JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 6, 685687. 2005 DOI: 10.1263/jbb.100.

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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 -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
* Corresponding author. e-mail: nakajima@fhw.oka-pu.ac.jp phone/fax: +81-(0)866-94-2157
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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 l of tempeh extract obtained with 70% (v/v) methanol under the following conditions: YMC-Pack column, ODSAM-303 (4.6 250 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, 1535% 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 (048 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-

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TABLE 1. 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 15.7 17.4 8.0 6.8 0.5 0.7 7.2 9.9 87.0 85.8 1.7 3.3 0.4 1.0 6.4 18.7 30.2 14.3 1.4 2.7 41.8 38.5 0.9 1.3 2.6 0.2 1.6 5.8 102.7 103.2

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, wet) are the mean of three replicates. b After 24 h fermentation. FIG. 1. Time course of changes in composition and content of isoflavone in the yellow-soybean tempeh. The values of isoflavone determined along with the fermentation time (048 h) are expressed in mg per 100 g (wet) of tempeh and are the means of three replicates. Method of isoflavone analysis is described in the text. Symbols: open circles, total isoflavone (aglycon and glycoside); open triangles, total glycoside (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, acetyldaidzin, acetylglycitin, and acetylgenistin); closed circles, total aglycon (daidzein, glycitein, and genistein).

TABLE 2. 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 382.6 121.5 206.1 65.9 144.5 31.0 32.0 24.6 494.5 155.2 25.5 8.6 59.7 14.1 21.0 13.6 171.2 47.2 98.3 23.9 44.0 27.4 20.8 5.6 10.6 2.3 43.4 12.5 877.1 276.7

Isoflavone

tion. We compared also the isoflavone content in tempeh depending on the soybean variety. Depending on the variety, year of harvest, and method of boiling soybeans, slight differences in the isoflavone content were observed (9). The typical contents of isoflavone in the yellow-soybean tempeh and black-soybean tempeh after 24 h fermentation are shown in Table 1. 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, and those of the nine types of glucoside ranged from 86 to 87 mg. Thus, the total isoflavone content was approximately 103 mg. On the basis of our present results, no essential difference in the isoflavone content in tempeh was observed between the two soybean varieties, that is, the yellow-soybean and black-soybean. Next, 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. As shown in Table 2, an exceedingly high isoflavone content (aglycon and glucoside) of approximately 880 mg was observed in the defatted-yellow-soybean-germ tempeh. In addition, the proportion of aglycon in total isoflavone was approximately 44%. On the other hand, 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). 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, wet) are the mean of three replicates. b After 24 h fermentation. 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 (%).

vone-enriched tempeh was then prepared by keeping the same proportion of aglycon described above (Table 2). The new tempeh was prepared by combining the defatted-soybean germ with the defatted-soybean cotyledon at a ratio of 20 : 80 (%). The total isoflavone content in the isoflavoneenriched tempeh was approximately threefold that in the traditional soybean tempeh. In addition, the proportion of aglycon in total isoflavone of the isoflavone-enriched tem-

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peh was 44%, whereas that in the soybean tempeh was 15%; the aglycon content in the isoflavone-enriched tempeh was approximately eightfold that in the soybean tempeh. From these results, 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. Currently, the maximum possible consumption of isoflavone for an adult, at which estrogen-like effects can be expected, is considered to be approximately 40 mg/d. It has also been reported that isoflavone with the aglycon form has a higher systemic absorptivity than that with the glucoside form (10). From the comparison of aglycon content, approximately 250 g of the soybean tempeh should be consumed per day, whereas one-eighth or less of this amount of the new isoflavone-enriched tempeh is sufficient. 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), the final proportions of the three types of aglycon become almost equal in the isoflavone-enriched tempeh made by adding the soybean germ. The taste of the isoflavone-enriched tempeh was not significantly different from that of the traditional soybean tempeh. Furthermore, since the defatted-soybean germ and cotyledon are granular, 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.
This work was supported in part by a program of Bioactive Okayama, Okayama Prefecture, Japan (20022004, H. Tsuji) and by the Takano Life Science Research Foundation, Japan (2005, K. Ishihara).

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