Antioxidative Activity of Fermented Soybean Products - ACS Publications

Chapter 29

Antioxidative Activity of Fermented Soybean Products 1

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H. Esaki , H. Onozaki , and Toshihiko Osawa

Downloaded by UNIV OF QUEENSLAND on April 28, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch029

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Department of Food and Nutrition, Sugiyama Jogakuen University, Chikusa, Nagoya 464, Japan Department of Food Science and Technology, Nagoya University, Chikusa, Nagoya 464-01, Japan

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The antioxidative activity and chemical components of the traditional fermented soybean products incubated with Aspergillus oryzae, Bacillus natto and Rhizopus oligosporous were investigated. These products are popular as "miso," "natto" and "tempeh," respectively, and have proved to be more stable against lipid peroxidation than steamed soybeans. This result indicates that antioxidative compounds can be produced by fermentation. Using H P L C analysis, no tocopherols were found to increase, but the amount of free isoflavones such as daidzein and genistein increased in miso and tempeh. These isoflavones are presumed to be the principal antioxidants in miso and tempeh. In the case of natto, there was little increase in the amount of free isoflavones, but the antioxidative activity of a water-soluble antioxidative fraction increased dramatically during incubation with Bacillus natto. From the evaluation of the antioxidative activities of aqueous ethanol (80%) extracts from soybeans fermented using 18 different kinds of Aspergillus strains, it was found that Aspergillus No. 13 and 14 showed the strongest antioxidative activity.

There are many traditional fermented soybean products, especially in Asian countries. Tempeh, a fermented product of Rhizopus oligosporous, is famous in Indonesia. On the other hand, natto — manufactured by fermentation of steamed soybeans using Bacillus natto — is a typical soybean food in Japanese diets. Traditional natto is eaten with hot rice, soy sauce and mustard. These fermented soybean products have been reexamined because of the increase in digestible protein content and other nutritive value. These soybean products can be used as good substitutes for meat in the diet. On the other hand, miso is very popular seasoning in traditional Japanese soup, and for many other Japanese dishes. Recently, some antioxidative components in the diet have attracted special interest because they can protect the human body from free radicals (1) which may cause many diseases, including

0097-6156/94/0546-0353$06.00/0 © 1994 American Chemical Society Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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cancer, and aging (2,3). With this background, we started our project to isolate and characterize the antioxidative components in fermented soybean products.


Lipid Stability of Fermented Soybean Products The lipid peroxidation rate of the lyophilized powder of natto, tempeh and miso was measured by a modified method of Ikehata et al. (4). Each powdered sample (400 mg) of natto, tempeh, miso or S.S.B. was divided into a 100 ml Erlenmeyer flask, and the flasks, open to the air, were stored at 40°C in the dark. The extent of lipid peroxidation in natto, tempeh, miso and S.S.B. was monitored at regular intervals by the thiocyanate method of Mitsuda et al. (5). The lipids in natto, tempeh and miso are much more stable against autoxidation than those in unfermented steamed soybeans (S.S.B.) (Figure 1).

Natto S.S.B. -#— Natto

Tempeh S.S.B. Tempeh

Miso S.S.B. Miso

Storage period (days) Figure 1. Lipid stability of fermented soybean products and steamed soybeans (S.S.B.). The extent of lipid peroxidation in natto, tempeh, miso compared to S.S.B. was determined at 500 nm by the thiocyanate method.

Chemical Analysis of Antioxidative Tocopherols The first attempt to evaluate the antioxidative activities of these three fermented soybean products was made by comparative quantitative analyses of the main antioxidative tocopherols. Tocopherols were extracted from each lyophilized powder with w-hexane containing B H T and 6-hydroxy-2,2,5,7,8pentamethylcroman (internal standard), and were determined by high performance liquid chromatography (HPLC) according to the method of Kanematsu et al. (6). H P L C analysis was carried out using a Develosil SI 60-5 column (φ4.6 χ 250 mm, Nomula Chem. Co., Ltd.) with a mobile phase of Λ-hexane, dioxane and isopropylalcohol (98.7:1.0:0.3) at a flow rate of 1.0 ml/min. A U V detector at 298

Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


29. ESAKI ET AL.

Antioxidative Activity of Fermented Soybean Products

nm was used for the determination of tocopherols. The amounts of tocopherols were determined by comparison to the peak areas of external standards which were injected after each analysis. From H P L C analysis of these three fermented soybean products, none of the tocopherols was found to increase (Figure 2). In natto, α-, β-, γ- and δ-tocopherol concentrations were 2.0, 0.4, 28.9 and 17.3 mg/100 g on dry basis, respectively, while in S.S.B. they were 2.2, 0.5, 29.0 and 17.6 mg/100 g on dry basis, respectively. This result agrees with the data reported by Kanno et al. (7). In addition, α-, β-, γ- and δ-tocopherol concentrations in tempeh were 1.1, 0.3, 11.9 and 9.2 mg/100 g on dry basis, respectively, while in S.S.B. they were 1.2, 0.3, 14.9 and 9.2 mg/100 g on dry basis, respectively. In the case of natto and tempeh, therefore, the tocopherols in S.S.B. were unmodified by the fermentation process. On the other hand, in miso, tocopherols in S.S.B. were inclined to decrease during the fermentation process. Concentrations of α-, β-, γ-, and δ-tocopherol in miso were 1.7, 0.2, 21.8 and 8.4 mg/ 100 g on dry basis, respectively, while in S.S.B. they were 2.4,0.3, 28.3 and 17.9 mg/100 g on dry basis, respectively. Chemical Analysis of Antioxidative Isoflavonoids Antioxidative free isoflavones in the three fermented soybean products were also quantified using H P L C according to the method of Murakami et al. (8). Isoflavones and their glucosides were extracted from each lyophilized powder with 80% methanol containing w-butyrophenone (internal standard). H P L C analysis was carried out using a Develosil ODS-7 column (φ4.6 χ 250 mm, Nomura Chem. Co., Ltd.) with a linear gradient of methanol in water from 20 to 60% in 60 min. The solvent flow rate was 0.7 ml/min and the absorption was measured at 262 nm. The amounts of isoflavones and their glucosides are shown in Figure 3. In natto, there was little increase in the amount of free isoflavones such as daidzein and genistein. On the other hand, the content of daidzein and genistein in tempeh increased by approximately 5 times compared with that in S.S.B. In miso, daidzein and genistein increased dramatically — by 25 times — from fermentation, and no isoflavone glucosides, daidzin and genistin, remained. Antioxidative Activity of Fermented Soybean Products Gyôrgy et al. (9) reported 6,7,4'-trihydroxyisoflavone as an antioxidant in tempeh. While this isoflavone has proved to be a potent antioxidant in aqueous solution at pH 7.4, it was not effective at preventing autoxidation of soybean oil and soybean powder (4). Murakami et al. (8) reported that the main isoflavones responsible for the antioxidative activity in tempeh were daidzein and genistein, which are liberated from daidzin and genistin in soybeans by β-glucosidase from Rhizopus oligosporous (Figure 4). Chemical analysis of the antioxidative components in tempeh showed tocopherol levels were not modified by fermentation, but lipophilic aglycones of isoflavone glucosides were liberated by β-glucosidase during fermentation. In the case of miso, some melanoidins (10) and peptides (77) are presumed to have antioxidative activity. But the principal antioxidants in miso have not been isolated. From H P L C analysis, tocopherols in S.S.B. tended to be metabolized during fermentation, but a large amount of lipophilic antioxidative aglycones can be produced in fermented product. These results suggest that the


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Figure 3. Changes in isoflavonoid content during fermentation. Levels were measured by H P L C analysis.

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lipophilic aglycones such as daidzein and genistein are mainly responsible for the stabilities of tempeh and miso. On the other hand, no significant change was observed in the amount of antioxidative tocopherols and free isoflavones in either natto or S.S.B, and the antioxidant potential in natto has not been ascertained. Each 80% methanol extract from natto and S.S.B. was fractionated by Toyopearl HW-40 chromatography, and the antioxidative activity of each fraction was determined by the buffer-ethanol system described by Osawa et al. (12). The water-soluble fraction exhibiting antioxidative activity increased dramatically in natto (13). Isolation and identification of the antioxidative substances present in natto are now in progress.

Genistin

Genistein

Figure 4. Scheme for the liberation of the antioxidants daidzein and genistein from daidzin and genistin.

Aspergillus strains have been used in manufacturing miso, "shoyu" (soy sauce), "sake," etc. S.S.B. was inoculated with 18 different kinds of Aspergillus strains and incubated at 30°C for 96 hrs. The fermented soybeans were dried and powdered. The antioxidative activities of aqueous ethanolic (80%) extracts from these fermented soybeans, in addition to natto, tempeh and miso groups were evaluated by the determination of lipid peroxidation in liposomes (14). Lipid peroxidation was induced by 2,2'-azobis(2-amidinopropane) hydrochloride and determined spectrometrically by an increase in thiobarbituric acid reacting substances (15). As may be seen in Figure 5, soybeans fermented with Aspergillus No. 13 and 14 have the strongest antioxidative activity.



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Figure 5. Antioxidative activities of the aqueous ethanolic (80%) extracts from soybeans fermented using various microorganisms. The T B A method in a liposome system was used to measure lipid peroxidation.

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Acknowledgements The authors wish to thank C. Matsuda, S. Suzuki, A . Ohori and Y . Yokoe for their cooperation. They also thank Fujisawa Pharmaceutical Co. Ltd., Marutake Co. Ltd., and Ichibiki Co. Ltd. for generously providing the fermented soybeans incubated with Aspergillus strains, natto and miso, respectively, used in this study. Literature Cited


1.

Osawa,T.; Kawakishi, S.; N a m i k i , M . In Antimutagenesis and Anticarcino-genesis Mechanisms II; Kuroda, Y.; Shankel, D . M . ; Waters, M . D . Eds.; Plenum: New York, 1990; pp 139-153. 2. Player, T. In Free Radicals, Lipid Peroxidation and Cancer; McBrien, D . C. H.; Slater, T. F., Eds.; Academic Press: London, 1982, pp 173-195. 3. Osawa, T.; Ide Α.; Su, J. D . and Namiki, M . J. Agric. Food. Chem. 1987, 35, 808-812. 4. Ikehata, H.; Wakaizumi, M . ; Murata, K. Agric. Biol. Chem. 1968, 32, 740- 746. 5. Mitsuda H.; Yasumoto K.; Iwami K . Eiyo To Shokuryo 1966, 19, 210-214. 6. Kanematsu H.; Ushigusa T.; Murayama T.; Niiya I.; Matsumoto T. Yakugaku 1983, 32, 51-53. 7. Kanno, Α.; Takamatsu, H . ; Tuchihashi, N . ; Watanabe, T.; Takai, Y . Nippon Shokuhin Kogyo Gakkaishi 1985, 32, 754-758. 8. Murakami, H.; Asakawa, T.; Terao, J.; Matsushita, S. Agic. Biol. Chem. 1984, 48, 2971-2975. 9. György, P.; Murata, K.; Ikehata, H . Nature 1964, 203, 870-872. 10. Yamaguchi N . ; Fujimaki M . Nippon Shokuhin Kogyo Gakkaishi 1973, 20, 507-512. 11. Okamoto M . ; Honma S.; Fujimaki M . Kaseigaku Zashi 1982, 33, 585-590. 12. Osawa T.; Namiki M . Agric. Biol. Chem. 1981, 45, 735-739. 13. Esaki, H . ; Nohara, Y . ; Onozaki, H . ; Osawa, T. Nippon Shokuhin Kogyo Gakkaishi 1990, 37, 474-477. 14. Pelle Ε.; Maes D.; Padulo G. Α.; K i m , E K . ; Smith, W.P. Arch. Biochem. Biophys. 1990, 283, 234-240. 15. Buege J. Α.; Aust S. D. Methods in Enzymology 1978, 52, 302-310. RECEIVED April 4, 1993


Antioxidative Activity of Fermented Soybean Products - ACS Publications

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