Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).     

Thermal stability of genistein and daidzein and its effect on their antioxidant activity

Soy isoflavones, present in many processed soy foods, are known for their phytoestrogenic and antioxidant activities. The aim of this work was to study the kinetics of genistein and daidzein degradation at elevated temperatures and to follow changes in their antioxidant activity. Daidzein and genistein in model solutions (pH 7 and 9) were thermally treated at 120 degrees C or incubated at 70, 80, and 90 degrees C. Isoflavone degradation was observed at all temperatures, with apparent first-order kinetics at 70-90 degrees C, and E(a) = 8.4 and 11.6 kcal/mol at pH 9, respectively. Microcalorimetric stability tests showed a similar pattern of degradation, however, with higher E(a) (genistein, 73.7 kcal/mol; daidzein, 34.1 kcal/mol) that may be attributed to the anaerobic conditions. The antioxidant activity of incubated isoflavone solutions, followed by the ABTS test, decreased rapidly at pH 9 for genistein, whereas only moderate reduction was observed for daidzein (pH 7 and 9) or genistein at pH 7. This may indicate different degradation mechanisms for genistein and daidzein.     

Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein

The oxidative metabolism of the major soy isoflavones daidzein and genistein was investigated using liver microsomes from Aroclor-treated male Wistar rats. Both daidzein and genistein were extensively metabolized and are therefore excellent substrates for cytochrome P450 enzymes. The identity of the metabolites was elucidated using high-performance liquid chromatography (HPLC) with diode array detection, gas chromatography-mass spectrometry (GC/MS), and HPLC/atmospheric pressure ionization electrospray mass spectrometry (API-ES MS) as well as reference substances. Daidzein was converted to nine metabolites, comprising four monohydroxylated, four dihydroxylated, and one trihydroxylated metabolite. Genistein was metabolized to four monohydroxylated and two dihydroxylated products. With both isoflavones the additional hydroxy groups are exclusively introduced into the ortho positions of existing phenolic hydroxy groups. The major metabolites of daidzein were identified as 6,7,4'-trihydroxyisoflavone, 6,7,3',4'-tetrahydroxyisoflavone, 7,8, 4'-trihydroxyisoflavone, and 5,6,7,4'-tetrahydroxyisoflavone. The main microsomal metabolites of genistein were 5,6,7, 4'-tetrahydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. Furthermore, the GC/MS and HPLC/API-ES MS analysis support the conclusion that one monohydroxylated metabolite of daidzein and genistein is hydroxylated at the aliphatic position C-2 of the C-ring. The UV-vis, GC/MS, and HPLC/MS data of all detected metabolites as well as the derived chemical structure of the metabolites are presented. Most metabolites are reported in this paper for the first time. On the basis of these findings it is suggested that hydroxylation reactions may also play an important role in the in vivo metabolism of the soy isoflavones daidzein and genistein.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.     

Changes of astringent sensation of soy milk during tofu curd formation

The effect of isoflavone on soy milk and tofu astringency was investigated, and no consistency was found between an undesirable astringent taste and isoflavone contents. Isoflavone-enriched extract (approximately 39% isoflavones) showed no astringency. Soybean foods having high amounts of isoflavones showed less astringency. About 80% of isoflavones exist freely in both soy milk and tofu, but 55% of phytates (which play an important role in the formation of the tofu curd network) exist freely in the soy milk, and 6-13%, on the basis of coagulation, existed freely in the tofu curds. A 1% potassium phytate solution at pH 7 showed the very same astringency as soy milk; however, calcium phytate at the same concentration and pH showed no undesirable sensation. Thus, it is assumed that the astringent characteristics caused by phytic ions in soy milk are lost upon conversion of phytic ions to their insoluble salt forms during soy milk coagulation.     

Changes in the volatile profile of oats induced by processing

Samples of an Australian oat cultivar, Echidna, were pilot-scale processed. At each stage of the processing (raw oats, groats, kiln dried dehulled oats (KDHO), and rolled (flaked)) samples were removed for later sensory and GC-MS analysis of the flavor components. Mean taste panel scores from a trained taste panel were calculated according to attributes (cereal, burnt, toasted, floury, and yeasty). Attributes were generally similar for both KDHO and flaked oats except in the yeasty attributes. Panelists were able to differentiate between groats, KDHO, and flaked oats (raw oats were not included). The largest effects of heat processing were found for the attributes toasted and yeasty aroma; toasted, cereal, and yeasty flavor; and toasted and yeasty aftertaste. A multi-organoleptic sensor analyzer was able to differentiate all samples when the output was subjected to discriminant function analysis. A reintroduced sample was recognized with a confidence level better than 96%. Solid-phase microextraction (SPME) of headspace followed by GC--MS was used to identify volatiles after either dry or slurry heating. Several SPME fiber types were evaluated as to their ability to sorb oat volatiles. A 100-microm poly(dimethylsiloxane) SPME fiber was found to provide the best adsorption profile as measured by number of compounds sorbed and peak area response. A range of alcohols, aldehydes, alkyl benzenes, dienes, and ketones was identified in the processed samples.     

Development of a simple method for the determination of genistein, daidzein, biochanin A, and formononetin (biochanin B) in human urine.

A simple method was developed for the determination of free and/or total isoflavones daidzein, genistein, and their respective 4'-methoxy derivatives biochanin A and formononetin (biochanin B) at low levels in human urine. A solid-phase extraction on octadecyl silica (C(18)) columns was used for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElut 1010 column connected on-line to a Florisil cartridge by a Teflon stopcock was used for effective eluate purification. A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers using GC/MS-SIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, whereas robigenin was used in the case of total isoflavone determination. Recoveries for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL(-)(1) level and from 63.5 to 89. 2% at the 5 ng mL(-)(1) level in urine. Analytical curves were linear between 5 and 25 ng mL(-)(1). Detection limits varied from 1 ng mL(-)(1) for formononetin to 2.3 ng mL(-)(1) for daidzein. Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL(-1) level.     

Accumulation of genistein and daidzein, soybean isoflavones implicated in promoting human health, is significantly elevated by irrigation

To circumvent drought conditions persisting during seed fill in the mid-south U.S. soybean production region, researchers have developed the early soybean (Glycine max [L.] Merr.) production system (ESPS), which entails early planting of short-season varieties. Because soybean supplies a preponderance of the world's protein and oil and consumption of soy-based foods has been associated with multiple health benefits, the effects of this agronomic practice on seed quality traits such as protein, oil, and isoflavones should be investigated. Four cultivars of soybean, two from maturity group IV and two from maturity group V, were planted in April (ESPS) and May (traditional) in a two-year study at Stoneville, MS. Near-infrared analysis of soybean seed was utilized to determine the percentages of protein and oil. Dependent upon variety, the oil content of the early-planted crop was increased by 3-8%, whereas protein was not significantly changed. Visualization of protein extracts fractionated by sodium dodecyl sulfate-polyacrylamide electrophoresis and fluorescence two-dimensional difference gel electrophoresis revealed that early planting did not affect the relative accumulation of the major seed-storage proteins; thus, protein composition was equal to that of traditionally cultivated soybeans. Maturity group IV cultivars contained a higher percentage of oil and a lower percentage of protein than did the maturity group V cultivars, regardless of planting date. Gas chromatographic separation of fatty acids revealed that the percentages of saturated and unsaturated fatty acids were not significantly altered by planting date. Methanol extracts of seed harvested from different planting dates when analyzed by high-performance liquid chromatography showed striking differences in isoflavone content. Dependent upon the variety, total isoflavone content was increased as much as 1.3-fold in early-planted soybeans. Irrigation enhanced the isoflavone content of both early- and late-planted soybeans as much as 2.5-fold. Accumulation of individual isoflavones, daidzein and genistein, was also elevated by irrigation. Because this cultural practice improves the quality traits of seeds, ESPS provides an opportunity for enhancing the quality of soybean.     

Rhizosphere isoflavones (daidzein and genistein) levels and their relation to the microbial community structure of mono-cropped soybean soil in field and controlled conditions

Despite an increase in the understanding of the soybean isoflavones involved in root-colonizing symbioses, relatively little is known about their levels in the rhizosphere and their interactions with the soil microbial community. Based on a 13-year experiment of continuous soybean monocultures, in the present study we quantified isoflavones in the soybean rhizosphere and analyzed the soil microbial community structure by examining its phospholipid fatty acid (PLFA) profile. Two isoflavones, daidzein (7, 4′-dihydroxyisoflavone) and genistein (5,7,4′- trihydroxyisoflavone), were detected in the rhizosphere soil of soybean plants, with the concentrations in the field varying with duration of mono-cropping. Genistein concentrations ranged from 0.4 to 1.2 μg g⁻¹ dry soil over different years, while daidzein concentrations rarely exceeded 0.6 μg g⁻¹ dry soil. PLFA profiling showed that the signature lipid biomarkers of bacteria and fungi varied throughout the years of the study, particularly in mono-cropping year 2, and mono-cropping years 6-8. Principal component analysis clearly identified differences in the composition of PLFA during different years under mono-cropping. There was a positive correlation between the daidzein concentrations and soil fungi, whereas the genistein concentration showed a correlation with the total PLFA, fungi, bacteria, Gram (+) bacteria and aerobic bacteria in the soil microbial community. Both isoflavones were easily degraded in soil, resulting in short half-lives. Concentrations as small as 1 μg g⁻¹ dry soil were sufficient to elicit changes in microbial community structure. A discriminant analysis of PLFA patterns showed that changes in microbial community structures were induced by both the addition of daidzein or genistein and incubation time. We conclude that daidzein and genistein released into the soybean rhizosphere may act as allelochemicals in the interactions between root and soil microbial community in a long-term mono-cropped soybean field.     

Determination of spore inactivation during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

The efficacy of microbial inactivation techniques is currently tested using time-consuming and labor-intensive plate count methods, which are the principal rate-limiting steps in developing inactivation kinetic parameters for alternative food processing technologies. Fourier transform infrared (FT-IR) spectroscopy combined with multivariate analysis was used to quantify viable spores and identify some biochemical changes in samples treated by autoclaving, pressure-assisted thermal processing (PATP), and thermal processing (TP). Spore suspensions ( approximately 109 CFU/mL) of Bacillus amyloliquefaciens TMW 2.479 Fad 82, B. amyloliquefaciens TMW 2.482 Fad 11/2, B. sphaericus NZ 14, B. amyloliquefaciens ATCC 49764, and Clostridium tyrobutyricum ATCC 25755 were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and by TP (121 degrees C) for 0, 10, 20, and 30 s. The concentrations of spores in treated samples were determined by plating (reference method). Models developed using partial least-squares regression (PLSR) for predicting spore levels in treated samples had correlation coefficients (r) of >0.99 and standard errors of cross-validation ranging between 100.2 and 100.5 CFU/mL. Changes in dipicolinic acid (DPA) and secondary structure of proteins were found to occur during inactivation of spores by PATP and TP. FT-IR spectroscopy could rapidly estimate viable bacterial spore levels in PATP- and TP-treated spore suspensions, providing an accurate analytical tool for monitoring the efficacy of sterilization techniques in inactivating spore-forming microorganisms.     

Soy isoflavonoid aglycons genistein and daidzein Do not increase the cytochrome P-450 content of the liver microsomes of mice

Mice (4-week-old, male ddy) were fed four isonitrogenic diets for 21 days: purified diet (C diet); fermented soybean (400 mg of soy isoflavonoids/kg; FSB); fermented soybean extract (400 mg of soy isoflavonoid aglycones/kg; FSBE); C with indole-3 carbinol (I3C) (2500 mg of I3C/kg; I3C). The I3C and FSB diets significantly increased the cytochrome P-450 content of hepatic microsomes in comparison with the C diet, while the FSBE diet did not. Other mice were fed seven diets for 21 days: C; C with 100 mg or 200 mg of genistein, 100 mg or 200 mg of daidzein, or 100 mg of genistein + 100 mg of daidzein/kg; I3C diet. Genistein and daidzein did not change the liver cytochrome P-450 content. There was no synergistic effect of the combined feeding of genistein and daidzein. The increase in the cytochrome P-450 content with the FSB diet depends on chemicals other than genistein and daidzein. Genistein and daidzein do not induce cytochrome P-450.     

Assessment of soy genotype and processing method on quality of soybean tofu

Protein quality in six soybean varieties, based on subunit composition of their protein, was correlated with quality of the produced tofu. Also, protein changes due to a pilot plant processing method involving high temperature/pressure and commercial rennet as coagulant were assessed. In each soybean variety, glycinin (11S) and β-conglycinin (7S) as well as 11S/7S ratio significantly changed from beans to tofu. Between varieties, the 11S/7S protein ratio in seed indicated genotypic influence on tofu yield and gel hardness (r = 0.91 and r = 0.99, respectively; p < 0.05). Also, the 11S/7S ratio correlated with soymilk pH (r = 0.89, p < 0.05), leading to a relationship between soymilk pH with protein recovery and yield of tofu (r = 0.94 and r = 0.91, respectively; p < 0.05). The soybean β'-subunit of 7S protein negatively influenced tofu hardness (r = -0.91, p < 0.05). Seed protein composition and proportion of 7S protein subunits under the applied production method had an important role in defining tofu quality.     

Changes in the physical state of sucrose during dark chocolate processing

Dark chocolate tablets were manufactured using 100% crystalline sucrose, 50% crystalline and 50% amorphous sucrose, and 100% amorphous sucrose. The physical state of sucrose was determined by differential scanning calorimetry (DSC) and X-ray diffraction. DSC scans of dark chocolate samples containing amorphous sucrose were characterized by a glass transition at 63 degrees C, a sucrose crystallization peak at 105 degrees C, and a melting endotherm at 188 degrees C. Independent of the amount of amorphous or crystalline sucrose used for the preparation of dark chocolate, all final chocolate products provided a single melting endotherm at 188 degrees C and a crystalline X-ray diffraction pattern. These results indicated that sucrose crystallized during production of dark chocolate and that no amorphous sucrose was present in the final chocolate products.     

茭白冷藏期间蛋白质表达谱的变化

为探讨茭白冷藏期间衰老的分子机理,应用蛋白质组学技术,研究了茭白冷藏期间蛋白质表达谱的变化。结果显示,双向电泳胶上共检测到大约650个蛋白点,其中35个蛋白表达量存在2.0倍以上显著(p0.05)差异,经过串联飞行时间质谱分析,成功鉴定出29个蛋白,根据其功能可分为6类,即代谢(20.7%)、细胞结构(27.6%)、抗胁迫(20.7%)、衰老(6.9%)、蛋白质合成(13.8%)和功能未知蛋白(10.3%);其中:代谢相关蛋白3个上调表达、3个下调表达,细胞结构相关蛋白6个上调表达、2个下调表达,抗胁迫相关蛋白4个上调表达、2个下调表达,衰老相关蛋白2个上调表达,蛋白质合成相关蛋白4个及功能未知蛋白3个均下调表达。这些差异表达蛋白的功能分析表明,茭白采后衰老机理可能涉及物质代谢过程的调整、能量代谢途径的改变、活性氧清除能力的减弱以及细胞结构的解体。     

Changes in contents of carotenoids and vitamin E during tomato processing

The aim of this study was to investigate the effect of different types of tomato processing on contents of lycopene, beta-carotene, and alpha-tocopherol. Samples of tomato sauce, tomato soup, baked tomato slices, and tomato juice were taken at different times of heating, respectively, after each step of production. HPLC was used to analyze contents of carotenoids and vitamin E. Due to the loss of water during thermal processing, contents of lycopene, beta-carotene, and alpha-tocopherol on a wet weight basis increased. On a dry weight basis, contents of lycopene increased or decreased depending on the origin of the tomatoes used, whereas the beta-carotene contents decreased or were quite stable. In contrast to lycopene, beta-carotene isomerized due to thermal processing. The alpha-tocopherol contents significantly rose during short-term heating. The increase was not caused by release of alpha-tocopherol from the seeds containing predominantly gamma-tocopherol and accounting for 2% of total alpha-tocopherol content only.     

Monitoring biochemical changes in bacterial spore during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.     

Analysis of the peptide profile of milk and its changes during thermal treatment and storage

In this study a new method was developed for analysis of the low molecular weight protein fraction of milk, allowing a simple and fast overview of the peptide profile of various milk samples. For this purpose, immobilized metal affinity chromatography (IMAC) was coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By this technique, two major peptides in milk could be identified as fragments of alpha-s1-casein. During heat treatment of raw milk, five new peptides were generated, the origin of which could be assigned to the casein fraction. Storage experiments with extended shelf life milk at 4 degrees C did not show any changes in the peptide profile, whereas in ultra high temperature milk stored at room temperature, one peptide increased significantly, which was identified as the N-terminus of alpha-s1-casein. The peptide was assumed to be formed in an enzymatic reaction, which was confirmed in a storage experiment with sterilized milk. Analyses of different commercially available milk samples confirmed the results obtained with the heated and stored milk. Furthermore, differences in the peptide profiles of the samples, probably due to different cow breeds or lactation stages, were observed. These results establish IMAC prior to MALDI-TOF-MS as a valid tool for the rapid analysis of the peptide profile of milk.     

Changes in the cell wall network during the thermal dehydration of alfalfa stems

The effects of heat treatments used to dry alfalfa stems were investigated. Heating at 70 or 100 degrees C caused no major change in the cell wall composition, but xylanase had lower activity on the cell wall of heated material and the amount of xylose released varied with the temperature used. Chemical fractionation of cell wall carbohydrates showed that the main changes occurring during stem dehydration concerned pectic polymers and probably hemicelluloses. There was less material soluble in ammonium oxalate from alfalfa heated at 100 degrees C than from fresh alfalfa. The results suggest that heat processing causes some changes in the cell wall network. Environmental scanning electron microscopy was used to examine fully hydrated tissues at high resolution. There was cell distortion without disruption of cell walls as water was lost.