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.     

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     

Enrichment of bioactive isoflavones in soymilk fermented with β-glucosidase-producing lactic acid bacteria

This study was undertaken to investigate the possible application of β-glucosidase-producing lactic acid bacteria as a functional starter cultures to obtain the bioactive isoflavones, genistein and daidzein, in fermented soymilk. Four strains – Lactobacillus plantarum KFRI 00144, Lactobacillus delbrueckii subsp. lactis KFRI 01181, Bifidobacterium breve K-101 and Bifidobacterium thermophilum KFRI 00748 – among the 31 lactic acid bacteria tested for β-glucosidase activity using ρ-nitrophenyl-β-d-glucopyranoside as the substrate were selected. Acid development, viable populations, and quantification of isoflavones using HPLC were performed at 0, 24, and 48 h of incubation at 37 °C. The significant bioconversion (P < 0.001) of the glucoside isoflavones into their bioactive aglycones in soymilk fermented with four β-glucosidase-producing strains, with an average 7.1-fold increase of aglycones (daidzein + genistein) was observed. There appeared to be correlations between the level of growth and β-glucosidase activity of each strain, and the hydrolysis of conjugated isoflavones in soymilk fermentation. Lactobacillus sp. were able to readily proliferate in soymilk than Bifidobacterium sp. (P < 0.05) and therefore completed more rapidly the hydrolysis of glucoside isoflavones.The present study indicates that four β-glucosidase-producing lactic acid bacteria have great potential for the enrichment of bioactive isoflavones in soymilk fermentation.     

Production of beta-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk

ABSTRACT:  The study determined β-glucosidase activity of commercial probiotic organisms for hydrolysis of isoflavone to aglycones in fermenting soymilk. Soymilk made with soy protein isolate (SPI) was fermented with Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and Lactobacillus casei LAFTI^® L26 at 37 °C for 48 h and the fermented soymilk was stored for 28 d at 4 °C. β-Glucosidase activity of organisms was determined using ρ-nitrophenyl β-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94, and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4 °C for 28 d was 20%, 15%, and 11% greater ( P < 0.05) than initial cell counts, respectively. All the bacteria produced β-glucosidase, which hydrolyzed isoflavone β-glycosides to isoflavone aglycones. The decrease in the concentration of β-glycosides and the increase in the concentration of aglycones were significant ( P < 0.05) in the fermented soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.     

Soymilk fermented with Lactobacillus rhamnosus CRL981 ameliorates hyperglycemia,lipid profiles and increases antioxidant enzyme activities in diabetic mice

Interest in soybeans and soy-based products has grown significantly in the last decades due to their reported nutritional and health-promoting effects. In soybean and non-fermented soy foods, isoflavones are predominantly found as glucosides, which must be hydrolyzed in order to exert their documented beneficial effects. The objective of this study was to evaluate the effect of soymilk fermented with Lactobacillus rhamnosus CRL981, a strain that can completely hydrolyze glucoside isoflavones due to its high beta-glucosidase activity. Using a diabetes animal model induced with streptozotocin, it was shown that the fermented soymilk was able to significantly decrease glucose levels, total cholesterol concentrations, triacylglycerols and increase antioxidant enzyme activities compared to animals that received unfermented soymilk. This study clearly shows that the adequate selection of starter cultures could be used to develop novel soy based products intended for the prevention or adjuvant treatment of diseases such as diabetes.     

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.     

Conversion of isoflavone glucosides to aglycones in soymilk by fermentation with lactic acid bacteria

ABSTRACT:  Four lactic acid bacteria (LAB), Lactobacillus paraplantarum KM, Enterococcus durans KH, Streptococcus salivarius HM and Weissella confusa JY, were isolated from humans and tested for their capabilities of converting isoflavone glucosides to aglycones in soymilk. Changes in growth, pH, and titratable acidity (TA) were investigated during fermentation at 37 °C for 12 h. After 6 to 9 h of fermentation, each population of 4 LAB reached 10⁸ to 10⁹ CFU/mL. The initial pH of 6.3 ± 0.1 decreased while the TA of 0.13%± 0.01% increased as fermentation proceeded, resulting in the final range between 4.1 ± 0.2 and 4.6 ± 0.1 for pH and between 0.51%± 0.02% and 0.67%± 0.06% for TA after the 12 h of fermentation. The glucoside concentrations were significantly decreased in soymilks fermented with either L. paraplantarum KM, S. salivarius HM, or W. confusa JY with fermentation time ( P < 0.05). L. paraplantarum KM was the best in percent conversion of glucosides to corresponding aglycones, resulting in 100%, 90%, and 61% hydrolysis of genistin, daidzin, and glycitin, respectively, in 6 h. Consequently, the aglycone concentrations in soymilk fermented with L. paraplantarum KM were 6 and 7-fold higher than the initial levels of daidzein and genistein, respectively, after 6 h of fermentation. Changes in the daidzin and genistin levels were not significant in soymilk fermented with E. durans KH. The rates of hydrolysis of glucosides varied depending on the species of LAB. Especially, L. paraplantarum KM seems to be a promising starter for bioactive-fermented soymilk based on its growth, acid production, and isoflavone conversion within a short time.     

Mutagenicity and antimutagenic effect of soymilk fermented with lactic acid bacteria and bifidobacteria

In this study, soymilk was first fermented with lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus) and bifidobacteria (Bifidobacterium infantis, Bifidobacterium longum) both individually and simultaneously. Mutagenicity and the suppression of fermented soymilk against the mutagenesis induced by 4-nitroquinoline-N-oxide (4-NQO), a direct-acting mutagen, and 3,2'-dimethyl-4-amino-biphenyl (DMAB), an indirect-acting mutagen, on Salmonella typhimurium TA 100, was then investigated. It was found that the fermented soymilk shows no mutagenic activity on Sal. typhimurium TA 100. Fermentation, in general, significantly (p<0.05) enhanced the antimutagenicity of soymilk. The levels of increased antimutagenicity of fermented soymilk varied with the starter organism and the type of mutagen tested. Although unfermented soymilk exerted lower antimutagenic activity against DMAB than 4-NQO, the fermented soymilk, generally, showed a higher antimutagenic activity against DMAB than 4-NQO. Among the various fermented soymilk tested, soymilk fermented with both Str. themophilus and B. infantis simultaneously exhibited the highest antimutagenicity of 85.07% and 85.78%, respectively, against 4-NQO and DMAB Further investigation on this fermented soymilk revealed that both the antimutagenic factors formed during fermentation and the cells of the starter organisms contributed to the increased antimutagenic activity against DMAB, while the former led to the increased activity against 4-NQO.     

Using of Lactobacillus and Bifidobacterium to product the isoflavone aglycones in fermented soymilk

The study determined isoflavone aglycone contents in soymilk and sugar-soymilk fermented with a single culture of two strains of Lactobacillus paracasei, two strains of Lactobacillus acidophilus, and one strain of Bifidobacterium longum respectively at 37 degrees C for 24 h. Isoflavone concentration was analyzed by HPLC. The viable count of bacteria in all of the fermented soymilk ranged from 7 to 9 log CFU/ml. The isoflavone aglycone had a significant increase of 62%-96% of isoflavone in all of the fermented soymilk compared to 17% in non-fermented soymilk (P<0.05). The five strains of microorganisms produced lesser amount of isoflavone aglycones in fermented sucrose-soymilk than in other fermented soymilk.     

Fermentation of calcium-fortified soymilk with Lactobacillus: effects on calcium solubility, isoflavone conversion, and production of organic acids

ABSTRACT:  The objective of this study was to enhance calcium solubility and bioavailability from calcium-fortified soymilk by fermentation with 7 strains of Lactobacillus , namely, L. acidophilus ATCC 4962, ATCC33200, ATCC 4356 , ATCC 4461 , L. casei ASCC 290, L. plantarum ASCC 276, and L. fermentum VRI-003. The parameters that were used are viability, pH, calcium solubility, organic acid, and biologically active isoflavone aglycone content. Calcium-fortified soymilk made from soy protein isolate was inoculated with these probiotic strains, incubated for 24 h at 37 °C, then stored for 14 d at 4 °C. Soluble calcium was measured using atomic absorption spectrophotometry (AA). Organic acids and bioactive isoflavone aglycones, including diadzein, genistein, and glycetein, were measured using HPLC. Viability of the strains in the fermented calcium-fortified soymilk was > 8.5 log₁₀ CFU/g after 24 h fermentation and this was maintained for 14-d storage at 4 °C. After 24 h, there was a significant increase ( P < 0.05) in soluble calcium. L. acidophilus ATCC 4962 and L. casei ASCC 290 demonstrated the highest increase with 89.3% and 87.0% soluble calcium after 24 h, respectively. The increase in calcium solubility observed was related to lowered pH associated with production of lactic and acetic acids. Fermentation significantly increased ( P < 0.05) the level of conversion of isoflavones into biologically active aglycones, including diadzein, genistein, and glycetein. Our results show that fermenting calcium-fortified soymilk with the selected probiotics can potentially enhance the calcium bioavailability of calcium-fortified soymilk due to increased calcium solubility and bioactive isoflavone aglycone enrichment.     

Enhancements of isoflavone aglycones, total phenolic content, and antioxidant activity of black soybean by solid-state fermentation with Rhizopus spp.

In the present study, a solid-state fermentation process of black soybean was developed. Three filamentous fungi including Rhizopus oligosporus (BCRC 31996), R. oligosporus (NTU-5), and Rhizopus oryzae (BCRC 30894) were cultivated with steam-cooked intact and dehulled black soybeans. It was found that fermentation enhanced the total phenolic and isoflavone aglycone content as well as antioxidant activity of the black soybean methanol [80 % (v/v)] extracts. Among the three candidates, R. oligosporus BCRC 31996 was chosen as the working strain, and dehulled Tainan NO.3 black soybeans were used as the substrate based on the relatively high total phenolic content, isoflavone aglycone accumulation, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect of the fermented extracts. In a 6-day fermentation, extracts of the fermented dehulled black soybeans yielded higher total phenolic content (2,876.2 μg/g) which was 1.16 times of the result from the intact soybean extracts. The DPPH scavenging effect of the fermented dehulled black soybean extract reached its maximum on the third day. The activity of β-glucosidase of dehulled black soybean samples increased with time and resulted in accumulation of isoflavone aglycones. The product of solid-state black soybean fermentation serves as a functional ingredient in the products of nutritional supplements and health foods.     

Physicochemical characteristics and production of whole soymilk from Monascus fermented soybeans

Monascus fermented soybeans (MFS) were prepared by solid state fermentation. MFS showed higher solubility than the unfermented control within the acidic pH region but showed the opposite trend at pH 7 and 9. Although the emulsifying activity index of MFS was lower than that of the control, MFS had significantly higher emulsion stability at pH 7 and 9. Significant protein hydrolysis took place during fermentation, and the proportion of peak area less than 1.35 kDa was greatly increased in MFS. Whole soymilk prepared from MFS contained monacolin K (475 μg/g), and was enriched in isoflavone aglycones. Neither homogenisation nor pasteurisation caused significant changes in the isoflavone and monacolin K contents of the Monascus fermented soymilk. Total phenol content and ABTS radical scavenging activity were significantly higher in the Monascus fermented soymilk than in the control soymilk.     

植物乳杆菌58发酵豆乳产大豆异黄酮苷元条件的优化

本研究通过测定植物乳杆菌58在不同碳源中的生长和产β-葡萄糖苷酶情况,筛选菌株的最适碳源,并确定接种至豆乳的最佳时间.在接种量、糖含量、发酵时间和发酵温度等单因素实验基础上,根据Box-Behnken中心组合原理进行响应面实验设计,以大豆异黄酮苷元含量为指标,进一步优化菌株58发酵豆乳产大豆异黄酮苷元条件.结果显示,植...     

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。     

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。     

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.     

Biotransformation of Isoflavones by Bifidobacteria in Fermented Soymilk Supplemented with D-Glucose and L-Cysteine

ABSTRACT: Soymilk prepared using soy-protein isolate supplemented with D-glucose and L-cysteine was fermented with 4 strains of Bifidobacterium. Enumeration of bifidobacteria and quantification of isoflavones using HPLC were performed at 0, 12, 24, 36, and 48 h of incubation. Supplementation did not significantly enhance ( p > 0.05) the growth of bifidobacteria between 0 and 12 h, but did after 12 h. The increase in concentration of isoflavone aglycones and equol was significantly lower ( p < 0.05) in supplemented soymilk after 24 h when compared to plain soymilk. Supplementation increased the concentration of aglycones by 0.796 mg/100 mL in soymilk fermented with B. animalis between 12 and 24 h, and the population by 1.27 log₁₀ CFU/mL ( p < 0.05).     

Fermentation with Bacillus spp. as a bioprocess to enhance anthocyanin content,the angiotensin converting enzyme inhibitory effect,and the reducing activity of black soybeans

Food possessing anthocyanins, angiotensin converting enzyme (ACE) inhibitory activity or reducing activity show beneficial effect on human health. To develop healthy food, black soybeans were fermented with either Bacillus subtilis BCRC 14715 or Bacillus sp. CN11, or a mixture of both Bacillus spp. in the present study. The anthocyanin content, the ACE inhibitory activity and the reducing power of the fermented black soybean were then examined. It was found that the ACE inhibitory activity of the extracts of bean and viscous material from the fermented black soybeans varied with extraction solvents and starter organism, yet increased as the fermentation period was extended, regardless of starter organism. After 18 h of fermentation, the water extract of bean showed less ACE inhibitory activity than did the respective 80% ethanol extract. While the water extract of viscous material showed a higher ACE inhibitory activity than the respective ethanol extract. With respect to extraction yield, it was found that the ACE inhibitor in the fermented black soybean could be extracted more efficiently with water than 80% ethanol. Fermentation with B. subtilis BCRC 14715 was also found to increase the anthocyanin content of black soybean and the reducing activity of the extracts. Finally, the 80% ethanol extract showed a higher reducing activity than the water extract.     

Fermentation of commercial soy beverages with lactobacilli and bifidobacteria strains featuring high β-glucosidase activity

An increase of isoflavone aglycone content in soy foods can be attained through fermentation with food-grade bacteria. In this study, two commercial soy beverages with distinctive chemical composition (AS and VS) were fermented by strains of lactobacilli (eight strains) and bifidobacteria (two strains) with a high β-glucosidase activity. Along fermentation, growth of the strains and isoflavone deglycosylation in the soy beverages were monitored. Large differences in growth, aglycone content and chemical parameters in AS and VS beverages fermented by different species and strains were observed. Isoflavone glycosides were completely transformed into their corresponding aglycones by most strains during fermentation of AS beverage, whereas large amounts of undeglycosilated isoflavones were still present in fermented VS. Four strains showing strong deglycosylation activity and appropriate technological properties (Lactobacillus casei LP71, Lactobacillus plantarum E112, Lactobacillus rhamnosus E41 and Bifidobacterium pseudocatenulatum C35) were proposed as industrial starters to improve functionality in soy-based fermented foods.     

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.