Tracking isoflavones: From soybean to soy flour,soy protein isolates to functional soy bread

Soybean seeds with three different levels (low, intermediate and high) of isoflavones were processed to soy flour and soy protein isolates (SPIs) and developed into functional soy breads. The effect of factors involved in all steps of the process was investigated by tracking the composition and concentration of native forms of isoflavones. The total isoflavone contents were 8033.3, 10570.1 and 15169.0 nmol/g DM (dry matter) in the three soybeans; 13201.5, 20034.4 and 26014.3 nmol/g DM in defatted soy flours; 9113.2, 13274.6 and 17918.3 nmol/g DM in the SPI; 2782.7, 4081.4 and 5590.3 nmol/g DM in soy breads, respectively. The bread making processes did not affect the total isoflavone content, but changed glucosides/acetylglucosides to aglycones. Malonylglucosides were stable prior to baking but degraded to acetylglucosides and further to glucosides during baking. Our results provide critical information for the production of functional soy breads that contain varying amounts of soy isoflavones.     

Determination of optimal acid hydrolysis time of soybean isoflavones using drying oven and microwave assisted methods

The aim of the present research was to determine an optimal acid hydrolysis condition using drying oven and microwave assisted methods to estimate isoflavone contents by RP-HPLC in soybean. All isoflavone glucosides were completely converted to their aglycones at 120 min for drying oven and 50 min for microwave. Optimal extraction time of the highest isoflavone aglycone content after acid hydrolysis was achieved in 3 h. These results indicated that the optimised hydrolysis and extraction conditions of isoflavones in soybean were: soybean (1 g) hydrolysed by 10 ml of 1 N HCl at 100 °C for 50 min using microwave assisted acid hydrolysis method, and then 15 ml of EtOH was added to the mixture which leaved alone for 3 h at room temperature for complete extraction. Thus, microwave is an easy, consumed less time, and reliable acid hydrolysis method to estimate soybean isoflavones in comparison with drying oven method.     

Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomycesthermophila

The β-glucosidase from Paecilomyces thermophila J18 was found to be capable of hydrolysing daidzin and genistin in a previous study. This report further evaluated the thermostability and hydrolysis of soybean isoflavone glycosides. The enzyme was found to be very stable at 50 °C, and retained more than 95% of its initial activity after 8 h at 50 °C. It converted isoflavone glycosides, in soybean flour extract and soybean embryo extract, to their aglycones, resulting in more than 93% of hydrolysis of three isoflavone glycosides (namely, daidzin, genistin and glycitin) after 4 h of incubation. Also, addition of the β-glucosidase greatly increased the contents of isoflavone aglycones in the suspended soybean flour and soymilk. The results indicate that the thermostable β-glucosidase may be used to increase the isoflavone aglycones in soy products. This is the first report on the potential application of fungal β-glucosidases for converting isoflavone glycosides to their aglycones in soy products.     

青方腐乳发酵过程中大豆异黄酮的转化研究

本文比较了青方、红方、白方和低盐红腐乳中大豆异黄酮组成和含量差异,并对青方腐乳发酵过程中大豆异黄酮含量和构型变化规律进行研究。结果表明,四种类型腐乳中大豆异黄酮基本以苷元形式存在,青方腐乳大豆异黄酮含量明显低于其他类型腐乳,仅为红方腐乳的33.01%,从单一异黄酮来看,大豆苷元和染料木素在四种类型腐乳中的含量明显高于黄豆黄素;青方腐乳发酵过程中大豆异黄酮转化研究发现,白坯中大豆异黄酮以糖苷型为主,染料木苷含量高于大豆苷和黄豆黄苷,前酵过程中糖苷型大豆异黄酮转化为苷元型大豆异黄酮,盐腌过程中糖苷型大豆异黄酮含量有轻微降低,发酵过程中苷元型大豆异黄酮总量在后酵前30 d显著下降,其中大豆苷元可能部分转化为雌马酚,导致青方腐乳大豆异黄酮含量明显低于其他类型腐乳,对其转化物质需进一步鉴定。     

HYDROLYSIS OF ISOFLAVONE PHYTOESTROGENS IN SOYMILK FERMENTED BY LACTOBACILLUS AND BIFIDOBACTERIUM COCULTURES

This study evaluated the hydrolysis of isoflavones in soymilk fermented at 37C for 48 h by four different Lactobacillus and Bifidobacterium cocultures. The hydrolysis of isoflavone β-glucosides significantly increased ( P <  0.05) the bioactive aglycones from 36 to over 90% of total isoflavones in soymilk fermented with any of the four Lactobacillus and Bifidobacterium cocultures as compared with unfermented soymilk. Compared with three other cocultures of Lactobacillus and Bifidobacterium, fermentation of soymilk with the Lactobacillus paracasei/Bifidobacterium longum cocultures yielded better isoflavone hydrolytic potential (Otieno-Shah index) and the highest β-glucosidase activity after 12 h of incubation.

PRACTICAL APPLICATIONS

Isoflavones are known as phytoestrogens because they are present in soy products and have estrogen-like activity. During fermentation, the majority of glucoside isoflavones in soymilk are converted to bioactive aglycones via microorganism-derived β-glucosidase. In human intestines, aglycone isoflavones are absorbed faster and in greater amounts than their glucosides. Using probiotic Lactobacillus and Bifidobacterium cocultures to ferment soymilk efficiently increases the bioactive aglycone concentrations. Hence, fermenting soymilk with this coculture could enhance the nutritional value of the product.     

Preparative column chromatography of four groups of isoflavones from soybean cake

Various adsorbents and solvent systems were compared with respect to the separation efficiency of four classes of isoflavones, malonylglucosides, acetylglucosides, glucosides and aglycones, in soybean cake by preparative column chromatography. The various fractions of isoflavones collected from column were monitored by HPLC. Soybean cake was found to contain malonylglucosides (803.70 g/ml), acetylglucosides (85.57 g/ml), glucosides (728.25 g/ml) and aglycones (53.02 g/ml). Both malonylglucosides and glucosides were separated using Diaion HP-20 as adsorbent and a solvent system of deionized water-ethanol with a ratio of 85:15 (v/v) and 73:27 (v/v), respectively. Whereas, both acetylglucosides and aglycones were separated using silica gel as adsorbent and a solvent system of n-hexane-isopropanol-ethanol (8:9:1, v/v/v). A total amount of 244.26, 21.25, 179.04 and 11.88 g/mg were obtained for malonylglucosides, acetylglucosides, glucosides and aglycones, respectively.     

Content of phytoestrogens in soy-based dietary supplements

The isoflavone content of 14 soy-based dietary supplements intended to help alleviate perimenopausal and menopausal symptoms on sale in Italy were analysed using HPLC with UV detection. The aim was to quantify soy isoflavones after hydrolysis as aglycones, which are the bioactive part of isoflavone molecules. In the examined products, the amounts of isoflavones were frequently expressed ambiguously, and none of the products stated whether the isoflavone content of the product was expressed as aglycones or as conjugates. Each product revealed a different aglycone concentration profile. These supplements have different “fingerprints”, probably due to different sources of raw materials and to methods used in processing and preparation of extracts. In more than half the supplements tested, the actual values contained were below those stated and below those expected to help alleviate perimenopausal and menopausal symptoms.     

Evaluation of enzymic potential for biotransformation of isoflavone phytoestrogen in soymilk by Bifidobacterium animalis,Lactobacillus acidophilus and Lactobacillus casei

Three strains of Lactobacillus acidophilus, two of Lactobacillus casei and one of Bifidobacterium were screened for β-glucosidase activity using ρ-nitrophenyl-β-d-glucopyranoside as a substrate and their potential for the breakdown of isoflavone glucosides to the biologically active aglycones in soymilk. Isoflavones quantification with HPLC and β-glucosidase activity were performed after 0, 12, 24, 36, and 48 h of incubation in soymilk at 37 °C. All six micro-organisms produced β-glucosidase, which hydrolysed the predominant isoflavone β-glucosides. There was a significant increase and decrease (P < 0.05) in the concentration of isoflavone aglycones and glucosides, respectively, in fermented soymilk. Based on the concentration of isoflavones during peak β-glucosidase activity, the hydrolytic potential was calculated. L. acidophilus 4461 had the highest aglycone concentration of 76.9% after 24 h of incubation, up from 8% in unfermented soymilk (at 0 h). It also had the best isoflavone hydrolytic index of 2.01, signifying its importance in altering the biological activity of soymilk.     

A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: Application to analyze isoflavone levels in soybean cultivars

Since only isoflavone aglycones are considered to be bioactive, the determination of total aglycones that are released from conjugated isoflavones after hydrolytic treatment may facilitate an objective alternative for quantifying isoflavone contents in soy products. Given this major benefit, a new ultra-high pressure liquid chromatography (UV-UPLC?) method was developed for the fast and reliable determination of total aglycones in soybeans (daidzein, glycitein, and genistein) after enzymatic hydrolysis applying helix pomatia digestive juice. Capitalizing on the enhanced performance of UPLC?, aglycones were separated within 3 min only, with a total runtime of 8 min till the next injection. Thus, especially compared to HPLC protocols, UPLC? proved to be superior due to significantly shorter runtimes and accordingly increasing sample throughput. Additionally, regarding the performed validation (linearity, precision, recovery, selectivity, and robustness), the established method proved to be suitable for quantifying total aglycones in soybeans. Moreover, method applicability was demonstrated by analyzing 23 commercial soybean cultivars for their isoflavone contents. Cumulative aglycone levels ranged from 100 to 255 mg per 100 g, hence implying an average ratio of 52%, 41%, and 7% of total isoflavones for genistein, daidzein and glycitein, respectively. However, for some soybeans, other distinct aglycone distributions were observed as well.     

Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone

The reduced incidence of prostate cancer (PCa) in Asia countries has been attributed to high soy diets, and major soy isoflavones, in particular daidzein and genistein, are thought to be the source of the beneficial and anti-cancer effects of soy foods. However, attention has been drawn to the safety of using high levels of soy isoflavones in humans, which is especially the concern for consumers taking regular soy isoflavone dietary supplements. The main objective of this study is thus to identify a soy isoflavone combination with lower levels of daidzein and genistein to be a more efficacious and safer chemo-preventive agent for PCa. The anticancer effects of daidzein and genistein, and their combinations on early-stage androgen-dependent PCa cells (LNCaP) and bone metastatic LNCaP-derivative PCa cells (C4-2B) were compared. Cells were treated with varying concentrations of daidzein, genistein (25–200 μM) or their combinations (25 or 50 μM) and cell proliferation, apoptosis, cell cycles and cellular uptakes of the isoflavones were measured after 48 h. Daidzein and genistein showed a synergistic effect on inhibiting cell proliferation and inducing apoptosis of both PCa cells. Twenty-five μM daidzein/50 μM genistein and 50 μM daidzein/50 μM genistein significantly increased the apoptotic effects on C4-2B cells although they did not show any effect when used individually. Except 50 μM daidzein/50 μM genistein, all other combinations had no impacts on cell cycles. For treatment with soy isoflavone combination, genistein was always better taken up than daidzein by both LNCaP and C4-2B cells.     

Transformation of Isoflavones During Sufu (A Traditional Chinese Fermented Soybean Curd) Production by Fermentation With Mucor flavus at Low Temperature

This study evaluated the changes in contents and composition of soybean isoflavone as well as the β-glucosidase production during sufu preparation by Mucor flavus (M. flavus) at low temperature. The results showed that sufu processing not only led to the losses of isoflavones but also caused the redistribution of isoflavone isomers. The levels of aglycones increased during sufu fermentation, while the corresponding levels of glucosides decreased. β-Glucosidase accumulation by M. flavus mainly contributed to the transformation of isoflavone glucosides into aglycones, which was affected by the NaCl supplementation. The highest content of aglycone isoflavones occurring was recorded as 99.4% at 15% NaCl content under investigated conditions. This work suggested that M. flavus had possible application as an alternative starter to produce sufu with the enhancement of the physiological function at lower temperature.     

Isoflavone phytoestrogen degradation in fermented soymilk with selected beta-glucosidase producing L. acidophilus strains during storage at different temperatures

Soymilk fermented with 3 selected Lactobacillus acidophilus strains were stored at various temperatures (-80 degrees C, 4 degrees C, 25 degrees C and 37 degrees C) for 8 weeks and the concentration of isoflavones determined weekly using RP-HPLC. The decreasing concentration of isoflavones in soymilk during storage due to degradation was found to fit the first order kinetics model. Isoflavone aglycones as well as isoflavone glucosides largely appeared to be stable during storage (P<0.01). Interestingly, the aglycone forms showed much smaller degradation as compared to glucoside forms at all the storage temperatures studied. Of the isoflavone aglycones, daidzein was found to be the most stable followed by genistein, while glycitein was least stable. Isoflavone aglycones such as glycitein, daidzein and genistein showed smaller degradation constants in fermented soymilk at lower storage temperatures (-80 degrees C and 4 degrees C) and higher degradation constants at higher storage temperatures (25 degrees C and 37 degrees C) with each strain. In contrast, glucosides glycitin and daidzin showed higher degradation at lower storage temperatures (-80 degrees C and 4 degrees C) and lower degradation at higher storage temperatures (25 degrees C and 37 degrees C). Storage temperature was therefore found to be very important in regulating the rate of degradation soy isoflavones in fermented soymilk.     

Use of Bacillus subtilis to enrich isoflavone aglycones in fermented natto

BACKGROUND: Natto is a food made by fermenting cooked soybeans with Bacillus subtilis. Soybean isoflavones are reported to provide many health benefits, including oestrogenic effects. However, isoflavone aglycones may be absorbed faster and in higher amounts in the human intestine than their glucosides. This study aimed to investigate the content of isoflavone components in commercial natto products as well as the use of B. subtilis strains to ferment cooked soybeans to produce a high level of isoflavone aglycones in natto. RESULTS: The content and composition of isoflavones in commercial natto products were predominantly (>76%) isoflavone glucosides. Fermentation of cooked soybeans with B. subtilis BCRC 14718 at 37 °C for 48 h was more effective in converting glucosides to aglycones than with other strains of B. subtilis, increasing the proportion of isoflavone aglycones from 12 to 68% of the total isoflavones in the fermented natto. The proportions of the isoflavone aglycones daidzein and genistein in cooked soybeans fermented with B. subtilis BCRC 14718 for 48 h increased from 6 to 54% and from 5 to 13% respectively. CONCLUSION: Bacillus subtilis BCRC 14718 incubated with cooked soybeans produces higher levels of isoflavone aglycones, which may enhance health benefits over traditional fermented natto. Copyright © 2008 Society of Chemical Industry     

Functional attributes of soybean seeds and products,with reference to isoflavone content and antioxidant activity

The concentrations of isoflavones, especially daidzein and genistein, were determined by high performance liquid chromatography in 4 soybean cultivars and 26 soybean products. The total isoflavone content of the soybean cultivars was in the range of 525–986 mg per kg, and for soy products it was 32.9–795 mg per kg. Amongst the soybean products, the isoflavone content decreased in the order: soy sprouts, soy seeds, soy flour, soy milk, soy meals and soy sauce. Significant differences in the concentration of genistein and daidzein were observed between the commercial soy products and also within the soybean cultivars. The antioxidant activity of soybean and soy products correlated well with total phenolic content (TPC) and total isoflavones (TI), whereas TPC showed higher correlation with TI.     

Enrichment of isoflavone aglycones in soymilk by fermentation with single and mixed cultures of Streptococcus infantarius 12 and Weissella sp. 4

Soymilk was fermented with either Streptococcus infantarius 12 (Si 12), Weissella sp. 4 (Ws 4), or their mixed cultures with different mixing ratios (Si 12:Ws 4 = 1:1, 1:3, 1:5, and 1:10, v/v) for 12 h at 37 °C. All cultures in soymilk readily proliferated and reached about 10^8–9 CFU/mL. After 12 h, pH and titratable acidity of soymilk ranged 4.19–4.47 and 0.57%–0.64%, respectively. The pH of soymilk fermented with Si 12 was the lowest while that obtained with Ws 4 the highest. A sharp increase in β-glucosidase (β-glu) activity corresponded well with a rapid decrease in isoflavone glucosides and an increase in aglycone contents. The rate of hydrolysis of isoflavone glucosides was the least with Si 12 while the highest with Ws 4, resulting in about 23%–33% and 98%–99% hydrolysis of the glucosides with Si 12 and Ws 4, respectively, after 12 h. Mixed cultures with 1:3, 1:5, and 1:10 ratios seem to be more effective starters for bioactive fermented soymilk with more aglycones and appropriate acidity in a short time than single cultures.     

Changes of isoflavone in soybean cotyledons soaked in different volumes of water

Soybean cotyledons, recently dehulled, were soaked at 50 °C for 12 h as a pre treatment to obtain defatted soy flour enriched in aglycones. Grains of cultivar BRS 213 from the crop years 2004 and 2005 were used and initially had 1.4 and 1.2 mg g⁻¹ of total isoflavones, respectively. The molar mass of malonyl and β-glycosides decreased after soaking (33% and 56.5%, in grains from the crop years 2004 and 2005, respectively), while the aglycones daidzein and genistein, that were previously undetectable, increased to 0.5 and 0.8 μmol g⁻¹ in grains from the crop years 2004 and 2005, respectively. Cotyledons treated with the reduced volume of water had a reduction of 4% of the total isoflavone molar mass while, for the cotyledons treated with higher volume of water, there was a reduction of 14%, due to the leaching of isoflavones to the soaking water.     

Stability of isoflavone phytoestrogens in fermented soymilk with Bifidobacterium animalis Bb12 during storage at different temperatures

Soy isoflavones in fermented soymilk with Bifidobacterium animalis Bb12 were stored at various temperatures (?80, 4, 24.8 and 37 °C) for 8 weeks and the concentration of isoflavones determined weekly using reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The first‐order kinetic model was used to assess the degradation of each isomer at each storage temperature. Soymilk predominantly possesses high concentrations of isoflavone glucosides and very low concentration of bioactive aglycone component. During storage at various temperatures, concentrations of individual isoflavone isomers appeared to be significantly stable (P < 0.01). Interestingly, aglycones showed much smaller degradation constants compared with glucosides at all the storage temperatures studied. Genistein and daidzein were much more stable than glycitein and had almost similar degradation patterns, despite differences in their concentrations in the fermented soymilk. It was, however, observed that 4 °C was the most suitable storage temperature for the product in order to guarantee minimal degradation of bioactive isoflavone aglycones.     

Method development and validation for isoflavones quantification in coffee

This paper reports the development and validation of an analytical method to quantify three isoflavones (daidzein, genistein and formononetin) in coffee beans and coffee brews, by HPLC with diode-array detection. The aglycones were released by methanolic acid hydrolysis in the presence of the antioxidant BHT and the internal standard 2′-methoxyflavone. The method showed high correlation coefficients (r > 0.999) for standards subjected to the entire procedure. For samples, good intra- and inter-day precisions (<8%), and accuracies (recoveries of 95 ± 1% for ground coffee and 92 ± 2% for coffee brew) were achieved. Detection and quantification limits ranged from 5 to 8 ng/mL and from 14 and 25 ng/mL, respectively. The proposed method proved to be sensitive, precise and accurate.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

Effect of the fermentation of whole soybean flour on the conversion of isoflavones from glycosides to aglycones

The effect of fermentation of whole soybean flour (WSF) on the conversion of isoflavone glycosides to the aglycone form was analysed by liquid chromatography. WSF (200 g) with 35% moisture, was autoclaved at 121 °C for 20 min (AWSF), cooled and inoculated with 2 mL of spores of the fungus Aspergillus oryzae CCT 4359, and then incubated for 24 h and 48 h. The fermented flour was dried in a vacuum oven and 10 g of each flour, non-fermented and fermented, sieved and defatted. One gram of each flour was used for extraction of the isoflavones with 10 mL of an 80% methanol solution. Aliquots were injected into the HPLC under the following conditions: C18 column, 30 °C, gradient elution, mobile phase of (A) water: 5% acetic acid (v/v) and (B) methanol. The results showed that the FAWSF-48 h contained predominantly isoflavone aglycones (75.51%) when compared to the AWSF (6.94%) and WSF (2.67%).