Formation of Succinyl Genistin and Succinyl Daidzin by Bacillus Species

ABSTRACT:  6"- O -Succinyl-4'-hydroxyisoflavone-7- O -β-D-glucopyranoside (succinyl-β-daidzin) and 6"- O -succinyl-6,4'-dihydroxyisoflavone-7- O -β-D-glucopyranoside (succinyl-β-genistin), 2 new isoflavone metabolites, are found in  cheonggukjang  or  natto , traditional soy-based foods fermented with  Bacillus  species. Standard isoflavones including daidzin, genistin, daidzein, and genistein, and mixtures of isoflavones extracted from roasted soybeans were added to the medium growing  Bacillus subtilis  or  B. subtilis  natto and formation of succinyl-β-daidzin and succinyl-β-genistin were monitored by high-performance liquid chromatography (HPLC). Samples containing  Bacillus  with daidzin and genistin produced succinyl-β-daidzin and succinyl-β-genistin, respectively, while those with daidzein and genistein did not produce succinyl derivatives. Daidzin in samples with  B. subtilis  and  B. subtilis  natto decreased by 39.7% and 10.7%, respectively, for 4 h incubation while genistin decreased by 66.8% and 17.6%, respectively. Genistein decreased faster than daidzein during incubation with  B. subtilis  or  B. subtilis  natto without formation of succinyl derivatives. In the case of mixture of isoflavones, succinyl derivatives increased and β-glucosides and aglycones of isoflavones decreased significantly for 8 h incubation ( P  < 0.05).     

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.     

Isoflavone during protease hydrolysis of defatted soybean meal

The study examined the effects of proteases on the isoflavones during enzymatic hydrolysis of soybean flour. Protease itself did not affect the isoflavones during hydrolysis, whereas the applied conditions and contaminated β-glucosidase in the enzyme could greatly affect the content and composition of isoflavones. Soybean flour hydrolysis by ENZECO Alkaline Protease L-FG at high pH (10) resulted in complete loss of malonylglucosidic and acetylglucosidic conjugates in the hydrolysate. However, these conjugates such as 6″-O-malonylgenistin and 6″-O-malonyldaidzin remained as the principal compounds accounting for 66.2, 58.3 and 70.5% of the total isoflavones in the Protease M “Amano”, Alcalase 2.4L, and neutral enzyme ENZECO Neutral Protease-NBP-L hydrolysates, respectively, compared to that of 57.8% in the original soybean flour. The residue prepared by Protease M contained 10 times higher aglycones than that of soy flour, which was due to the contaminated β-glucosidase activity in the enzyme preparation. Our result showed that β-glucosidase contaminated in Protease M has a unique selectivity compared to that of the purified almond β-glucosidase. Results from the study indicated that hydrolysis of soybean flour may provide another alternative approach to enrich aglycone isoflavones in soybean-containing products.     

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.     

Optimisation of soybean hydrothermal treatment for the conversion of β-glucoside isoflavones to aglycones

The objective of this work was to optimise the conditions for the soaking process of soybean for maximal conversion of β-glucoside isoflavones into aglycones using response surface methodology. The investigated variables were the time (3, 4, 6, 8, and 9 h) and temperature (41, 45, 55, 65, and 69 °C ± 1 °C) for the soaking of soybean in 1:1.5 (g:g) ratio with a buffer solution pH 6. The maximum aglycones content (1.22 μmol g⁻¹) in soaked soybeans was estimated and validated at 55 °C ± 1 °C and 6 h of soaking. In this condition, the hydrothermally treated soybean grains showed a minimum content of β-glucoside isoflavones and low β-glucosidase activity. A 30% reduction in total isoflavones content of the system was estimated at 69 °C ± 1 °C not depending on the soaking time, attributed to the thermal instability of malonylglucosides. This work may contribute to the preparation of soy products rich in aglycones from hydrothermally treated soybean.     

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。     

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.     

基于UPLC-Q-TOF-MS技术对高盐稀态酱油发酵过程中代谢产物变化的分析

采用超高效液相色谱-四极杆飞行时间串联质谱技术,结合主成分分析和正交偏最小二乘法分析研究高盐稀态酱油发酵过程中代谢物质的变化。结果表明,发酵第90天酱油的代谢物质种类及总峰面积分别是第1天的1.47 倍和6.57 倍。发酵过程中共鉴定了155 种化合物,其中含有34 种显著差异代谢物质（VIP＞1）,包含10 种氨基酸衍生物,9 种脂肪酸,5 种异黄酮,3 种酯,2 种有机酸,2 种生物碱等。其中,氨基酸衍生物、异黄酮、生物碱的峰面积占所有物质总峰面积百分比分别从0.44%、6.82%、0.02%增长到20.11%、14.88%和10.81%,是酱油发酵过程中的关键差异代谢产物。本研究对酱油发酵过程中代谢规律的探索以及发酵品质调控具有重要意义。     

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.     

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...     

Hydrolysis of the toxic constituents (vicine and convicine) in fababean (Vicia faba L.) food preparations following treatment with β-glucosidase

Several experiments were carried out to determine whether the concentrations of vicine and convicine in fababean food preparations were reduced following heat and β-glucosidase treatment. Heating fababeans to relatively high temperatures was not effective in reducing the concentration of these glycosides. Treatment of cooked fababeans (fababean paste) with ground raw almond powder, which is a rich source of β-glucosidase (EC 3.2.1.21) activity, resulted in the hydrolysis of vicine and convicine. The degree of hydrolysis ranged from partial to complete and was dependent on the duration of the treatment period (1 to 24 h), the temperature at which treatment was carried out (1 to 30°C), the pH (6.4 to 4.6) as adjusted by the addition of lemon juice and the amount of ground almonds added to the fababean paste. For example, 88 to 89% of the glycosides were hydrolysed when 1 g of fababean paste was mixed with 0.1 g of almond powder and 0.1 ml of lemon juice and incubated at 30°C for 3 h. This study demonstrated that the concentration of vicine and convicine and their corresponding aglycones can be greatly reduced in fababean food preparations by enzymic hydrolysis.     

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.     

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.     

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.     

Effect of Lactic Acid Fermentation on Enrichment of Antioxidant Properties and Bioactive Isoflavones in Soybean

ABSTRACT: The antioxidant properties of 80% ethanolic extracts from soybean obtained with lactic acid fermentation using ABTS•⁺ [2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt] free radical decolorization assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay were measured, and the relationship between the observed antioxidant properties and the compositional changes in the isoflavone isomers was evaluated. Fermentation of soybean with 4 bacterial strains producing β-glucosidase for 48 h at 37°C— Lactobacillus plantarum KFRI 00144, Lactobacillus delbrueckii subsp. latisKFRI01181, Bifidobacteriathermophilum KFRI00748, and Bifidobacteria breve K-101—resulted in a significant increas (P < 0.05) in the antioxidant capacity expressed as Trolox equivalent antioxidant capacity (TEAC) (mM) and % scavenging activity. The significant bioconversion (P < 0.001) of the isoflavone glucosides (daidzin + genistin) into their responding bioactive aglycones (daidzein + genistein) during soy fermentation was observed. There was a good linear correlation between the concentration of isoflavone aglycones and the scavenging activity of ABTS ( R = 0.9045) assay, and DPPH ( R = 0.8299) assay in each extract. Among each extract, B. thermophilum KFRI 00748 showed a particularly high antioxidant activity (19.8 mM TEAC) and increased by 4.1 times compared with that of the control (4.8 mM TEAC), which fermented without strains. These results indicated that fermented soybean could be regarded as a potent antioxidant and radical scavenging dietary source due to their remarkable content of bioactive isoflavones.     

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 bioactive compounds and bioactivities of soybean dried by different methods and conditions

Soybean has attracted significant research and commercial interests due to its many health-promoting bioactive compounds, especially isoflavones (β-glucosides, malonyl-β-glucosides, acetyl-β-glucosides and aglycones). Isoflavones possess antioxidant activity and α-glucosidase inhibitory activity, which has proved effective in the treatment of type 2 diabetes mellitus. Prior to its use, however, soybean needs to be dried to extend its storage life and to prepare the material for subsequent food or pharmaceutical processing. The present study investigated the effects of drying methods and conditions on the drying characteristics, isoflavones, antioxidant activity and α -glucosidase inhibitory activity of dried soybean. Hot-air fluidized bed drying (HAFBD), superheated-steam fluidized bed drying (SSFBD) and gas-fired infrared combined with hot air vibrating drying (GFIR-HAVD) were carried out at various drying temperatures (50, 70, 130 and150 °C). The results showed that higher drying temperatures led to higher drying rates and higher levels of β-glucosides and antioxidant activity, but to lower levels of malonyl-β-glucosides, acetyl-β-glucosides and total isoflavones. At the same drying temperature GFIR-HAVD resulted in the highest drying rates and the highest levels of β-glucosides, aglycones and total isoflavones, antioxidant activity as well as α-glucosidase inhibitory activity of dried soybean. A drying temperature of 130 °C gave the highest levels of aglycones and α-glucosidase inhibitory activity in all cases. The relationships between all the studied parameters were monitored and simple correlations between them were determined.     

Enhancement of Biological Properties of Soymilk by Fermentation

Soymilk has attracted much interest in the food industry because of its health- promoting properties. Fermentation with lactic acid bacteria is known to provide value addition to soymilk by reducing the beany flavor and content of indigestible oligosaccharides and by enhancing the bioavailability of isoflavones, resulting in a nutritious probiotic food product. In the present study, Soymilk fermentation was studied using Lactobacillus acidophilus strain isolated from ragi, and effect of soymilk supplementation on the fermentation characteristics was also investigated. Viable count of 1.99 × 10⁹ CFU/mL sample and 630.1 U/g of β-glucosidase activity were determined in soymilk after 12 h of fermentation. Soymilk supplementation with skimmed milk powder at 5% level gave best results with viable counts of 22.9 × 10⁹ CFU/ml sample or 212.9 × 10⁹ CFU/g solids; 764 U/g of β-glucosidase activity, 1.44% la titratable acidity and pH of 3.9. Daidzein and genistein profile in fermented soymilk showed that 45.8% and 57.5% of respective isoflavones existing as aglycone form, indicating enhancement of the biological property of soymilk through improvement of health-relevant bioactive forms.     

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.