Comparison of three thermostable β-glucosidases for application in the hydrolysis of soybean isoflavone glycosides

A novel thermostable β-glucosidase (Te-BglA) from Thermoanaerobacter ethanolicus JW200 was cloned, characterized and compared for its activity against isoflavone glycosides with two β-glucosidases (Tm-BglA, Tm-BglB) from Thermotoga maritima. Te-BglA exhibited maximum hydrolytic activity toward pNP-β-d-glucopyranoside (pNPG) at 80 °C and pH 7.0, was stable for a pH range of 4.6-7.8 and at 65 °C for 3 h, and had the lowest K(m) for the natural glycoside salicin and the highest relative substrate specificity (k(cat)/K(m))((salicin))/(k(cat)/K(m))((pNPG)) among the three enzymes. It converted isoflavone glycosides, including malonyl glycosides, in soybean flour to their aglycons more efficiently than Tm-BglA and Tm-BglB. After 3 h of incubation at 65 °C, Te-BglA produced complete hydrolysis of four isoflavone glycosides (namely, daidzin, genistin and their malonylated forms), exhibiting higher productivity of genistein and daidzein than the other two β-glucosidases. Our results suggest that Te-BglA is preferable to Tm-BglA and Tm-BglB, but all three enzymes have great potential applications in converting isoflavone glycosides into their aglycons.     

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.     

Comparison of extraction methods for the quantification of selected phytochemicals in peanuts (Arachis hypogaea)

Peanuts have been reported to contain bioactive phytochemicals, particularly isoflavones (genistein, daidzein, and biochanin A) and trans-resveratrol. Currently, limited data are available regarding the levels of these bioactive compounds in peanuts with variations in reported levels. The purpose of this study was to compare four methods of extraction [stirring, sonication, Soxtec, and microwave-assisted sonication (MAS)] for runner peanuts. Quantification of the selected compounds was conducted by reverse-phase high-performance liquid chromatography (RP-HPLC). The results showed that the MAS and Soxtec methods extracted significantly higher amounts of the phytochemicals. Also, the defatted peanuts gave significantly higher amounts of the phytochemicals compared to the nondefatted peanuts. The high levels of the isoflavones may be attributed to heat-induced conversion of conjugate glycosides to aglycons. The MAS and Soxtec methods may be used for total isoflavone content quantitation, while sonication or stirring may be the method of choice for quantitation of isoflavone composition (aglycons and glycoside conjugates) in peanuts.     

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.     

Effects of lactulose supplementation on the growth of bifidobacteria and biotransformation of isoflavone glycosides to isoflavone aglycones in soymilk

Bifidobacterium animalis subsp. lactis bb12 and B. longum 20099 were used to hydrolyze isoflavone glycosides (IG) to biologically active forms, which are isoflavone aglycones (IA), in soymilk (SM) prepared from soy protein isolate (SPI) and SM supplemented with 0.5% (w/v) of lactulose (SML). Supplementation of lactulose significantly ( p < 0.05) enhanced the viable counts of B. animalis subsp. lactis bb12 and B. longum 20099 up to 2.34 and 2.15 log CFU/mL, respectively. Bifidobacterium animalis subsp. lactis bb12 and B. longum 20099 utilized 3.32 and 3.75 mg/mL of lactulose at 24 h of incubation, respectively. Supplementation of lactulose also appeared to be a key factor in decreasing the pH of SML. The biotransformation of IG to IA was enhanced significantly by 6.8-17.1% and 12.8-13.5% in SML by B. animalis subsp. lactis bb12 and B. longum 20099, respectively. However, the presence of lactulose in SML showed the stimulating effect on B. longum 20099 only after 12 h of incubation. Isoflavone aglycones ranged from 69.5 to 77.1% of total isoflavone compounds in SML after incubation.     

Heat and pH effects on the conjugated forms of genistin and daidzin isoflavones

Isoflavones occur primarily as glycosides (namely, malonyl-, acetyl-, and non-conjugated beta-glycosides) and a small percentage as the bioactive aglycon. The different chemical structures of isoflavones can dictate their stability during processing. Therefore, our objective was to determine the effects of pH and thermal treatments on conjugated isoflavones with regard to interconversions and loss. Conjugated daidzin and genistin were heated at 25, 80, and 100 degrees C under neutral, acidic, and basic conditions. Changes in isoflavone derivatives were monitored using high-performance liquid chromatography. Along with interconversions, considerable loss in total known isoflavone derivatives was noted for each isoflavone, especially under elevated pH and temperature. The malonylglycosides showed more stability than acetylglycosides, especially under acidic conditions. Overall, loss in isoflavone derivatives was significantly higher for daidzin than for genistin glycoside forms. Our results highlighted the significance of chemical structure with regard to stability, which is a key factor in determining soy processing conditions.     

Hydrolysis of isoflavone glycosides by a thermostable β-glucosidase from Pyrococcus furiosus

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.     

Retention and changes of soy isoflavones and carotenoids in immature soybean seeds (Edamame) during processing

Isoflavones and carotenoids in four experimental genotypes and Hutcheson cultivar soybeans were evaluated as a function of processing treatments and maturity. Total isoflavone and carotenoid contents were affected by genotypes and maturity stages (p < 0.0001). Total isoflavones ranged from 472 microg/g (in NTCPR93-40) to 2280 microg/g (in Hutcheson). Lutein contents ranged from 895 (in NTCPR93-286) to 2119 (in Honey Brown), and beta-carotene ranged from 291 (in Hutcheson) to 491 (in NICPR92-40) microg/100 g. Mean total isoflavone retention percentages in immature Hutcheson soybeans were 46% (boiling), 53% (freezing), and 40% (freeze-drying). Mean retentions of lutein and beta-carotene, respectively, were 92 and 73% in frozen, 62 and 62% in boiled, and 34 and 27% in freeze-dried soybeans. Boiling caused a substantial increase in daidzin, genistin, and genistein. The results show that post-harvest changes in total isoflavones and carotenoids in soybeans are influenced by processing methods, but genotype has an effect on isoflavone and carotenoid profiles during seed development.     

Isoflavones in soy-based foods consumed in Brazil: levels,distribution, and estimated intake

The isoflavone content and profile in processed soy-based products consumed in Brazil were determined by high-performance liquid chromatography and photodiode array detection of the intact isoflavones (naturally occurring aglycons, malonyl, acetyl, and beta-glycosides derivatives). Total isoflavone content varied significantly among products, from 2 to 100 mg/100 g (wet basis, expressed as aglycons), with the lowest content being found for soy-based enteral/oral diets and the highest found for textured soy proteins. For soy beverages isoflavone content varied from 12 to 83 mg/L. Soy sauce, miso, and tofu had isoflavone contents of 5.7 mg/L, 20 mg/100 g, and 7 mg/100 g, respectively. The beta-glycosides were the predominant form of the isoflavones in the products analyzed, except for miso, shoyu, and "Diet Shake" in which the aglycons were present in the highest proportions. On the basis of these data, the daily intake of isoflavone from soy products was estimated: the highest values were found for infants fed soy-based formulas, from 1.6 to 6.6 mg/kg of body weight.     

Group B saponins in soy products in the U.S. Department of Agriculture--Iowa State University isoflavone database and their comparison with isoflavone contents

Isoflavones in soy protein foods are thought to contribute to the cholesterol-lowering effect observed when these products are fed to humans. The group B saponins are another ethanol-soluble phytochemical fraction associated with soy proteins and isoflavones and have also been associated with cholesterol-lowering abilities. We measured the group B soyasaponin concentrations in a variety of soy foods and ingredients in the U.S. Department of AgricultureIowa State University Isoflavone Database. We compared the isoflavone and soy saponin concentrations and distributions in intact soybeans, soy ingredients, and retail soy foods. Group B saponins occur in six predominant forms. There appears to be no correlation between saponin and isoflavone concentrations in intact soybeans ranging from 5 to 11 mumol isoflavones/g soybean and from 2 to 6 mumol saponin/g soybean. Depending upon the type of processing, soy ingredients have quite different saponins/isoflavones as compared to mature soybeans. In soy foods, the saponin:isoflavone ration ranges from 1:1 to 2:5, whereas in soy protein isolates, the ratio is approximately 5:3. Ethanol-washed ingredients have very low saponins and isoflavones. These very different distributions of saponins and isoflavones in soy products may affect how we view the outcome of feeding trials examining a variety of protective effects associated with soy consumption.     

Pharmacokinetics of a slow-release formulation of soybean isoflavones in healthy postmenopausal women

Pharmacokinetic studies of soybean isoflavones have shown that following oral ingestion, the two major isoflavones, daidzin and genistin, are hydrolyzed in the intestine, rapidly absorbed into the peripheral circulation, and eliminated from the body with a terminal half-life of 7-8 h. These characteristics make maintenance of steady-state plasma isoflavone concentrations difficult to attain unless there is repeated daily ingestion of foods or supplements containing isoflavones. In an attempt to sustain more constant plasma isoflavone concentrations, a new slow-release formulation of a soybean isoflavone extract was prepared by microencapsulation with a mixture of hydroxypropylcellulose and ethylcellulose to alter its dissolution characteristics. In vitro experiments confirmed slow aqueous dissolution of isoflavones from this formulation when compared with the conventional isoflavone extract. The pharmacokinetics of this slow-release isoflavone extract was studied in 10 healthy postmenopausal women after oral administration of a single capsule containing the equivalent of 22.3 mg of genistein and 7.47 mg of daidzein expressed as aglycons. A comparison of the key pharmacokinetic parameters obtained in this study with those established in extensive studies performed previously in this laboratory indicated that the mean residence time of genistein and daidzein increased 2-fold with microencapsulation. These findings are indicative of a decreased rate of absorption, consistent with the observed slow in vitro dissolution rate. These findings show that it is feasible to employ polymer matrices that slow the aqueous dissolution for preparing sustained-release formulations of soy isoflavones. Further studies to optimize such formulations are warranted.     

Red clover Trifolium pratense L. phytoestrogens: UV-B radiation increases isoflavone yield, and postharvest drying methods change the glucoside conjugate profiles

Isoflavone extracts of red clover Trifolium pratense L. (cv. Pawera) with dissimilar glucoside conjugate profiles were obtained by employing different postharvest drying methods. The most prominent isoflavones found were formononetin and biochanin A and their corresponding glucosides and malonyl glucoside esters. Postharvest freeze drying inhibited the conversion of the glycosides to the aglycones, while vacuum drying allowed for maximum conversion of the glycosides to their corresponding aglycones. Air drying produced a low level of the aglycones formononetin and biochanin A, and oven drying promoted decarboxylation of the malonyl glucosides to the acetyl glucosides. Exposure to enhanced UV-B radiation resulted in an increase in total formononetin and biochanin A isoflavone levels, indicating that harvest during a period of high ambient UV-B radiation may be appropriate for maximum yield. The levels of caffeic acid and flavonols also increased by about 40 and 250%, respectively, on exposure to enhanced UV-B radiation.     

Isoflavone aglycon and glucoconjugate content of high- and low-soy U.K. foods used in nutritional studies

The isoflavone aglycon and glucoconjugate content of commercially prepared and "home-prepared" high- and low-soy foods selected for use in an on-going nutritional study were measured by LC-MS. The daidzin, daidzein, 6"-O-malonyldaidzin, 6"-O-acetyldaidzin, genistein, genistin, 6"-O-malonylgenistin, 6"-O-acetylgenistin, glycitin, glycitein, 6"-O-malonylglycitin, and 6"-O-acetylglycitin content are expressed in terms of individual isoflavones, total isoflavone equivalents, and milligrams of isoflavones per portion served. Soybeans (774 mg x kg(-1) total isoflavones) and soybean-containing foods had the highest isoflavone content of the foods examined. The low-soy foods all contained very low concentrations (<8 mg x kg(-1) total isoflavones) of the isoflavone aglycons and glucoconjugates. High- and low-soy 11 day rotating menus were constructed from the analyzed foods to deliver 100.0 and 0.5 mg of isoflavones per day, respectively.     

Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis

Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 +/- 0.6 nmol/g total genistein; 3.0 +/- 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.     

Heating affects the content and distribution profile of isoflavones in steamed black soybeans and black soybean koji

Steamed black soybeans and black soybean koji, a potentially functional food additive, were subjected to heating at 40-100 degrees C for 30 min. It was found that steamed black soybeans and black soybean koji after heating at 80 degrees C or higher generally showed reduced contents of malonylglucoside, acetylglucoside, and aglycone isoflavone and an increased content of beta-glucoside. A lower reduction in malonylglucoside and acetylglucoside isoflavone but greater reduction in aglycone content was noted in steamed black soybeans compared to black soybean koji after a similar heat treatment. After 30 min of heating at 100 degrees C, steamed black soybean retained ca. 90.3 and 83.8%, respectively, of its original malonylglucoside and acetylglucoside isoflavone, compared to lower residuals of 80.9 and 78.8%, respectively, for black soybean koji. In contrast, the heated black soybeans showed an aglycone residual of 68.0%, which is less than the 80.0% noted with the heated black soybean koji.     

Effects of high temperature stress at different development stages on soybean isoflavone and tocopherol concentrations

Soybean contains a range of compounds with putative health benefits including isoflavones and tocopherols. A study was conducted to determine the effects on these compounds of high temperature stress imposed at specific development stages [i.e., none, pre-emergence, vegetative, early reproductive (R1-4), late-reproductive (R5-8), or all stages]. Two cultivars (AC Proteina and OAC Champion) were grown in growth chambers set at contrasting temperatures [i.e., stress conditions of 33/25 °C (day/night temperature) and control conditions of 23/15 °C] in order to generate these treatments. Isoflavone and tocopherol concentrations in mature seeds were determined using high-performance liquid chromatography. In both cultivars isoflavone response was greatest when stress occurred during the R5-8 stages and during all development stages, these treatments reducing total isoflavone concentration by an average of 85% compared to the control. Stress imposed at other stages also affected isoflavone concentration although the response was smaller. For example, stress during the vegetative stages reduced total isoflavones by 33% in OAC Champion. Stress imposed pre-emergence had an opposite effect increasing daidzein concentration by 24% in AC Proteina. Tocopherol concentrations were affected the most when stress was imposed during all stages of development, followed by stress restricted to stages R5-8; response to stress during other stages was limited. The specific response of tocopherols differed, α-tocopherol being increased by high temperature by as much as 752%, the reverse being observed for δ-tocopherol and γ-tocopherol. The present study demonstrates that while isoflavone and tocopherol concentrations in soybeans are affected the most by stress occurring during seed formation, concentrations can also be affected by stress occurring at other stages including stages as early as pre-emergence.     

Stability of isoflavones in soy milk stored at elevated and ambient temperatures

Soy isoflavones are widely recognized for their potential health benefits. The increased use of traditional and new food products calls for the assessment of their stability during processing and storage. The present study examines the stability of genistein and daidzein derivatives in soy milk. Soy milk was stored at ambient and elevated temperatures, and the change in isoflavone concentration was monitored with time. Genistin loss in time showed typical first-order kinetics, with rate constants ranging from 0.437-3.871 to 61-109 days(-1) in the temperature ranges of 15-37 and 70-90 degrees C, respectively. The temperature dependence of genistin loss followed the Arrhenius relation with activation energies of 7.2 kcal/mol at ambient temperatures and 17.6 kcal/mol at elevated temperatures. At early stages of soy milk storage at 80 and 90 degrees C, the 6' '-O-acetyldaidzin concentration increased, followed by a slow decrease. The results obtained in this study can serve as a basis for estimating the shelf life of soy milk as related to its genistin content.     

Variations in isoflavone levels in soy foods and soy protein isolates and issues related to isoflavone databases and food labeling

The reliability of databases on the isoflavone composition of foods designed to estimate dietary intakes is contingent on the assumption that soy foods are consistent in their isoflavone content. To validate this, total and individual isoflavone compositions were determined by HPLC for two different soy protein isolates used in the commercial manufacture of soy foods over a 3-year period (n = 30/isolate) and 85 samples of 40 different brands of soy milks. Total isoflavone concentrations differed markedly between the soy protein isolates, varying by 200-300% over 3 years, whereas the protein content varied by only 3%. Total isoflavone content varied by up to 5-fold among different commercial soy milks and was not consistent between repeat purchases. Whole soybean milks had significantly higher isoflavone levels than those made from soy protein isolates (mean +/- SD, 63.6 +/- 21.9 mg/L, n = 43, vs 30.2 +/- 5.8 mg/L, n = 38, respectively, p < 0.0001), although some isolated soy protein-based milks were similar in content to "whole bean" varieties. The ratio of genistein to daidzein isoflavone forms was higher in isolated soy protein-based versus "whole bean" soy milks (2.72 +/- 0.24 vs 1.62 +/- 0.47, respectively, p < 0.0001), and the greatest variability in isoflavone content was observed among brands of whole bean soy milks. These studies illustrate large variability in the isoflavone content of isolated soy proteins used in food manufacture and in commercial soy milks and reinforce the need to accurately determine the isoflavone content of foods used in dietary intervention studies while exposing the limitations of food databases for estimating daily isoflavone intakes.     

Isoflavone transformation during soybean koji preparation and subsequent miso fermentation supplemented with ethanol and NaCl

Soybeans were soaked with water for 4 h, steam-cooked, inoculated with the conidia of Aspergillus oryzae, and incubated for 3 days for koji preparation. The koji was then mixed with water-soaked and steam-cooked soybeans (1:2, w/w), ground into paste, and supplemented with 15% ethanol and 12.5% NaCl or 3% ethanol and 6% NaCl for miso fermentation at 30 degrees C. Daidzin, genistin, daidzein, and genistein contents were extracted from the lyophilized and pulverized soybean powder or from the miso homogenate by a developed one-tube procedure and analyzed with an HPLC. After water soaking, daidzein and genistein contents increased markedly, whereas daidzin and genistin contents decreased. Further increases of daidzein and genistein contents and decreases of daidzin and genistin contents were observed after koji mold growth. During fermentation, fungal and lactic acid bacterial (LAB) growth in the miso products was inhibited, whereas soluble protein contents increased much more rapidly in the low-salt miso products supplemented with 3% ethanol and 6% NaCl than the other products. When the 4- and 8-week-fermented miso products were cooked with tofu for sensory evaluation, flavor ratings of the low-salt products were higher than that of a popular commercial product. In both products, the most daidzins and genistins were hydrolyzed after 4 weeks of fermentation. The hydrolytic enzymes contributing to isoflavone transformation originated from soybeans after water soaking and from koji with mold growth. It was of merit that the low-salt fermented products were fairly acceptable in flavor rating and rich in daidzein and genistein contents after 4 weeks of fermentation.     

MALDI-TOF MS analysis of isoflavones in soy products

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a new technique having a number of advantages for food analysis. This study is the first to demonstrate the use of MALDI-TOF MS to identify isoflavones in soy samples. 2',4',6'-Trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHB) were both good matrices for isoflavones, but DHB was chosen as the best because it worked well for sample extracts, with good spot-to-spot repeatability. Isoflavones were predominantly ionized in a protonated form with a very small amount of sodium or potassium adduct ions. Fragmentation occurred only through loss of glycosidic residues. Daidzin showed more than twice the response of genistin using MALDI-TOF MS. A simple solid phase extraction of isoflavones from soy samples was developed for MALDI-TOF MS analysis. MALDI-TOF MS can provide an isoflavone profile in 2 min and serves as a powerful tool to identify and study processing changes of isoflavones in soy products.