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© 2005, The Society for Biotechnology, Japan
Analysis of Isoflavone Content in Tempeh, a Fermented Soybean, and Preparation of a New Isoflavone-Enriched Tempeh
Nobuyoshi Nakajima,1,2* Nobuyuki Nozaki,3 Kohji Ishihara,4 Akiko Ishikawa,1 and Hideaki Tsuji1
Graduate School of Health and Welfare Science, Okayama Prefectural University, Kuboki, Soja, Okayama 719-1197, Japan,1 Collaborative Research Center, Okayama Prefectural University, Kuboki, Soja, Okayama 719-1197, Japan,2 Industrial Technology Center of Okayama Prefecture, Haga, Okayama 701-1296, Japan,3 and Department of Life Science, Okayama University of Science, Ridai-cho, Okayama 700-0005, Japan4
Received 23 June 2005/Accepted 9 September 2005
To produce a tempeh-like functional food containing a high level of isoflavone with a high absorptivity, we analyzed changes in the composition of isoflavone during tempeh fermentation and the difference in isoflavone content depending on the soybean variety and particular tissue. By adding soybean germ (hypocotyl) that contained a large amount of isoflavone, we prepared a new isoflavone-enriched tempeh in the form of a granular fermented soybean-based food, which can serve as a nutritious supplement for the elderly.
[Key words: soybean, tempeh, fermented soybean, isoflavone, Rhizopus]
Because isoflavone, which is abundant in soybeans, exhibits estrogen-like function, it is expected to alleviate symptoms of osteoporosis occurring after menopause (1, 2). In addition, isoflavones can suppress the onset of arteriosclerosis because it also improves the metabolism of lipids such as cholesterol (3, 4). Tempeh, a traditional Indonesian fermented soybean food, has recently been focused on among the many fermented soybean foods, because of its superior nutritive qualities and metabolic regulatory functions (5, 6). Recently, a method of producing γ-aminobutyric acid-enriched tempeh, which has antihypertensive effects, has been reported (7, 8). However, traditional soybean tempeh (9) is usually formed into a block during the fermentative production process. Since tempeh is produced using boiled and dehulled soybean without the soybean germ (hypocotyl) that contains a high level of isoflavone, the isoflavone content in tempeh produced tends to be low. In addition, isoflavone with the aglycon form has a higher absorptivity than that with the glucoside form (10). To produce a tempeh-like functional food containing a high level of isoflavone with a high absorptivity, we analyzed changes in the composition of isoflavone during tempeh fermentation and the difference in isoflavone content depending on the soybean variety and particular tissue. On the basis of these results, we developed a production method for a granular isoflavone-enriched tempeh, which can be easily consumed by the elderly. Commercially available soybean seeds (yellow-soybean, Tamahomare and black-soybean, Sakushuu-kuro) harvested in Okayama prefecture, Japan, were used in this study. The yellow-soybean tempeh and black-soybean tempeh were
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produced by Ukan-Farmers-Factory (Okayama). The defatted-soybean germ and defatted-soybean cotyledon from the yellow-soybean made in USA were provided by KatohSeiyu (Okayama). Rhizopus sp., a mold used for tempeh fermentation, was purchased from Akita-Konno (Akita). Tempeh made of soybeans or their particular tissues was produced as previously described (9). The contents of 12 different derivatives of isoflavone (three types of isoflavoneaglycon and the corresponding nine types of isoflavone-glucoside) in tempeh (per 100 g, wet) were determined by HPLC according to a previously reported method (11) using 10 µl of tempeh extract obtained with 70% (v/v) methanol under the following conditions: YMC-Pack column, ODSAM-303 (φ4.6 × 250 mm); compositions of solvent A, acetonitrile: H2O :acetic acid = 15 : 85 : 0.1 (v/v/v) and solvent B, acetonitrile : H2O : acetic acid = 35 : 65 : 0.1 (v/v/v); gradient elution, 15–35% acetonitrile/60 min; flow rate, 1 ml/min; detection at 254 nm. Twelve derivatives of isoflavone provided by Fujicco (Kobe) were used as standards. First, we investigated the time course of changes in the composition and content of isoflavone along with the fermentation time (0–48 h) of a yellow-soybean tempeh as shown in Fig. 1. The content of the three types of isoflavone-aglycon increased with fermentation time, and became approximately twofold after 24 h fermentation compared with that before fermentation (0 h). Among the nine types of isoflavone-glucoside, the content of malonylglucoside increased transiently from 12 to 24 h of fermentation. However, the contents of monoglucoside and acetylglucoside decreased with fermentation time. As a result, the total content of isoflavone (both aglycon and glucoside) decreased as the fermentation process progressed. Therefore, a sufficient increase in the isoflavone content, especially that of its aglycon form, could not be expected during tempeh fermenta-
5 0. wet) are the mean of three replicates.686 NAKAJIMA ET AL. the yellow-soybean and black-soybean. malonylgenistin.0 13. As shown in Table 2. that is. the isoflavone content in tempeh made of the defatted-yellow-soybean cotyledon was approximately the same as that in tempeh made of whole soybean (data not shown). the proportion of aglycon in total isoflavone of the isoflavone-enriched tem- .1 21. an exceedingly high isoflavone content (aglycon and glucoside) of approximately 880 mg was observed in the defatted-yellow-soybean-germ tempeh. b After 24 h fermentation.6 0.7 14.4 12. The contents of the three types of the aglycon in isoflavone in the yellow-soybean tempeh and black-soybean tempeh ranged from 16 to 17 mg. the total isoflavone content was approximately 103 mg. Comparison and content of the isoflavone in the defatted-yellow-soybean-germ tempeh and the isoflavone-enriched tempeha Isoflavone contentb (mg) IsoflavoneDefatted-yellowenriched soybean-germ tempehc tempeh 382. Method of isoflavone analysis is described in the text.8 5. The typical contents of isoflavone in the yellow-soybean tempeh and black-soybean tempeh after 24 h fermentation are shown in Table 1. J. Symbols: open circles.7 Isoflavone tion.6 121. c The isoflavon-enriched tempeh was prepared by combining the defatted-yellow-soybean germ with the defatted-yellow-soybean cotyledon in the ratio of 20: 80 (%). open triangles. Next. In addition.7 30.3 43. vone-enriched tempeh was then prepared by keeping the same proportion of aglycon described above (Table 2). The total isoflavone content in the isoflavoneenriched tempeh was approximately threefold that in the traditional soybean tempeh. year of harvest. On the basis of our present results.8 1.6 59.3 2. FIG. On the other hand.8 102. The new tempeh was prepared by combining the defatted-soybean germ with the defatted-soybean cotyledon at a ratio of 20 : 80 (%). The values of isoflavone determined along with the fermentation time (0–48 h) are expressed in mg per 100 g (wet) of tempeh and are the means of three replicates.0 27.2 98. total isoflavone (aglycon and glycoside). BIOSCI..7 3.8 0.4 20.9 44.5 8.3 1.7 41.7 17. genistin.0 32.5 31.4 2.4 1. malonylglycitin. wet) are the mean of three replicates.6 10. total aglycon (daidzein. Depending on the variety.0 85. glycitin.4 18. TABLE 1.3 23. and those of the nine types of glucoside ranged from 86 to 87 mg. In addition.9 1.7 103.6 2.6 171. TABLE 2.4 8. Comparison of the isoflavone content in the yellow-soybean tempeh and the black-soybean tempeha Isoflavone Isoflavone contentb (mg) Yellow-soybean Black-soybean tempeh tempeh 15. b After 24 h fermentation. closed circles. and acetylgenistin).1 276.2 47.2 9.6 5.5 877. acetyldaidzin.0 6.5 155.2 Total aglycon Daidzein Glycitein Genistein Total glucoside Daidzin Glycitin Genistin Malonyldaidzin Malonylglycitin Malonylgenistin Acetyldaidzin Acetylglycitin Acetylgenistin Total isoflavone (aglycon + glucoside) a Values (mg per 100 g of tempeh. Time course of changes in composition and content of isoflavone in the yellow-soybean tempeh.5 0.9 144.9 87. malonyldaidzin. the proportion of aglycon in total isoflavone was approximately 44%. glycitein. and method of boiling soybeans.2 1.0 24. no essential difference in the isoflavone content in tempeh was observed between the two soybean varieties.7 7.0 6. total glycoside (daidzin. acetylglycitin. slight differences in the isoflavone content were observed (9). we measured the composition and the content of isoflavone in tempeh (24 h fermentation) made of the granular germ of defatted-yellow soybean after the extraction of soybean oil.1 65. 1. and genistein). Thus.8 38.2 25. We compared also the isoflavone content in tempeh depending on the soybean variety.2 14. A new isofla- Total aglycon Daidzein Glycitein Genistein Total glucoside Daidzin Glycitin Genistin Malonyldaidzin Malonylglycitin Malonylgenistin Acetyldaidzin Acetylglycitin Acetylgenistin Total isoflavone (aglycon + glucoside) a Values (mg per 100 g of tempeh.3 0.6 494. BIOENG.5 206.
: Tempeh: a moldmodified indigenous fermented food made from soybeans and/or cereal grains. M. A. Arch. 43. Endo. Y. The taste of the isoflavone-enriched tempeh was not significantly different from that of the traditional soybean tempeh. B. S. M. Uchida. Rev. Endo.: A randomized trial comparing the effects of casein with that of soy protein containing varying amounts of isoflavones on plasma concentrations of lipids and lipoproteins. Aoki. Currently. J. D... V. D.. 2. K. Okayama Prefecture. Biol. Crit. 89–94 (1997). and Fujimoto.. Wiley Europe. Japan (2005... U. and Fujimoto.. whereas that in the soybean tempeh was 15%. 100.. I. Shmueli. 159.. Furuya. 1–5 (2001). A. Tanos. 292–298 (2000). K. K. Biochem. Med. A. I. Furuya. D. Assoc. 14–19 (1999). 2070–2076 (1999). K.. since the defatted-soybean germ and cotyledon are granular. Dechema Biotechnology Conference.. Stein. Ishihara). A. Welti. 4. Mizushima. This work was supported in part by a program of “Bioactive Okayama”. Chem. Y.. J. G.. 67. 6. Biotechnol. Y.. approximately 250 g of the soybean tempeh should be consumed per day. Aoki. 55. Magnolato. 137–188 (1993). Tsuji) and by the Takano Life Science Research Foundation.. REFERENCES 1. Brezezinski.: Short-term effects of phytoestrogen-rich diet on postmeno pausal women.: Effects of soybean-isoflavone aglycons. It has also been reported that isoflavone with the aglycon form has a higher systemic absorptivity than that with the glucoside form (10). and Burke. the final proportions of the three types of aglycon become almost equal in the isoflavone-enriched tempeh made by adding the soybean germ. C. Kudou. and Soda. New Food Ind. V. H... G. Terry. A. 4.: Malonyl isoflavone glycosides in soybean seeds (Glycine max Merrill). Since glycitein content among the three types of aglycon is low in the soybean tempeh and genistein content is low in the soybean germ (11).. Biosci... Epidemiol.. Uda. the isoflavone-enriched tempeh was found to have not only a higher total isoflavone content but also a higher aglycon content than the soybean tempeh. vol. Am. R. (in Japanese) 11. Diet.. 10. 2005 NOTES 687 peh was 44%... and Imai. Agric. R. Obata.: The production of a new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid by anaerobic incubation with Rhizopus. 1806– 1808 (2003). Y.: Changes in chemical composition of tempe during fermentation. (ed. and Rehm. 130–138 (1990). 205–208.. 19.VOL. From the comparison of aglycon content. L. D. Rosler. Matsumoto. 68. Biosci. 2227–2233 (1991). Vitolins. G. (in Japanese) 7. Biochem. K... K.: FDA approves health claim labeling for foods containing soy protein. the maximum possible consumption of isoflavone for an adult.: Dietary soybeans intake and bone mineral density among 995 middle-aged women in Yokohama. From these results. 9. Kitamura.. 100. Baumann. Fleury. J. Menopause. J. Weinheim (1991). Tagami.. H. Shaoul. J.. K. Nippon Shokuhin Kogyo Gakkaishi.: Effects of γ-aminobutyric acid-enriched tempeh-like fermented soybean (GABA-tempeh) on the blood pressure of spontaneously hypertensive rats. J.. the aglycon content in the isoflavone-enriched tempeh was approximately eightfold that in the soybean tempeh. . is considered to be approximately 40 mg/d. K. at which estrogen-like effects can be expected. 9. K. and Okubo.). the isoflavone-enriched tempeh made of these materials may be applicable as a functional food for the elderly to suppress the onset of arteriosclerosis and alleviate osteoporosis symptoms which occur after menopause.. T. K. 5. Bisping.. S. Japan (2002–2004. 8. H. Biotechnol. Furthermore.... Tsuchida. 4. 3. D.. T. Toba. Hachmeister. Crouse. p. Morgan.. whereas one-eighth or less of this amount of the new isoflavone-enriched tempeh is sufficient. In Behrens. S. and Schenker. Adlerceutz. J. H. Ellis..: Content and release of amino acids during the fermentation of tempe by several strains of Rhizopus sp. Intern. 37. and Fung. Microbiol. Y. 1018–1023 (2003).
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