Isoflavone Aglycone Content Research Articles

Method development and optimization for isoflavone quantification in dry soy extract containing products

Isoflavones are secondary metabolites belonging to flavonoids found in a variety of plants, especially soy (Glycine max L.). Due to the estrogen-like activity of these compounds several soy products are on the market and applied to relieve menopausal symptoms. The aim of our study was to develop a simple method for the determination of isoflavones in soy extract containing products. The main isoflavones of soybean are genistin, glycitin, daidzin and their respective acetyl, malonyl and aglycone forms. Quantitative measurement of each individual isoflavone is difficult because some of the reference compounds are not widely commercially available [1]. Keeping in mind that the biological effects of soy isoflavones depend upon aglycone form, hydrolysis of glycosides is a practical method for the quantitative determination of total isoflavones and assessment of biological value of extracts [2]. The objective of our investigation was to establish an acid hydrolysis method for the quantitative HPLC determination of total isoflavones including daidzein, genistein and glycitein. To optimize the extraction and hydrolysis of isoflavones, the effect of HCl concentration (1.5–6 N), hydrolysis time (25–210min) and temperature (30–100°C) on total isoflavone aglycone content was studied using an RP-HPLC-DAD method. By mathematical fitting and optimization methods optimum hydrolysis conditions for maximizing the quantification of isoflavones were determined (t=94min, cHCl=5.09 N, T=80°C). The experimentally verified model has a good coefficient of R2 (0,9928) and the recovery of isoflavones was 97.81–102.76%. The developed method allows a reliable and maximum determination of isoflavones in dry soy extract containing products.

Bioconversion of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast

To study the role of β-glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High-performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97.49 to 98.49% and 62.71 to 92.31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.

Isoflavone content and antioxidant activity of Thai fermented soybean and its capsule formulation

Soybeans (Glycine max) are usually eaten as processed foods. Fermented soybeans are among the most popular of these processed foods. The aim of this study was to determine the effect of fermentation duration on isoflavone content and antioxidant activity of fermented soybeans. Capsule formulation of fermented soybeans was also studied. The Thai soybean variety, Rajamangala60, was fermented with Aspergillus oryzae. Isoflavone content and antioxidant activity were studied at 0, 12, 18, 36, 48, 96, 120, 168, 240, 360 and 480 h of fermentation duration. The results showed that isoflavone glycones (daidzin and genistin) decreased during fermentation, but aglycones (daidzein and genistein) increased. The highest amount of isoflavone aglycones was 384.30 ± 4.60 and 116.50 ± 1.56 mg/100 g fermented soybeans for daidzein and genistein, respectively. Antioxidant activity of fermented soybeans was evaluated by ABTS cation radical scavenging and ferric reducing antioxidant power (FRAP) methods. Antioxidant activity of fermented soybeans is increased during fermentation. Increases in isoflavone aglycones content and antioxidant activity were related to fermentation duration. The highest antioxidant activity of fermented soybean was found at the 240 h of fermentation with trolox equivalent antioxidant capacity (TEAC) 1.98 ± 0.09 ig trolox/g fermented soybean and FRAP value of 0.623 ± 0.002
g FeSO4/g fermented soybean. Soybeans fermented for 240 h were then formulated as capsules by a wet granulation method. They were then assessed for appearance, weight variation, disintegration time and antioxidative properties. The results showed that fermented soybean capsules conformed to USP32/NF27 criteria on weight variation and disintegration. Their antioxidant activity was lower than 240 h fermented soybeans, but still higher than the non-fermented ones (p < 0.05).

Comparison of hormonal activity of isoflavone-containing supplements used to treat menopausal complaints

The isoflavones present in red clover and soy are used as an alternative treatment for menopausal complaints and are commercially available as high-dose food supplements. These preparations contain varying amounts of active ingredients, often without detailed specifications. Thus, it is difficult to derive a recommended daily dose, and the reliability of these products is rather low. We quantified the isoflavone content of 19 different isoflavone-containing preparations and compared their binding and transactivational activities with regard to estrogen receptor alpha, estrogen receptor beta, androgen receptor, progesterone receptor, peroxisome-proliferator-activated receptor, and aryl hydrocarbon receptor. The food supplements that we tested bound to and transactivated both the estrogen receptors and the other receptors. After comparing the isoflavone content quantified by us with the isoflavone content specified on the package labels, we found that at least the specified isoflavone content or more could be detected in only 5 of the 19 food supplements that we tested. Preparations containing isoflavones should be standardized for the isoflavone aglycone content to facilitate the prediction of theoretical hormonal activity, facilitate the intake of a controlled amount of isoflavones, and ensure greater product reliability.

Performance of Starter in Yogurt Supplemented with Soy Protein Isolate and Biotransformation of Isoflavones during Storage Period

In this study, soy protein isolate (SPI) (4%, v/w) was supplemented to the yogurt mix to increase the amount of biologically active isoflavone in yogurt (SY). The control yogurt was without any SPI supplementation (USY). The supplementation significantly (P < 0.05) increased the lactose metabolism by the yogurt starter including Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 (Lb 11842) and Streptococcus thermophilus ST 1342 (ST 1342) during the fermentation process by 4.7%. The starter produced more acetic acid and less lactic acid in SY than that in USY and altered the ratio of lactic and acetic acid during the entire storage period. The viability of both Lb 11842 and ST 1342 in SY was significantly (P < 0.05) lower than that in USY from 14 d of the storage period, however, their concentration still remained high (8.11 to 8.84 log CFU/g). The starter transformed 72.8% of total inactive isoflavone glycosides (IG) to active isoflavone aglycones (IA), increasing the IA content from 1.35 to 15.01 mg/100 g sample. During the storage period, IA concentration slowly rose from 15.02 to 15.51 mg/100 g sample.

Hydrolysis of soybean isoflavone glycosides by a thermostable β-glucosidase from Paecilomyces thermophila

Abstract The β -glucosidase from Paecilomyces thermophila J18 was found to be capable of hydrolysing daidzin and genistin in a previous study. This report further evaluated the thermostability and hydrolysis of soybean isoflavone glycosides. The enzyme was found to be very stable at 50 °C, and retained more than 95% of its initial activity after 8 h at 50 °C. It converted isoflavone glycosides, in soybean flour extract and soybean embryo extract, to their aglycones, resulting in more than 93% of hydrolysis of three isoflavone glycosides (namely, daidzin, genistin and glycitin) after 4 h of incubation. Also, addition of the β -glucosidase greatly increased the contents of isoflavone aglycones in the suspended soybean flour and soymilk. The results indicate that the thermostable β -glucosidase may be used to increase the isoflavone aglycones in soy products. This is the first report on the potential application of fungal β -glucosidases for converting isoflavone glycosides to their aglycones in soy products.

Effect of β-Glucosidase as a Feed Supplementary on the Growth Performance, Digestive Enzymes and Physiology of Broilers

The effects of -glucosidase on the overall growth performance and a set of physiological parameters of broilers were investigated. 240 male, one-day old Avine broiler chickswere randomly allocated to four treatment groups and fed with a corn-soybean meal supplemented with 0% (control), 0.2%, 0.4% and 0.6% -glucosidase. The 0.2% -glucosidase group, but not the 0.4% and 0.6% -glucosidase groups, showed a significantly increased average daily weight gain (p-glucosidase feed groups showed significantly higher feed conversion ratios than the control group (p-glucosidase significantly raised the contents of serum isoflavone aglycones as shown by decreases of genistin and daizin (p-glucosidase feeding significantly increased the intestinal amylase activity while it had little effect on lipase and trypsin activities (p>0.05). 0.2% -glucosidase feeding also significant elevated the levels of highdensity lipoprotein cholesterol and malate dehydrogenase while lowering the level of low-density lipoprotein cholesterol (LDL-C). Finally, -glucosidase improved the anti-oxidative activities of the animals; the 0.2% -glucosidase feed group had higher activities of superoxide dismutase (p

Sensory properties of soy yoghurts prepared from yellow and black soymilk using Streptococcus infantarius 12 and Weisellia sp. 4

AbstractBACKGROUND: Soy yoghurts containing enhanced bioactive isoflavone aglycones were prepared from combinations of yellow (YS) and black soymilk (BS) (YS:BS = 100:0, 70:30, 50:50 and 0:100, v/v) by fermentation with Streptococcus infantarius 12 and Weisellia sp. 4 at 37 °C for 10 h. Sensory properties of the bioactive soy yoghurts were investigated. Furthermore, other characteristics including the contents of isoflavones, viable cell numbers, pH, colour and DPPH radical scavenging activity were investigated.RESULTS: One hundred percent YS yoghurt showed the highest scores of all sensory attributes while 100% BS yoghurt was the lowest for colour and smoothness. The overall acceptability scores were 5.9 and 5.2 for 100% YS and 100% BS yoghurts, respectively (9‐point scale). The L‐values of soy yoghurts decreased as the percentage BS increased, indicating darkening of soy yoghurts. The contents of bioactive isoflavone aglycones (daidzein and genistein) in soy yoghurts remarkably increased (&gt;1300 mg kg−1) by fermentation. Viable cell numbers and pH of soy yoghurts ranged from 8.4 × 108 to 9.6 × 108 CFU g−1 and 4.43 to 4.54, respectively. DPPH radical scavenging activity significantly increased as the percentage BS increased (P &lt; 0.05).CONCLUSION: One hundred percent YS yoghurt was better for overall sensory properties while 100% BS yogurt was more bioactive in terms of the isoflavone aglycone contents and DPPH radical scavenging activity compared to other yoghurts. Copyright © 2008 Society of Chemical Industry

An Improved Method for Basic Hydrolysis of Isoflavone Malonylglucosides and Quality Evaluation of Chinese Soy Materials

Basic hydrolysis procedure is often included in the sample preparation in order to quantify malonylglucosides or acetylglucosides of soy materials. However, it is preferable not to use NaOH as a hydrolytic reagent considering the effect of its alkalinity on the successive injection to HPLC and low acidity of soy isoflavones. This paper presents an improved method for basic hydrolysis using ammonia as a hydrolytic reagent without the additional neutralization step. Moreover, by means of HPLC and LC-MS methods, a systematic quality evaluation of natural soy materials from Chinese markets were established and discussed, inclusive of soybeans, black soybeans, defatted soy flours, as well as the distribution of isoflavones in the seed coat, hypocotyl and cotyledon. The results indicate that HPLC profiling patterns of originating various isoflavone constituents of Chinese soybeans was similar to those of Japanese ones, and those of Chinese black soybeans was similar to those of American ones. The average content level of total soy isoflavones of Chinese soybeans and black soybeans were a little lower than that of American and Japanese ones. Additionally, the thorough analysis for Semen Sojae Praeparatum, a Chinese herbal medicine made from fermented black soybeans or soybeans was done for the first time and the characteristic of its HPLC profiling patterns shows the higher content of isoflavone glucosides and aglycones than those of natural soy materials.

Fermentation of Calcium‐Fortified Soymilk with Lactobacillus: Effects on Calcium Solubility, Isoflavone Conversion, and Production of Organic Acids

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 degrees C, then stored for 14 d at 4 degrees 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 log(10) CFU/g after 24 h fermentation and this was maintained for 14-d storage at 4 degrees 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.

Production of β‐Glucosidase and Hydrolysis of Isoflavone Phytoestrogens byLactobacillus acidophilus,Bifidobacterium lactis, andLactobacillus caseiin Soymilk

The study determined beta-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 degrees C for 48 h and the fermented soymilk was stored for 28 d at 4 degrees C. beta-Glucosidase activity of organisms was determined using rho-nitrophenyl beta-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 degrees C for 28 d was 20%, 15%, and 11% greater (P < 0.05) than initial cell counts, respectively. All the bacteria produced beta-glucosidase, which hydrolyzed isoflavone beta-glycosides to isoflavone aglycones. The decrease in the concentration of beta-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.

Extraction and Purification of Isoflavones from Soybeans and Characterization of Their Estrogenic Activities

Soybean isoflavones have multiple beneficial health effects especially on estrogen-deficient diseases such as menopausal symptoms. In this study, isoflavones were produced from soybean flour, and the extraction and purification parameters were optimized to give a high yield of total isoflavones, about 0.62 mg of aglycones/g of soybean flour, which is >2 times the initial yield. HPLC analysis and MTT cell proliferation assay using MCF-7 cells revealed that the product thus obtained not only contained a high content of isoflavone aglycones but also had estrogenic activity. MTT data also revealed that both genistein and daidzein exhibited estrogenic effects at lower concentrations and antiproliferative effects at higher concentrations, and 1 microM genistein and 10 microM daidzein exerted significant estrogenic activities, which were not more than that of the endogenous level of 17beta-estradiol (E2). The production method developed can be used as a guideline for manufacturing soy isoflavones, and the MTT assay was demonstrated to be suitable for quality control on isoflavone products. The results on the estrogenic properties of isoflavones can be used as reference data for their effective and safe usages in estrogenic therapy.

The Potential Antioxidant Capacity and Angiotensin I‐Converting Enzyme Inhibitory Activity of Monascus‐Fermented Soybean Extracts: Evaluation of Monascus‐Fermented Soybean Extracts as Multifunctional Food Additives

The potential antioxidant capacity and angiotensin I-converting enzyme (ACE) inhibitory activity of Monascus-fermented soybean extracts (MFSE) were investigated. The average antioxidant capacities of 70% ethanol extracts from soybean after fermenting for 15 d at 30 degrees C were increased by a 5.2 to 7.4-fold (0.26 mM trolox equivalent/g dry weight, 91.7% 1,1-diphenyl-2-picrylhydrazyl [DPPH] radical scavenging effect at 3 mg/mL) when compared with those of the unfermented soybean extracts (P < 0.01). The potentially significant antioxidant properties of MFSE are associated with its content of bioactive mevinolins (r= 0.85) and isoflavone aglycones (r= 0.98), which were derived from the soybean during Monascus-fermentation. It was also found that the water extract having a molecular mass 1 to 3 kDa showed the highest ACE inhibitory activity (65.3%), which was remarkably greater (6.5 times) than the control.

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 °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.

Role of sucrose in the development of Fusarium wilt in lupine embryo axes

The response of embryo axes of Lupinus luteus L. cv. Polo to invasion by Fusarium oxysporum Schlecht f.sp. lupini was studied 84, 96 and 108 h after inoculation and under various trophic conditions. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). The exogenous sucrose caused an accumulation of sucrose, glucose and fructose in +Sn embryo axes. Pathogen invasion generally resulted in a sharp decline in soluble carbohydrate content of +Si embryo axes. Symptoms of Fusarium wilt included growth inhibition of the embryo axes and a decrease in their fresh weight, which was particularly strong in −Si axes. While the addition of exogenous sucrose to the medium 24 h prior to inoculation significantly alleviated the disease, its application 48 h after inoculation did not restrict disease development. In +Si embryo axes, the activity of peroxidases (EC 1.11.1.7), covalently and ionically bound to the cell walls, increased considerably, mainly up to 96 h after inoculation. This increase was significantly higher than that seen in +Sn embryo axes. These results are in contrast to those of an earlier study [Morkunas I, Marczak Ł, Stachowiak J, Stobiecki M. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005;43:363–73], which showed a pronounced decrease in the flavonoid glycoside content of +Si tissues. Furthermore, starting 96 h after inoculation, a decrease in phenylalanine ammonialyase activity (PAL, EC 4.3.1.5) was observed in inoculated tissues, in comparison to non-inoculated tissues. The decrease was especially pronounced in +Si tissues. However, a significant increase in the free isoflavone aglycones content of +Si embryo exes, i.e. genistein, 2′-hydroxygenistein, wighteone and luteone. Simultaneously, a strong increase in the activity of β-glucosidase (EC 3.2.1.21), hydrolyzing isoflavone glycosides, was observed in inoculated tissues. The activity of these enzymes was much higher in −Si embryo axes than in +Si embryo axes. The results suggest that soluble carbohydrates may play a role in the restriction of Fusarium wilt development, as they may be related both to intensified lignification and to the antimicrobial role of free aglycones, which may be products of the hydrolysis of isoflavone glycosides generated prior to the invasion stage.