Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4′-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Revision received: 9 September 1995 / Accepted: 21 September 1995     

Metabolism of the soy isoflavones daidzein and genistein by fungi used in the preparation of various fermented soybean foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, (1)H-, and (13)C-NMR spectra.     

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4'-O-carboxymethyl-daidzein and 4'-O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4'-O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95-0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0-6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma values (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8+/-6.8 and for genistein, 3.2+/-7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).     

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.     

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a β-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin. Received 10 September 1996 / Received revision: 22 November 1996 / Accepted: 7 December 1996     

Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases. Soy and soy foods are rich sources of isoflavones, which have been shown to possess several biological activities. In this study, the metabolism of soy isoflavones daidzein, genistein and glycitein was investigated in human subjects. The aim was to find and identify urinary phase I metabolites of isoflavones, which have an intact isoflavonoid skeleton, and which might possess some bioactivity. Six volunteers included three soy bars per day into their normal western diet for a 2-week period. Daily urine samples were collected before, and after the supplementation period. Urine samples were hydrolyzed with Helix pomatia, extracted with diethyl ether, purified with Sephadex LH-20 chromatography, and analyzed as trimethylsilyl derivatives using gas chromatography–mass spectrometry (GC–MS). The structures of the isoflavone metabolites were identified using authentic reference compounds. The metabolites, for which authentic reference compounds were not available, were identified by the interpretation of mass spectra. Several new isoflavone metabolites were identified, and the presence of previously reported metabolites confirmed. The metabolic pathways of daidzein, genistein and glycitein are presented on the basis of the identification of the metabolites in human urine after soy supplementation.     

Pharmacokinetics of isoflavones,daidzein and genistein,after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women

Background  

Isoflavones from soybeans may provide some beneficial impacts on postmenopausal health. The purpose of this study was to compare the pharmacokinetics and bioavailability of plasma isoflavones (daidzein and genistein) after a single dose of orally administered soy beverage and soy extract capsules in postmenopausal Thai women.     

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.     

Ethanol recovery from corn fiber hydrolysate fermentations by pervaporation

Corn fiber, a byproduct of corn wet milling, is an attractive feedstock for biomass ethanol production. Corn fiber was hydrolyzed by dilute sulfuric acid and neutralized by one of two methods: conventional lime treatment or neutralization by strongly basic anion exchange. The anion exchange neutralized (AEN) hydrolysate contained substantially lower levels of the inhibiting compounds furfural, 5-hydroxymethylfurfural, and acetic acid compared to the lime neutralized hydrolysate. In batch fermentations the ethanol yields and final ethanol concentration of the two hydrolysates were similar at 0.32-0.43 g/g and 29-44 g/l, respectively. Sugar consumption in the AEN fermentations was superior. Coupling of a membrane pervaporation unit to a fed-batch fermentation of AEN hydrolysate maintained the ethanol concentration below 25 g/l with complete sugar utilization for approximately 5 days. A concentrated ethanol stream of 17 wt.% ethanol was produced by the pervaporation unit.     

Spectroscopy and molecular docking study on the interaction of daidzein and genistein with pepsin

The interaction of pepsin with daidzein (Dai) or genistein (Gen) was investigated using spectroscopic techniques under simulated physiological conditions. Dai and Gen can quench the fluorescence of pepsin and the quenching mechanism was a static process. The binding site number n and apparent binding constant K were measured at different temperatures. The thermodynamic parameters ΔΗ, ΔG and ΔS were calculated. The results indicated that van der Waals forces and hydrogen bond formation played major roles in the interaction of Dai or Gen with pepsin. The binding distance between pepsin and Dai or Gen was calculated according to energy transfer theory. The results of synchronous fluorescence spectra showed that the microenvironment and conformation of pepsin were changed. UV absorption and 3D fluorescence spectra showed that the binding interaction disturbed the microenvironment of amino acid residues and induced conformational changes in pepsin. Molecular docking results showed that Dai and Gen entered into the hydrophobic cavity of pepsin and two hydrogen bonds formed between Dai or Gen and pepsin. The results demonstrated that the interaction behavior between Dai and Gen with pepsin was slightly different, which denoted that the 5‐hydroxyl group of Gen, to a certain extent, had an effect on ligand binding to proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison between daidzein and genistein antioxidant activity in primary and cancer lymphocytes

The main objective of this study was to compare the protective effect of daidzein and genistein against induced oxidative damage in Jurkat T-cell line and in peripheral blood lymphocytes of healthy subjects. After supplementation of cells with isoflavones (from 2.5 to 20micromol/L in Jurkat T-cell and from 0.01 to 2.5micromol/L in primary lymphocytes, 24h), we determined DNA damage induced by hydrogen peroxide using the comet assay and lipid peroxidation evaluating malondialdehyde (MDA) production after ferrous ion treatment. Supplementation of Jurkat cells and primary lymphocytes with both isoflavones significantly increased DNA protection from oxidative damage at concentrations between 0.1 and 5micromol/L (P<0.05), and with just daidzein, at concentrations higher than 2.5micromol/L, there was a decrease in the production of MDA (P<0.05). Our results seem to support that daidzein is just as effective as genistein in protecting cells against oxidative damage especially with respect to DNA. Moreover, since the protective effect was found at concentrations reachable in plasma after soy consumption (less than 2micromol/L), it can be assumed that the antioxidant activity of isoflavones could really contribute to the healthy properties of soy.     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

牛瘤胃分离菌株静息细胞培养体系生物转化黄豆苷原

从牛瘤胃胃液中分离了一株在厌氧条件下能利用其生长细胞将大豆异黄酮黄豆苷原高效还原为二氢黄豆苷原的革兰氏阳性细菌菌株Niu-O16。研究了菌株Niu-O16静息细胞体系转化黄豆苷原的最佳转化条件,通过单因素试验确定菌株Niu-O16静息细胞转化黄豆苷原的最佳条件是:初始pH6.0～8.0,静息细胞浓度32～64mg/mL(湿重),加入底物浓度0.8～1.2mmol/L。通过正交试验确定了静息细胞浓度、加入底物浓度及转化时间的最佳组合为:静息细胞浓度32mg/mL、加入底物浓度0.8mmol/L、转化时间24h;最佳转化条件下底物转化率最高为63.9%。该结果为厌氧菌的静息细胞转化及工业应用提供了参考。     

In situ biobutanol recovery from clostridial fermentations: a critical review

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid–liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.     

Variations in inocula and their influence on the productivity of antibiotic fermentations

Summary Inocula, used for the production of penicillin and griseofulvin, giving different yields, were investigated. Biochemical factors, such as the level of enzyme activity and efficiency, were at least as important as morphology in determining yield, being carried forward from the inoculum to the production stage.