Thermal Effects on the Conversion of Isoflavones in Soybean

Chapter 11

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Thermal Effects on the Conversion of Isoflavones in Soybean Hsi-Mei Lai* and Pei-Yin Lin Department of Agricultural Chemistry, National Taiwan University No. 1, Roosevelt Rd., Sec. 4, Taipei 10617, Taiwan *[email protected]

Soybeans and soy-based products are getting popular in the world due to their nutritional and health-promoting benefits for human beings. The most human health related compounds in soys are isoflavones. There are twelve isoflavone conjugates existed in soybeans with different bioavailabilities and aglycones are reported to be the most effective one. The isoflavone profiles of soybeans can be changed by many factors, including the varieties of seed, pretreatments and thermal processing conditions. In this study, the effects of variety, pretreatment and thermal treatment on isoflavone profile of soybeans were evaluated. Two soybeans (KS1 and KS8) and two black soybeans (TN3 and TN6) grown domestically were soaked or germinated and followed by moist-thermal (121 °C up to 60 min) or dry-thermal (195 °C up to 30 min) treatments. The soaking and germination processes significantly increased the total amounts of isoflavones for 9-85%, depending on the soybean variety. During thermal processing, in general, malonylglucosides were significantly lost, corresponding with the increases of acetylglucosides and glucosides at early heating stage. Higher conversion ratio was found in dry-thermal process than in moist-thermal process. Aglycones could be effectively converted in matured soybeans after short dry-thermal treatment (10-20 min). For matured soybeans, the thermal processing is recommended for the conversion of aglycone conjugates from malonylglucosides, acetylglucosides, and glucosides. For soaked and germinated soybeans, dry thermal processing is not suggested due to its significant loss of total isoflavones and © 2010 American Chemical Society In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

insignificant increase of aglycone conjugates after long heating time.


Introduction Soybeans and soy-based products are consumed daily by Asian people for centuries because of rich sources of proteins, unsaturated fatty acids, dietary fibers and phytochemicals. In the last decades, soy foods are widely promoted and consumed in the whole world due to their reported nutritional and health-promoting benefits. More and more studies have shown soy or its phytochemicals, especially, isoflavones, as effective cancer-preventive agents for several hormone-related diseases (1, 2). Soybean isoflavones had been evidenced to be beneficial for the releasing menopausal symptoms (3) and preventing from osteoporosis of menopausal women (4–6) by stimulating bone formation and suppressing bone resorption (7). Isoflavones are a subclass of flavonoids and also called phytoestrogen compounds due to their structural similarity with human hormone estradiol. There are three types of isoflavones in soybean, and each type exists in four different chemical forms, which include aglycoside conjugates (daidzein, genistein, and glycitein) and their β-glucoside conjugates: glucosides (daidzin, genistin and glycitin), malonylglycosides (6”-O-malonyldaidzin, 6”-O-malonylgenistin and 6”-O-malonylglycitin), and acetylglycosides (6”-O-acetyldaidzin, 6”-O-acetylgenistin and 6”-O-acetylglycitin). The bioavailability and bioactivity of isoflavones are affected by their chemical structures. It appears that aglycones of daidzein and genistein are more bioavailable than their conjugated forms in humans (8, 9). The various bioavailabilities were also investigated among isoflavone families, as daidzein was found to be more bioavailable than genistein in adult woman (10). These observations indicated that deglycosylation of soy isoflavones achieved by applying optimal pretreatment or processing may significantly beneficial for human consumption. The total content of isoflavones in unprocessed soybeans is dependent on the varieties with the ranges of 1.2 to 4.2 mg/g wet basis reported by Wang and Murphy (11, 12) and 1.8 to 4.3 mg/g dry basis reported by Lin and Lai (13). The profiles of isoflavone isomers were similar among varieties, in which the malonylglycosides are the major components (56-76%) while the aglycones are trace (13). During processing, the content and profile of isoflavone isomers of the soybeans were variously changed depending on the types and degrees of processing. Soybean sprouts, the germinated soybeans, were reported contained high amounts of total isoflavones and aglycones, which were resulted from the bioconversion of seeds during germination. Lin and Lai (13) reported that the total isoflavones and aglycones content increased 28.6% and 19.5 times for a soybean (KS1) after 1-day germination. Thermal processing is the most extensively used method of food preparation and preservation to destroy microorganisms thereby extending its shelf-life. The precooked soybean or soybean flour could be applied in many food items, such as instant drink mixes, desserts and snacks, for developing on the isoflavone 172 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


enriched functional soy-based foods. Vaidya et al. (14) indicated the differences in molecular structure between malonylgenistin and malonyldaidzin didn’t affect their conversion rates of malonyl isomers to β-glucosides, but did affect the degradation rates of malonyl isomers while pH and heat increased. Chien et al. (15) indicated that the moist heating was more susceptible to conversion and degradation of isoflavones than dry heating when pure genistein and its conjugates in methanol were tested at 100, 150 and 200 °C. In soy milk, the genistein showed higher stability to heat treatment than daidzein and glycitein (16). Coward et al. (1) indicated that the toasted soy flour contained large amounts of 6”-O-acetyl-β-glucoside conjugates, formed by heat inducing decarboxylation of the malonate group to acetate. Converting 6”-O-malonylgenistin to 6”-O-acetyl-genistin during drying process and converting to genistin during hot water extraction were also reported by Barnes et al. (17). More and more soy containing food products are continuously demanded because the beneficial effects of bioactive compounds in soybeans. The raw or precooked soybeans or soy flour could be used for several soy-based products. Thus, the conversion among various isoflavones, as affected by different combinations of pretreatments and thermal treatments, has to be investigated. The objective of this study was to evaluate the moist and dry thermal effects on the conjugated isoflavones with regard to interconversions in soybeans and black soybeans.

Materials and Methods Sample Preparations Two soybeans (Glycine max L.) (KS1 and KS8) and two black soybeans (TN3 and TN6) were bred and provided from Kaohsiung and Tainan District Agricultural Research and Extension Stations in Taiwan, respectively. Soybeans were pretreated with soaking and germination, followed by thermal treatments either by autoclave steaming (moist heating) or oven roasting (dry heating). For soaked soybeans, the matured seeds were soaked in tap water at 35 °C for 5 hr and then water was drained out. For germinated soybeans, the matured seeds were prepared as previous (13), which the soybeans and black soybeans were germinated for 1 day with 3-6 mm and 4 days with 3-6 cm length of sprouts in an incubator (GTH-150-20-CP-AR, Giant Force Co., Ltd., Taiwan) at 25 °C, 75% RH. The soaked and germinated soybeans were immediately frozen overnight at -30 °C and dried by freeze-dryer (FD-1240, PANCHUM Scientific Corp., Taiwan), or followed by the thermal treatments. The pretreated soybeans and black soybeans used for this study were shown in Figure 1. Thermal Treatments of Soybeans The pretreated soybeans, soaked and germinated, were followed by thermal treatment which was either steamed at 121 °C in an autoclave (CL-32L, ALP Co. Ltd., Japan) for 10 to 60 min or roasted at 195 °C in an oven (Chung Pu Baking Machinery Co. Ltd., Taichung, Taiwan) for 10, 20 and 30 min. After steaming, 173 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


the samples were lyophilized as described above, ground and stored in a desiccator until use. The roasted soybeans were ground immediately after roasting and stored in a desiccator, except for the 10 min-roasted soybeans. The short roasting time could not efficiently remove water from soybeans and caused the difficulty in grounding samples so that the roasted soybeans were lyophilized before grinding.

Figure 1. The pretreated soybeans (a) and black soybeans (b).

174 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


HPLC Analysis of Isoflavones The isoflavones of soybeans were extracted and analyzed according to the method of Lin and Lai (13) with some modifications. A Hitachi HPLC equipment (Hitachi Ltd., Japan) and a YMC-pack ODS-AM-303 column (250 mm × 4.6 mm i.d., 5 µm, YMC Co., Ltd., Japan) were used for the determinations. A linear gradient system of 0.01% TFA in acetonitrile (A) and 0.01% TFA in distilled water (B) is programmed as follows: from 0 to 20 min, eluent A is increased from 15 to 20%; from 20 to 30 min, eluent A was from 20 to 24%; from 30 to 34 min, eluent A was kept in 24%; from 34 to 44 min, eluent A was from 24 to 35%; from 44 to 60 min, eluent A was from 35 to 40%. The isoflavone isomers were identified by comparing the retention times and quantified by using standard curves with authentic standards; daidzein, daidzin, acetyldaidzin, genistein, genistin, acetylgenistin, malonylgenistin, malonylglycitin (LC Laboratories Co., Woburn, MA), glycitein, glycitin, acetylglycitin, and malonyldaidzin (Nagara Science Co., Ltd., Gifu, Japan).

Statistical Analysis All assays were run at least in duplicate and the results were analyzed by analysis of variance (ANOVA) using the general linear model and Duncan’s new multiple-range test (p

Thermal Effects on the Conversion of Isoflavones in Soybean

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