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     

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     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

Isoflavone profile of a high intensity pulsed electric field or thermally treated fruit juice-soymilk beverage stored under refrigeration

A fruit juice-soymilk (FJ-SM) beverage was treated by high intensity pulsed electric fields (HIPEF) (35 kV/cm with 4 μs bipolar pulses at 200 Hz for 800 or 1400 μs) or heated (90 °C, 60 s) in order to evaluate and compare the effects of both technologies on the isoflavone profile along the storage at 4 °C. Total isoflavone content was determined by quantification of aglycone and glucoside forms analyzed with HPLC. Immediately after treatments, all the evaluated samples showed a higher concentration of glucosides than aglycones, genistin being the most abundant glucoside. Heat treated samples showed higher initial concentration of glucosidic forms as compared to the untreated or HIPEF treated beverages. No significant changes were observed in total isoflavone content after the increasing HIPEF treatment time from 800 to 1400 μs. During the storage, genistein, daidzein and daidzin content increased; while genistin showed a slight decrease, irrespective of the treatment applied. In this way, total isoflavone content tend to increase throughout the time in all the evaluated samples. After 56 days of storage, there was no significant difference in HIPEF treated and fresh FJ-SM beverages, the latter being slightly higher in those thermally treated. As a result, HIPEF could be a good technology in order to obtain FJ-SM beverages with a high content of isoflavones and fresh-like characteristics. Considering the great concentration of isoflavones in untreated and treated FJ-SM beverages, their consumption could be a good way to increase the isoflavone daily intake of the population.Industrial relevanceHIPEF processing could be a competent technology in order to obtain FJ-SM beverages with fresh-like appearance and similar isoflavone content than just made products. The fruit juice-soymilk beverage evaluated in the present research has a great isoflavone content regarding to commercial soy based beverages. Its consumption could increase the current isoflavone intake of the population. Industry could apply this non-thermal technology in order to meet current consumers' claims for quality foods with high nutritional values.     

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.     

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.     

Soluble phenolics and antioxidant properties of soybean (Glycine max L.) cultivars with varying seed coat colours

This research was the first to investigate nutritional components, including soluble phenolics (isoflavones and anthocyanins), protein, oil, and fatty acid as well as antioxidant activities in different coloured seed coat soybeans (yellow, black, brown, and green) for two crop years. The soluble phenolics differed significantly with cultivars, crop years, and seed coat colours, while protein, oil, and fatty acid exhibited only slight variations. Especially, malonylgenistin and cyanidin-3-O-glucoside compositions had the most remarkable variations. Green soybeans had the highest average isoflavone content (3079.42 μg/g), followed by yellow (2393.41 μg/g), and black soybeans (2373.97 μg/g), with brown soybeans showing the lowest value (1821.82 μg/g). Anthocyanins showed only in black soybeans, with the average contents of the primary anthocyanins, cyanidin-3-O-glucoside, delphinidine-3-O-glucoside, and petunidin-3-O-glucoside, quantified at 11.046, 1.971, and 0.557 mg/g, respectively. Additionally, Nogchae of green soybean and Geomjeongkong 2 of black soybean may be recommended as potential cultivars owing to the highest average isoflavone (4411.10 μg/g) and anthocyanin (21.537 mg/g) contents. The scavenging activities of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals also differed remarkably, depending upon isoflavone and anthocyanin contents, with black soybeans exhibiting the highest antioxidant effects.     

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.     

Effect of time and temperature on bioactive compounds in germinated Brazilian soybean cultivar BRS 258

The objective was to determine the effect of time and temperature on the concentration of bioactive compounds during germination of Brazilian soybean cultivar BRS 258. The concentration of bioactive compounds was optimized using Response Surface Methodology, with a 2² central composite rotational design and germination time and temperature as independent variables. Germination was carried out in a germination chamber, using paper in trays containing 500 g of seeds. Germination temperature and time modified the concentrations of bioactive compounds within the ranges studied. An increase in germination time at 25 °C decreased the concentration of Bowman–Birk inhibitor, lectin and lipoxygenase. After 63 h of germination, a temperature increase from 20 °C to 30 °C resulted in a decrease of lipoxygenase activity by 22.5%. Optimal increases in the concentrations of isoflavone aglycones (daidzein and genistein) and saponin glycosides were observed with a 63 h germination time at 30 °C. Both germination time and temperature had an influence on the composition and concentration of bioactive compounds in germinated soybean flour. Equations determined can be used to predict concentrations of bioactive compounds in germinated soybeans in relation to climate changes and thus optimize their potential use in human health and nutrition.     

Antioxidant potential and radioprotective effect of soy isoflavone against gamma irradiation induced oxidative stress

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60 mg/kg) for 21 days followed by gamma irradiation exposure. Survival studies in rats exposed at 10 Gy and endogenous spleen colony forming unit assay (CFU) at 6.0 Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0 Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.     

Effect of soybean varieties on the content and composition of isoflavone in rice-koji miso

To clarify the effect of soybean varieties on isoflavone, a useful component for human health, in soybean products, we investigated changes in the isoflavone content and composition in rice-koji miso, after fermentation/aging for 6 or 12 months using varieties of soybeans (Tohoku-126, Tohoku-135, Tohoku-139, Suzuyutaka and Chinese soybeans), by high performance liquid chromatography. In soybeans, the total isoflavone content in Tohoku-126 was 444 mg/100 g, which was 1.2–2.0 times the content in the other soybean varieties. The malonyl glycosides and aglycones in soybeans accounted for more than 60% and only a few percent, respectively. As for rice-koji miso, the total isoflavone and aglycone contents were the highest in miso prepared from Tohoku-126. The ratios of glycosides to aglycones (80.1–92.6%) in miso were higher than those in the original soybeans. The time course of the isoflavone composition during the fermentation/aging process of rice-koji miso indicated that glycosides decreased from 86.4% to 44.9% after 6 months but aglycones increased from 9.6% to 53.3%.     

Thermal degradation kinetics of isoflavone aglycones from soy and red clover

Driven by their beneficial effects on human metabolism, isoflavonoids have gained considerable importance reflected by an increased number of isoflavone-rich foods, food supplements and pharmaceutical products on the market, mainly derived from soy and red clover. While it is well known that the genuine isoflavone pattern will be altered during processing, data on aglycone stability are rare. Therefore, a thorough study into the thermal sensitivities of biochanin A, daidzein, formononetin, flavone, genistein, glycitein and isoflavone was performed. Samples were heated at 150 degrees C over a period of 7 h at three different pH values, and degradation of the aglycones was monitored by HPLC-DAD analyses. Therefrom, structure-related stability characteristics could be established. While virtually no decay was observed at pH 7.0 and 5.6, degradation was most prominent at pH 3. 1. Individual aglycone retention was further dependent on heating time with daidzein being the most labile compound after any time interval. Curve fitting of the data revealed first-order degradation kinetics for flavone and glycitein, while the remaining aglycones exhibited a sigmoidal degradation pattern.