Evaluation of the composition and concentration of isoflavones in soy based supplements,health products and infant formulas

Isoflavones are weak estrogenic compounds found in soybeans and soy foods. The objective of the present study was to determine the content and composition of isoflavones in different soy isoflavone supplements, soy based health products and infant formulas. Commercially available samples of 13 different soy supplements and 11 soy based health products obtained from South East Asia were analyzed for their isoflavone concentrations by using high-performance liquid chromatography and their peak identities confirmed by LC–MS. The extent of daily intake of isoflavones from different products based on the directions for their consumption was further evaluated. The amount of isoflavones in dietary supplements, soy based health products and infant formulas ranged from 405 to 57,570, 46.32–1333.80 and 59.54–226.84 μg/g of the sample, respectively, expressed as aglycone equivalents.     

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     

Efficacy of isoflavones in relieving vasomotor menopausal symptoms – A systematic review

This review assessed the efficacy of isoflavone supplements to reduce vasomotor symptoms in menopausal women by reviewing all published randomized controlled trials. Systematic literature searches were carried out in 70 databases. Randomized and placebo controlled studies were included if they investigated the treatment of isoflavone supplements derived from soy or red clover on vasomotor symptoms in peri‐ or postmenopausal women for at least 12 wks. Data were analyzed concerning outcome and methodological quality of the study. Twenty‐three trials met the inclusion criteria, thereof 17 investigated soy isoflavones and 6 red clover isoflavones. Without exception, selected trials examining the effect of red clover isoflavones were already assessed in several meta‐analyses and were therefore excluded from this evaluation. As the soy isoflavone studies were very heterogeneous concerning interventions and outcome measures, meta‐analysis could not be performed and trials were systematically assessed in a structured approach. Included soy isoflavone studies had numerous quality deficiencies and did not consistently show a reduction of flushes after treatment with soy isoflavones. Therefore, there is no conclusive evidence, but only some indication of a benefit of soy isoflavones on hot flush frequency or severity.     

Modification of isoflavone profiles in a fermented soy food with almond powder

Isoflavone profiles of a fermented soy food, cheonggukjang, were modified using almond powder. Isoflavones were analyzed by high performance liquid chromatography (HPLC) with an ultraviolet detector. Malonyl derivatives of isoflavones decreased and aglycones of isoflavones increased in samples with almond powder for 48 h. As added, almond powder increased from 0%, 5%, and 10% (w/w), amounts of aglycones increased to 21.11%, 26.63%, and 32.45% for 48 h, respectively. β-Glucosidase activity in 5% and 10% almond added samples was significantly higher than samples without addition of almond (P < 0.05). The content of succinyl daidzin and succinyl genistin, new metabolites from isoflavones, in almond-added cheonggukjang was significantly lower than control samples, implying that β-glucosidase activity from almond affected negatively the formation of succinyl derivatives (P < 0.05). Principal component analysis (PCA) for isoflavone distribution showed that first principal component (PC1) and second principal component (PC2) expressed 64.78% and 22.26% of the data variability, respectively. Biotransformation of isoflavones in any fermented soy foods can be achieved using natural products containing high β-glucosidase activity such as almond. PRACTICAL APPLICATION: The results of this study can help to modify the structural transformation of phytochemicals in any fermented soy foods using natural products. Adjusting the content of almond powder can achieve wanted profiles, for example, high aglycones content. Also, content of metabolites such as succinyl derivatives can be controlled using proper amounts of almond and fermentation time.     

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.     

Determination of phytoestrogens in dietary supplements by LC-MS/MS.

Labelling data quantifying the exact content of individual phytoestrogen analytes in dietary supplements are generally poor. As these products are commonly used in the management of menopause symptoms, any clinical benefits would be dependent on the exact dosage of isoflavones received. Well-established extraction procedures and updated isotope dilution mass spectrometry liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) have been used to accurately quantify the concentrations of ten common isoflavones in 35 dietary supplement samples on sale in the UK, Canada and Italy. Concentration-specific ionization suppression is described for biochanin A and formononetin. All supplements contained phytoestrogens. The soya isoflavones (genistein, daidzein, glycitein) were present in all products and the majority also contained the red clover isoflavones (biochanin A, formononetin) and some the Kudzu isoflavones (daidzein, puerarin). The content of total isoflavones per dose ranged from <1 to 53 mg. Trace amounts of coumestrol were found in six products. Other less common analytes, the prenylnaringenins (6-prenylnaringenin, 8-prenylnaringenin, 6,8-diprenylnaringenin) were not found in any of the products. Only 14 of 35 supplements were found to deliver more than or equal to 40 mg day(-1) of aglycone isoflavones, a consensus dose value recognized as delivering therapeutic benefit. Eleven did not match label claims. Six delivered less than 10 mg day (-1) of isoflavones. There has been little improvement in the overall quality of industry labelling in the five years since this was last investigated. Consequently, the public, retailers and healthcare professionals should consider using standardized isoflavone supplements, which are supported by analytical measurements.     

Determination of phytoestrogens in dietary supplements by LC-MS/MS

Labelling data quantifying the exact content of individual phytoestrogen analytes in dietary supplements are generally poor. As these products are commonly used in the management of menopause symptoms, any clinical benefits would be dependent on the exact dosage of isoflavones received. Well-established extraction procedures and updated isotope dilution mass spectrometry liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) have been used to accurately quantify the concentrations of ten common isoflavones in 35 dietary supplement samples on sale in the UK, Canada and Italy. Concentration-specific ionization suppression is described for biochanin A and formononetin. All supplements contained phytoestrogens. The soya isoflavones (genistein, daidzein, glycitein) were present in all products and the majority also contained the red clover isoflavones (biochanin A, formononetin) and some the Kudzu isoflavones (daidzein, puerarin). The content of total isoflavones per dose ranged from <1 to 53 mg. Trace amounts of coumestrol were found in six products. Other less common analytes, the prenylnaringenins (6-prenylnaringenin, 8-prenylnaringenin, 6,8-diprenylnaringenin) were not found in any of the products. Only 14 of 35 supplements were found to deliver more than or equal to 40 mg day(-1) of aglycone isoflavones, a consensus dose value recognized as delivering therapeutic benefit. Eleven did not match label claims. Six delivered less than 10 mg day (-1) of isoflavones. There has been little improvement in the overall quality of industry labelling in the five years since this was last investigated. Consequently, the public, retailers and healthcare professionals should consider using standardized isoflavone supplements, which are supported by analytical measurements.     

Determination of phytoestrogens in dietary supplements by LC-MS/MS

Labelling data quantifying the exact content of individual phytoestrogen analytes in dietary supplements are generally poor. As these products are commonly used in the management of menopause symptoms, any clinical benefits would be dependent on the exact dosage of isoflavones received. Well-established extraction procedures and updated isotope dilution mass spectrometry liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) have been used to accurately quantify the concentrations of ten common isoflavones in 35 dietary supplement samples on sale in the UK, Canada and Italy. Concentration-specific ionization suppression is described for biochanin A and formononetin. All supplements contained phytoestrogens. The soya isoflavones (genistein, daidzein, glycitein) were present in all products and the majority also contained the red clover isoflavones (biochanin A, formononetin) and some the Kudzu isoflavones (daidzein, puerarin). The content of total isoflavones per dose ranged from <1 to 53 mg. Trace amounts of coumestrol were found in six products. Other less common analytes, the prenylnaringenins (6-prenylnaringenin, 8-prenylnaringenin, 6,8-diprenylnaringenin) were not found in any of the products. Only 14 of 35 supplements were found to deliver more than or equal to 40 mg day^?1 of aglycone isoflavones, a consensus dose value recognized as delivering therapeutic benefit. Eleven did not match label claims. Six delivered less than 10 mg day^?1 of isoflavones. There has been little improvement in the overall quality of industry labelling in the five years since this was last investigated. Consequently, the public, retailers and healthcare professionals should consider using standardized isoflavone supplements, which are supported by analytical measurements.     

混菌发酵豆豉中异黄酮含量及对小鼠肺癌细胞的抑制作用

选用高产蛋白酶菌株枯草芽孢杆菌D2和β-葡萄糖苷酶产生菌枯草芽孢杆菌U35进行单菌种、混合菌种发酵生产豆豉,测定大豆异黄酮含量及其抗肿瘤能力,分析异黄酮含量和抗肿瘤能力的关系。用HPLC法测定豆豉提取物异黄酮含量,采用MTT比色法检测异黄酮含量对细胞增殖的影响,并在荧光显微镜下观察小鼠肺癌(LLC)细胞形态变化。结果表明,D2U35混菌豆豉苷元型异黄酮含量分别比D2、U35单菌豆豉高101.95μg/mL(2.38倍)和35.48μg/mL(1.25倍),D2U35混菌豆豉对肿瘤细胞的抑制率分别比D2、U35单菌豆豉高15.79%和7.02%,说明混菌发酵不仅提高了苷元型异黄酮含量,还提高了豆豉提取物的抗肿瘤能力。对肿瘤抑制作用,苷元苷型异黄酮>总异黄酮>糖苷型异黄酮。     

Quantitative Determination of Isoflavones in Soy Based Nutritional Supplements by High-Performance Liquid Chromatography

An increasing number of nutritional supplements contain phytoestrogens, in particular isoflavones, which potentially alleviate climacteric complaints. Intention of the present study was the qualitative and quantitative determination of isoflavones in soy based nutritional supplements in order to compare the actual content with the labeling of these products. For the analysis high-performance liquid chromatography (HPLC) with photo diode array (PDA) detection was used. The amount of isoflavones was determined via external calibration functions. Our analyses revealed certain variations in the isoflavone content of soy based nutritional supplements, with regard to the individual and the total amount. Furthermore, the total amount of isoflavones per serving unit in some products was higher than declared on the labeling, although only the major isoflavones were taken into account. This study shows that there is the necessity to clearly specify the isoflavone composition on the labeling and - due to safety aspects - to standardize and control the isoflavone content.

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Zusammenfassung:  Nahrungserg?nzungsmittel mit Zusatz von Soja werden vermehrt als “Alternative” zur klassischen Hormonersatztherapie für Frauen im Klimakterium angeboten. Diese Pr?parate enthalten Isoflavone, welche zu den Phytoestrogenen z?hlen und eine estrogene Wirkung aufweisen. Die in Sojabohnen enthaltenen Isoflavone sollen unter anderem klimakterische Beschwerden wie z.B. Hitzewallungen oder Tachykardie lindern. In der vorliegenden Studie wird eine Methode beschrieben, mit der aus Nahrungserg?nzungsmitteln mit Zusatz von Soja-Extrakten der Gehalt an Isoflavonen bestimmt werden kann. Dazu wurde eine HPLC-Methode mitDioden-Array-Detektion entwickelt, mit welcher die Quantifizierung der in Soja vorliegenden Isoflavone über externe Kalibrierfunktionen erfolgen kann. Damit war es m?glich, die vom Hersteller gemachten Angaben bezüglich der Isoflavongehalte zu überprüfen. Unsere Untersuchungen wiesen deutliche Unterschiede sowohl im Gesamtgehalt an Isoflavonen als auch in der individuellen Zusammensetzung der einzelnen Produkte auf. In einigen F?llen war der Isoflavongehalt in einigen Produkten pro Verzehrseinheit deutlich h?her als vom Hersteller angegeben. Unsere Untersuchungen belegen die Notwendigkeit im Sinne des Verbraucherschutzes, die Isoflavongehalte in Nahrungserg?nzungsmitteln klar zu spezifizieren und zu deklarieren.

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Received: January 9. 2008; accepted: February 19. 2008     

Critical Issues in R&D of Soy Isoflavone-enriched Foods and Dietary Supplements

ABSTRACT: Increasing consumer awareness of soy as a healthy food ingredient has led to a tremendous growth in sales of soy isoflavone‐enriched foods and dietary supplements in the past 3 y. Because of their weak estrogenic activity, isoflavones are believed to have preventive effects for several hormone‐dependent diseases. This article discusses some critical issues to be considered in any R&D program of novel soy and isoflavone products for the healthy‐food market, such as (1) Which isoflavone compounds should be in the product? (2) Which individual isoflavones are the most beneficial ones? (3) Can certain isoflavone groups be targeted to specific disease prevention goals? (4) Are there any safety concerns in isoflavone consumption? (5) How are isoflavones affected by thermal processing and storage? (6) How should isoflavones be analyzed? These questions may affect the choice of isoflavone source, processing conditions, quality control procedures, and marketing considerations.     

Systematic evaluation of pre-HPLC sample processing methods on total and individual isoflavones in soybeans and soy products

There are mainly two protocols in isoflavone analysis, one that involves hydrolysis prior to HPLC analysis and the other direct HPLC analysis. In this study, three different hydrolysis methods were systematically re-evaluated, and compared with direct HPLC analysis. Acidic hydrolysis (1.2-3 M HCl in ethanol at 80 °C) showed a maximum conversion of ca. 92% from glucosides to aglycones in 2 h; however, longer reaction caused degradation of genistein. Alkaline hydrolysis using 2 M NaOH converted acetylglucosides and malonylglucosides to their respective glucosides within 10 min. Glucuronidase from H. pomatia effectively converted isoflavone glucosides and acetylglucosides to aglycones. Quantification of the total isoflavones in various soy food products showed no significant difference among direct injection and the three hydrolysis methods (P < 0.05). We conclude that direct analysis of isoflavones in crude extracts is a rapid and accurate method to obtain isoflavone profiles and compositions in soybean, soy foods and beverages.     

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.     

腐乳前发酵和盐制过程中大豆异黄酮组分的变化研究

研究腐乳前发酵和盐制过程中大豆异黄酮组成和含量的变化。研究结果表明:在腐乳前发酵和盐制过程中,大豆异黄酮的总含量逐渐减少;大豆异黄酮总含量的损失主要是由白坯的制作和盐制过程引起的。在腐乳前发酵和盐制过程中,大豆异黄酮组分也发生改变:随着大豆异黄酮糖苷(daidzin和genistin)含量的减少,其相应的苷元(daidzein和genistein)明显增加。盐制过程中较低盐含量下的大豆异黄酮的含量较高。     

Profiling and quantification of isoflavones in soymilk from soy protein isolate using extracted ion chromatography and positive ion fragmentation techniques

Dietary supplements on soy based foods and beverages are increasingly gaining prominence all over the world. In this study, liquid chromatography coupled with positive electrospray ionisation tandem mass spectrometry (LC-ESI-MS) and diode array detection was used for the quantitation and characterisation of isoflavones in fermented and unfermented soymilk made from soy protein isolate SUPRO 590. Bifidobacterium animalis ssp. lactis Bb12 was used for the fermentation of soymilk. The isoflavones were found to produce characteristic radical ions as well as molecules of H₂O, CO₂, a sugar unit, and an alcohol through collision-induced fragmentation. Product ion fragments revealed unique fragmentation pathways for each isoflavone compound. Characteristic fragmentation of different isoflavones were unequivocally identified and differentiated. The occurrence of aldehydes such as pentanal, ethanal and methanal was shown to be specifically linked with isoflavone aglycones, daidzein, genistein and glycitein, respectively. Main glycosides such as genistin, daidzin and glycitin as well as the acetyl-, and malonyl forms also showed respective aglycone ions in their spectra fragmentation. Thus positive ion fragmentation was important in the absolute confirmation of isoflavones and to reveal the occurrence of other related compounds such as aldehydes in soymilk.     

A New Simplified and Stability Indicating Liquid Chromatography Method for Routine Analysis of Isoflavones Aglycones in Different Complex Matrices

In this work, a stability indicating method for routine analysis of isoflavones in different matrices was developed and validated. In order to simplify the analytical method, the glycosides were previously hydrolyzed to the corresponding aglycone forms. The separation of all isoflavone aglycones was achieved in 23 min, with a total time of analysis of 30 min, using trifluoroacetic acid 0.1 % (v/v) and acetonitrile:trifluoroacetic acid (100:0.01, v/v) as mobile phase. The LC method specificity was evaluated by the analysis of isoflavone standards submitted to acidic, alkali, neutral, oxidative, and photolytic stress conditions. The isoflavones degraded in alkali at 60 °C or in alkali under ulraviolet C (UVC) radiation, forming, in both conditions, three degradation products D1, D2, and D3 which were analyzed by liquid chromatography mass spectroscopy (LC-MS/MS). The method showed linearity higher than 0.999 with the concentration ranged from 0.1 to 10 μg ml^-1 for all isoflavone aglycones. The limits of quantitation obtained using calibration curves were from 0.28 to 0.37 μg ml^-1 and the intermediary precision at three levels (2, 6, and 10 μg ml^-1) showed RSD values between 0.03 and 0.25 %. After the performed validation, the LC method was applied to compare the isoflavone aglycones content in three different matrices: Glycine max beans, Glycine max dry extract, and isoflavone aglycone loaded nanoemulsion. The repeatability data showed RSD values between 0.02 and 1.41 % and the intermediary precision at three levels showed RSD values between 0.05 and 1.99 %. The recovery data of the isoflavone aglycones standards in the matrices at three levels were between 90.74 and 106.43 %.     

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.     

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.     

大豆胚轴中异黄酮的提取及其抗血小板聚集作用的研究

从大豆胚轴提取总糖苷后，采用C18反相柱层析法分离大豆异黄酮糖苷，再将其水解制备成异黄酮苷元，并研究大豆异黄酮对人血小板聚集性的影响。结果：在体外人血小板聚集实验中，大豆异黄酮苷元均抑制ADP、胶原蛋白和肾上腺素诱导的血小板聚集。提示：大豆异黄酮可能有助于防止血栓形成。     

Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria

In the present study, soymilk is fermented with lactic acid bacteria (Streptococcus thermophilus BCRC 14085, Lactobacillus acidophilus BCRC 14079) and bifidobacteria (Bifidobacterium infantis BCRC 14633, B. longum B6) individually, and in combination. The change in the content of various isoflavones (aglycones, glucoside, acetyl- and malonyl-glucosides) and the beta-glucosidase activity in soymilk during fermentation is investigated. It is observed that fermented soymilk contains a lower total isoflavone content (81.94-86.61 microg/ml) than soymilk without fermentation (87.61 microg/ml). Regardless of starter organism employed, fermentation causes a major reduction in the contents of glucoside, malonylglucoside and acetylglucoside isoflavones along with a significant increase of aglycone isoflavones content. The level of change in the content of various isoflavones and beta-glucosidase activity after fermentation varies with the starter organism. Among all the fermented soymilks tested, soymilk fermented with S. thermophilus showed the highest beta-glucosidase activity and the greatest increase in the contents of aglycones. The percentage of daidzein, genistein and glycitein to total isoflavone content in S. thermophilus-fermented soymilk increases from an initial 14.24%, 6.89% and 2.45%, respectively, to 36.20%, 28.80% and 12.44% after 24h of fermentation. Finally, the increase of aglycones and decrease of glucoside isoflavones during fermentation coincides with the increase of beta-glucosidase activity observed in fermented soymilk.