance progressed with the reaction time. It has been reported that the characteristics of brown color in the enzymatic browning reactions depend on the nature of the reactants, the rate of reaction, and the photoabsorption characteristics of the reactants (23). The characteristic absorbance of CERPs at 280, 380 and 420 nm indicates the presence of dimers, procyanidins and melanin as reported previously (24-27). CERPs As an Oxidation Inhibitor of Linoleic Acid. Antioxidant activity of CERPs, measured by the inhibitory effect on the peroxides formation in the linoleic acid autooxidation system, is shown in Figure 3. The control (linoleic acid alone) produced peroxides very rapidly, reaching a maximum absorbance at 48 hr, and then decreasing thereafter, while the samples of CERPs added were lower in absorbance due to their inhibitory activity. There were significant differences in the antioxidant activity among CERPs obtained at different reaction times. The CERPs obtained during the early stages of the reaction (before 15 min) showed a higher level of activity as compared to that of the catechin alone (2 mM, original concentration), but those CERPs obtained after 15 min of reaction time exhibited lower activity that decreased with increased reaction time. Residual catechin content in CERPs at the 15 min reaction time was about onethird of the original catechin; therefore, antioxidant activity derived from the residual catechin in CERPs at 15 min should be minimal. The antioxidant activity of CERPs was shown to be concentration-dependant (Figure 4). Antioxidant activity of CERPs (at 120 min reaction time) at the level of 0.05% was higher than that at 0.01%. Antioxidant activity of various plant phenolic compounds has been well documented (28, 29). Recently, the chemical characteristics of phenolic compounds as a hydrogen donor or free radical scavenger and their structure-activity relationships have been reviewed extensively (29). However, information on the antioxidant activity of the phenolic compounds-polyphenol oxidase reaction products is limited (8-10, 30). Antioxidant Activity of CERPs as a Free Radical Scavenger. Free radical scavenging activity of CERPs using a DPPH method is shown in Figure 5. DPPH is reduced by cysteine, glutathione, tocopherol, polyhydroxy aromatic compounds, and others, converting the purple color to colorless. Therefore, DPPH has been used often to test for antioxidant, hydrogen donor, or free radical scavenging activities (8, 19, 20, 30). We observed a varied range of scavenging activity among CERPs obtained at the different reaction times. CERPs collected from the early stages of the oxidation reaction had a higher free radical scavenging activity than those from the later stages of oxidation (data not shown) and the pattern of the antioxidant activity was similar to those observed in linoleic acid oxidation (Figure 3). Comparison of the antioxidant activity of CERPs (120 min) and other commercial antioxidants, such as butylatedhydroxyanisole (BHA) and a-tocopherol, showed that CERPs at the 0.05% level exhibited comparable antioxidant activity as 0.005% BHA, and that 0.01% CERPs had the same level of antioxidant activity as 0.01% α-tocopherol (Figure 5).


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Figure 1. Changes in catechin content and the browning rate during the catechin-tyrosinase oxidation reaction at 25 °C, pH 6.5.

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Figure 2. Absorption spectra of catechin and CERPs.


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Figure 3. Antioxidant activity of CERPs at different reaction times on linoleic acid oxidation at 37 °C. Measurement was made on the formed peroxides.



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Enzymatic Browning Reaction of Catechin205

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Figure 5. Free radical scavenging activity of CERPs (at 120 min), BHA, and α-tocopherol measured by the DPPH method. In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Figure 6. Inhibitory effect of catechin and CERPs (at 120 min) on the linoleic acid oxidation catalyzed by lipoxygenase. Measurement was made on the formed conjugated dienoic acid.



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Antioxidant Activity of CERPs as a Lipoxygenase Inhibitor. Lipoxygenase catalyzes oxidation of linoleic acid and related compounds which have a cis, cis1,4 pentadiene structure and produces a conjugated dienoic acid. This oxidation product, dienoic acid, can be measured at 238 nm. The inhibitory effect of CERPs (120 min reaction time) added to the lipoxygenase-linoleic acid system at the level of 0.05% and 0.15% was higher than that of 0.15% catechin (Figure 6). Again, a higher inhibitory effect on lipoxygenase was noticed as the concentration of the added CERPs increased. The inhibitory effect of some phenolic compounds on lipoxygenase has been reported (57, 32) and is explained by the coupled oxidation theory or/and conversion of lipoxygenase to an inactive ferrous form (33, 34), but there is no report on inhibitory effect of polyphenol oxidation products on lipoxygenase. Conclusion The result of this study using catechin-PPO in a model system demonstrated that CERPs at the early stage of the reaction exhibited a high level of antioxidant activity but the activity decreased as the oxidation time increased. Likewise, the early stage reaction products showed a high free radical scavenging activity and high lipoxygenase inhibitory effect. In the tyrosine-enzyme system, a similar pattern of the antioxidant activity was observed. Although the additional antioxidant activity derived from the residual catechin in CERPs cannot be disregarded; it appears that dimers or trimers of catechin, which, are the predominant reaction products at the early stage of the enzyme catalyzed browning reactions (26, 27), made the most contribution to the antioxidant activity. A similar observation was made on inhibitory effect of CERPs on polygalacturonase (35) which dimers and trimers showed a higher inhibitory effect. This antioxidant activity of the CERPs may be explained by their ability to donate a hydrogen atom, free radical scavenging, and lipoxygenase inhibition. However, the exact mechanism of the antioxidant activity of CERPs cannot be explained until their chemical structures are elucidated. Further research in this area is deemed necessary.

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9. Omura, H.; Sonda, T.; Asada, Y.; Inatomi, Y. Nippon Shokuhin Kogyo Gakkashi 1975, 22, 387-394. 10. Sonda, T.; Aramaki, T.; Omura, H. Eiyo to Shokuryo 1975, 28, 151-158. 11. Hirose, Y.; Yamaoka, H.; Nakayama, M. J. Assoc. Off. Anal. Chem. 1991, 68, 131-132. 12. Matsuzaki, T.; Hara, Y. Nippon Nogeikagaku Kaishi 1985, 59, 129-134. 13. Hirose, Y.; Yamaoka, H.; Nakayama, M. Agric. Biol. Chem. 1990, 54, 567569. 14. Mori, Y.; Mitani, A. J. Home Econ. (Japan), 1978, 29, 14-17. 15. Nicolas, J.; Richard-Forget, F.; Goupy, P.; Aubert, S. CRC Crit. Rev. Food Sci. Nutr. 1994, 34, 109-157. 16. Jaworski, A; Lee, C. Y. J. Agric. FoodChem.1987, 35, 257-259. 17. Mitsuda, H.; Yasumoto, K.; Iwami, K. Eiyo to Shokuryo 1966, 19, 60-64. 18. Cheigh, H. S.; Lee, J. S.; Lee, C. Y. J. Korean Soc. Food Nutr. 1993, 22, 570-575. 19. Blos, M. S. Nature 1958, 26, 1199-1200. 20. Kim, M. W.; Choi, K. J.; Cho, Y. H. J. Korean Agric. Chem Soc. 1980, 23, 173-177. 21. Rogstad, Α.; Reintion, R. J. Am. Oil Chem. Soc. 1977, 54, 282-285 22. Bergmeyer, H. U. Methods of Enzymatic Analysis; Academic Press: New York, NY, 1974; Vol. 1, pp 483-484. 23. Sonda, T.; Inatomi, Y.; Asada, Y. Sci. Bull. Fac. Agric.; Kyushu University: Kyushu, Japan, 1974; Vol. 29, pp 71-77. 24. Mason, H. S. J. Biol.Chem.1948, 172, 83-99. 25. Omura, H.; Sonda, T. Eiyo to Shokuryo 1970, 23, 367-373. 26. Oleszek, W.; Lee, C. Y.; Price, K. R. Acta Soc. Bot. Poloniae 1989, 58, 273283 27. Oszmianski, J.; Lee, C. Y. J. Agric. FoodChem.1991, 39, 1050-1052. 28. Wang, P. F.; Zheng, R. L. Chem. Physics Lipids 1992, 63, 37-40. 29. Shahidi, F.; Janitha, P. K.; Wanasundra, P. D. CRC Crit. Rev. Food Sci. Nutr. 1992, 32, 67-102. 30. Ariga, T.; Koshiyama, I.; Fukushima, D. Agric. Biol. Chem. 1988, 52, 27172722. 31. King, D. L.; Klein, B. P. J. FoodSci.1987, 52, 220-224. 32. Oszmianski, J.; Lee, C. Y. J. Agric. Food Chem. 1990, 38, 688-690. 33. Yasumoto, K.; Yamamoto, Α.; Mitsuda, H. Agric. Biol. Chem. 1970, 34, 1162-1168. 34. Kemal, C.; Louis-Flamberg,P. Biochem. 1987, 26, 7064-7072. 35. Guyot, S.; Pellerin, P.; Brillouet, J. M.; Cheynier, V.; Moutounet, M. Abstracts of the 17th International Conference on the Groupe Polyphenols; 1994;p133. R E C E I V E D November 30, 1994


Enzymatic Browning and Its Prevention - American Chemical Society

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