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.     

Role of microbial strain and storage temperatures in the degradation of isoflavone phytoestrogens in fermented soymilk with selected β-glucosidase producing Lactobacillus casei strains

Soymilk fermented with 2 Lactobacillus casei strains were stored at various temperatures (80 °C, 4 °C, 24.8 °C and 37 °C) for 8 weeks and isoflavone concentration analysed at weekly intervals using RP-HPLC. The degradation of each isoflavone compound at each storage temperature was found to fit first order kinetic model. Aglycone as well as glucosides generally appeared to be stable during storage (P < 0.01) at the 4 storage temperatures. Aglycone forms had smaller degradation constants compared to glucosides at all storage temperature and in the presence of both microorganisms. Specifically, aglycones showed a unique trend of smaller degradation at lower storage temperatures (−80 °C and 4 °C) than at higher temperatures (24.8 °C and 37 °C). Glucoside genistin was least stable at all storage temperatures compared to other isoflavones in the fermented soymilk with each strain while aglycone daidzein was the most stable. L. casei 2607 in fermented soymilk stored at 4 °C after 8 weeks gave the least degradation for daidzein of a mere 3.78% loss from 9.53 to 9.17 ng/μL. L. casei 2607 showed greater hydrolytic potential than L. casei ASCC 290 as denoted by higher degradation of isoflavone glucosides in fermented soymilk at lower storage temperatures.     

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。     

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.     

Isoflavone Profiles and Kinetic Changes during Ultra‐High Temperature Processing of Soymilk

Isoflavone profile is greatly affected by heating process. However, kinetic analyses of isoflavone conversion and degradation using a continuous industry processing method have never been characterized. In this study, Proto soybean was soaked and blanched at 80 °C for 2 min and then processed into soymilk, which underwent UHT (ultra‐high temperature) at 135 to 150 °C for 10 to 50 s with a pilot plant‐scale Microthermics processor. The isoflavone profile was determined at different time/temperature combinations. The results showed that all isoflavone forms exhibited distinct changing patterns over time. In the soymilk under UHT conditions, the degradation (disappearance) of malonyldaizin and malonylgenistin exhibited first‐order kinetics with activation energies of 59 and 84 kj/mole, respectively. At all UHT temperatures, malonylgenistin showed higher rate constants than malonyldaidzin. However, malonylglycitin changed irregularly under these UHT temperatures. The increase of genistin, daidzin, glycitein and acetlydaidzin during heating demonstrated zero‐order kinetics and the rate constants increased with temperature except for the conditions of 145 to 150 °C for 50 s. Overall, genistein series exhibited higher stability than daidzein series. Under all UHT conditions, total isoflavone decreased from 12% to 24%.     

Original article: Thermal dynamic properties of isoflavones during dry heating

Thermal stabilities of major soya isoflavones at different dry‐heating temperatures were determined in this study. The conversion of glucoside isoflavones to aglycone isoflavones was monitored as well. The thermal degradation of the isoflavones: glucoside form daidzin, glycitin, and genistin and aglycone form daidzein, glycitein, and genistein followed first‐order reaction kinetics at heating temperatures 100, 150, and 200 °C. The degradation rate constants of the six isoflavones were not significantly different at 100 °C. However, the constants increased with increasing heating temperature. The half‐life for the glucoside and aglycone isoflavones was from 144 to 169 min and 139 to 176 min at 100 °C, respectively. They decreased rapidly to 15.7–54.7 and 36.0–90.7 min when temperature increased to 150 °C. When heated at 200 °C, they further decreased to 5.8–6.0 and 15.7–21.2 min, respectively. The order of thermal stability from lowest to highest was glycitin < genistin < daidzin < glycitein < genistein < daidzein at temperature below 150 °C. However, their thermal stabilities were not different at 200 °C. The conjugated glucosides were cleaved from the isoflavones to produce their corresponding aglycones when heated at 150 °C or higher. The production of glycitein increased constantly and was the highest among the three aglycone isoflavones.     

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     

大豆异黄酮组分在腐乳后酵过程中的变化

研究腐乳后酵过程中大豆异黄酮组成和含量的变化。结果表明：在腐乳后酵过程中,大豆异黄酮总含量呈下降趋势;当食盐含量较低时,糖苷(daidzin 和genistin)分解较快;大豆异黄酮糖苷形式(daidzin 和genistin)转化为苷元形式(daidzein 和genistein)。说明腐乳后酵有效提高了大豆异黄酮的生物活性。     

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.     

丹贝异黄酮的提取分离与结构鉴定

从丹贝中分离纯化了 3种异黄酮 ,它们分别是染料木素、大豆黄素、黄豆黄素 ,而未分离到 6 ,7,4’ -三羟基异黄酮。丹贝中异黄酮的组成主要是各种苷元 ,证明丹贝生物活性的提高是由于异黄酮由糖苷水解成苷元所致。同时还发现 ,使用甲醇作溶剂在分离纯化过程中可能使染料木素转变成黄豆黄素     

Effect of thermal processing on genistein,daidzein and glycitein content in soymilk

Soymilk was subjected to various heat treatments at 95, 121 and 140 °C for various lengths of time. The contents of the aglycones of isoflavone (daidzein, glycitein and genistein) of the soymilk were determined using C₁₈ reversed‐phase high‐performance liquid chromatography. Genistein showed greater stability to heat treatment than daidzein and glycitein. Both the daidzein and glycitein contents decreased rapidly during the early stage of heating, but on continued heating the rates of decrease were much slower. Heating may cause an increase or decrease in the genistein content of soymilk depending on the temperature and time used. Upon heating at 95 and 121 °C, there was an increase in the genistein content in the early stage of heating, possibly due the conversion of genistin to genistein. Heating at 140 °C for more than 15 s and prolonged heating at 95 and 121 °C, however, caused a slow decline in the genistein content. Copyright © 2006 Society of Chemical Industry     

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.     

Bioavailability of soy isoflavones in rats Part I: application of accurate methodology for studying the effects of gender and source of isoflavones

There are limited and controversial reports about the effects of gender and source of isoflavones on their bioavailability. Moreover, several previous studies have not used appropriate methodology to determine the bioavailability of soy isoflavones, which requires comparing the area under the plasma concentration-time curve after both oral and intravenous injection (IV) administration. Therefore, the present study was conducted to determine the bioavailability of isoflavones from different sources following both oral and IV administration in male and female rats. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycones (daidzein, genistein and glycitein) and a mixture of synthetic glucosides (daidzin, genistin and glycitin) were tested. Following administration, blood samples were collected at several time points (0, 10, 30 min and 1, 2, 8, 24, 48 h post oral gavage and 0, 10, 30, 45 min and 1, 2, 3, 4, 8 h post-IV dosing) and plasma isoflavones were measured by LC/MS. Bioavailability values for daidzein, genistein and glycitein were significantly (p <0.05) higher (up to sevenfold) in Novasoy and the glucoside forms of isoflavones compared with those of the aglycone forms. Moreover, significant (p <0.05) gender differences in the bioavailability of 7-hydroxyl-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein), glycitein and daidzein were observed for Novasoy, with higher values in male rats. In summary, the source of isoflavones and the sex of rats had significant effects on isoflavone bioavailability.     

Lesser in vitro anaerobic cecal isoflavone disappearance was associated with greater apparent absorption of daidzein and genistein in Golden Syrian hamsters

Our hypothesis in this study was that in vitro disappearance of isoflavones from fecal or cecal contents of Golden Syrian hamsters paralleled the apparent absorption of these compounds, comparable with previous findings from in vitro human fecal incubations. Two studies were conducted to test this idea: one on in vitro fecal (study 1, n = 20/sex) and the other on in vitro cecal contents (study 2, n = 10/sex) ability to degrade isoflavones. According to HPLC analysis, urinary isoflavone excretion was significantly less by 2-4 fold in males compared with females in both studies. Fecal isoflavone excretion was not significantly different between sexes or isoflavones (study 1) and was <0.5% of ingested dose. In vitro anaerobic fecal isoflavone degradation rate constants from study 1 were minimal with no significant correlation between urinary and fecal isoflavone excretion. However, in vitro anaerobic cecal isoflavone degradation rate constants (study 2) were greater and significantly correlated with urinary excretion of daidzein (R = 0.90; p = 0.01) and genistein (R = 0.93; p = 0.004), but not glycitein (R = 0.50; p = 0.3). Both male and female hamsters showed a pattern of urinary isoflavone excretion similar to that found in humans (daidzein > genistein). Hamster in vitro cecal isoflavone degradation rate constants seemed to be analogous to human in vitro fecal isoflavone degradation rate constants for genistein and daidzein. The sex difference in isoflavone excretion in hamsters and the instability in glycitein excretion across studies coupled with the paucity of human data on this isoflavone deserve further investigation.     

Content and Profile of Isoflavones in Soy-Based Foods as a Function of the Production Process

Soy has been traditionally incorporated in diet as processed foods, such as soymilk, tofu, miso, tempeh, etc., and the consumption is commonly associated with a reduction of the development of chronic diseases due to their antioxidant, anti-inflammatory, and anti-allergic properties, among others. Many of the health benefits of soy have been attributed to isoflavones. They comprise a group of naturally occurring flavonoids consisting of heterocyclic phenols. Soy contains three types of isoflavones in four chemical forms: the aglycones daidzein, genistein, and glycitein; the β-glucosides daidzin, genistin, and glycitin; their 6″-O-malonyl-β-glucosides (6OMalGlc); and their 6″-O-acetyl-β-glucosides (6OAcGlc) conjugates. Industrial processing methods of soy-based food products commonly lead to the loss of isoflavones through removal of undesirable fractions. On the other hand, isoflavones can be transformed into different conjugates, which may have significant effects on the food texture and on the bioavailability and pharmacokinetics of the isoflavones. This article reviews the effect of a number of soybean processing treatments on the isoflavone content and profile. The preparation and manufacturing of different soy-based food and food ingredients, fermented and non-fermented, has been analyzed in terms of content and distribution of the three major isoflavone derivatives, daidzein, genistein, and glycitein, and their respective conjugates.     

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.     

Suppression of arachidonic acid metabolism and nitric oxide formation by kudzu isoflavones in murine macrophages

Inhibitory effect of kudzu isoflavones on arachidonic acid metabolism and nitric oxide (NO) production in lipopolysaccharide activated RAW 264.7 macrophages were investigated. Isoflavone aglycones, such as daidzein, genistein, biochanin A, and formononetin significantly suppressed arachidonic acid release (50 microM). Biochanin A, which displayed the most active inhibition on arachidonic acid release in HT-29 human colon cancer cells, exhibited its most potent suppression in RAW 264.7 cell (by 86%) without showing cytotoxicity. However, isoflavone glucosides, puerarin and daidzin, showed lower inhibitory activities on the release of arachidonic acid and its metabolites. In NO formation, biochanin A showed marked inhibition, by 62% (50 microM), followed by genistein, daidzein, formononetin, and daidzin, 56, 39, 33, and 8%, respectively. 5,7-Dihydroxyl group in the A-ring of isoflavones could be a key functional group responsible for the strong inhibitory activity of biochanin A and genistein on NO production. These activities may contribute to the antiinflammatory and anticarcinogenic properties of kudzu isoflavones.     

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.     

食品胶对豆浆中活性成分异黄酮苷元热稳定性的影响

研究了羧甲基纤维素钠(CMC)、海藻酸钠、黄原胶等食品添加剂对豆浆热处理过程中活性成分异黄酮苷元热稳定性的影响。高效液相色谱分析表明,CMC、黄原胶、海藻酸钠都能减少黄豆苷元、大豆黄素、染料木黄酮三种异黄酮苷元的热损失,其中CMC效果最理想,海藻酸钠次之,黄原胶最差。食品胶的复配能够减少豆浆中异黄酮苷元热损失。CMC/海藻酸钠组合明显好于CMC/黄原胶和黄原胶/海藻酸钠两种组合,当CMC/海藻酸钠=3∶2时,在热处理条件为95℃,10min条件下,各异黄酮苷元热损失最少,黄豆苷元含量提高9·83%,大豆黄素12·67%,染料木黄酮18·63%。在三种食品胶共同复配组合中,CMC/黄原胶/海藻酸钠(4∶3∶3)组合的效果最好,黄豆苷元含量提高9·3%,大豆黄素10·13%,染料木黄酮18·26%。      

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.