Simultaneous detection of glycinin and ß-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides.

Glycinin and ß-conglycinin are the two main allergic proteins in soybean. Due to their complex structures and lack of protein standards, it is difficult to achieve quantitative determination of these proteins in soybeans. In this study, an HPLC-MS/MS method was developed for the simultaneous determination of five subunits of glycinin (G1, G2, G3, G4, and G5) and three subunits of ß-conglycinin (α, α', and ß) in processed soybean products based on 8 specific peptides and their stable isotope-labeled peptides. Here, each specific peptide was derived from one of the above 8 subunits. When soy protein was extracted and digested with trypsin, 8 specific peptides, and corresponding stable isotope-labeled peptides were analyzed by HPLC-MS/MS. The linear range for the specific peptides was between 3.2 and 1000 ng/mL (R2 > 0.9955). The recoveries of added peptides ranged from 83.4% to 117.8%, and the intra-day precisions (% CV) were below 17.4%. The limit of quantification of each subunit of glycinin and ß-conglycinin in processed soybean products (in terms of protein amount) was between 15.1 and 156.1 g/g. This method was successfully applied to the analysis of 8 subunits of glycinin and ß-conglycinin in 68 different processed soybean products, which provides technical support for processed product quality evaluation and monitoring soybean processing technology.

Different copper sources and levels affect growth performance, copper content, carcass characteristics, intestinal microorganism and metabolism of finishing pigs.

Copper (Cu) is an essential trace element in the production of swine. This study was conducted to investigate the effect of 3 different sources of Cu on growth performance, Cu metabolism, and intestinal microorganisms of finishing pigs, so as to estimate the bioavailability of the 3 sources for pigs. A total of 42 male finishing pigs (88.74 ± 5.74 kg) were randomly allocated to 7 treatments. The factors were 3 sources (CuSO4, Cu-glycine, Cu-proteinate) and 2 levels (5 and 20 mg/kg) of Cu, plus one negative control treatment (0 mg/kg added Cu level) for the entire 28-d experiment. The average daily gain (ADG) and feed to gain ratio (F:G) both increased when Cu was added. The Cu level in liver, bile, kidney, serum, lung, urine and feces rose (P < 0.001) with increasing dietary Cu level regardless of the source. Meanwhile, pigs receiving organic Cu (glycinate or proteinate) retained more Cu and excreted less Cu than those receiving inorganic Cu (CuSO4), which showed that organic forms were more bioavailable. At the transcriptional level, changes in the level and source of dietary Cu resulted in modulation of transporters. In the jejunal mucosa, import transporter high affinity copper uptake protein 1 (CTR1) and export transporter ATPase copper transporting alpha (ATP7A) in supplemental Cu treatments were down-regulated compared to the control. Also, peptide transporter 1 (PepT1) and lanine-serine-cysteine transporter, type-2 (ASCT2) were significantly (P < 0.01) up-regulated in 20 mg/kg Cu-proteinate and Cu-glycinate treatments, respectively. Microbial diversity was lowest in the 20 mg/kg CuSO4 treatment, and the ratio of Firmicutes to Bacteroidetes was higher in added Cu treatments, especially Cu-glycinate treatment. These results indicate that uptake of different Cu forms is facilitated by different transporters and transport mechanisms, and compared with inorganic Cu, organic Cu provides benefits to intestinal microflora and reduces Cu excretion.

Quantification of lectin in soybeans and soy products by liquid chromatography-tandem mass spectrometry.

Lectin is one of the major anti-nutritional factors in soybeans and inhibits digestion of dietary protein. Here, an absolute quantification method was developed to detect lectin using synthetic peptide 183TTSWDLANNK192 as reference standard and corresponding isotope labeled peptide TTSWDLANNK (Alanine-13C3,15N) as internal standard to normalize results. After the ground soybeans and soy products were defatted with n-hexane and extracted with extraction buffer, the crude protein extract was digested on filter membrane by trypsin. Further, the enzymatic hydrolysis peptides were quantified using liquid chromatography-tandem mass spectrometry. The synthetic reference peptide showed a detection limit of 0.27 ng/mL and a linear relationship in the range of 3.2-1000 ng/mL (r2 > 0.997). Correspondingly, the detect limit of lectin in soybean samples was 35.5 µg/g. The results showed that the recoveries of the lectin in spiked samples ranged from 80.9% to 108.7% with intra-day precisions (% CV) less than 9%. The method was successfully used to evaluate lectin levels in hundreds of soybean seeds from different varieties and soy products from different soybean processing techniques. Furthermore, the method may provide a potential application as a general method for the ultrasensitive detection of various protein anti-nutritional factors in food.

Soy Isoflavones and their Effects on Xenobiotic Metabolism.

BACKGROUND: Soy isoflavones, such as genistein and daidzein, are bioflavonoids found in soy products that are able to interact with various hormones such as estrogen. Epidemiological studies reveal a proper level of isoflavones in diet can prevent many diseases like cancers or diabetes. Therefore, it is important to study the biotransformation and xenobiotic metabolism of soy isoflavones. METHODS: A systematic review of published studies was carried out to investigate the characterization of isoflavones and their metabolites, sample pretreatment and quantitative analysis of isoflavones, and the influence of soy isoflavones on drug and xenobiotic metabolism. RESULTS: Aglycones with weak estrogen-like activities are the biologically active forms of the soy isoflavones in mammals. The most recent advances including extraction, purification and detection of isoflavones in soybean and soy products are discussed. The effects of soy isoflavones on drug and xenobiotic metabolism involve in regulation of phase I cytochrome P450 (CYPs) enzyme and phase I detoxifying enzymes expression and activity. At the molecular level, soy isoflavones have proved capable of estrogenic/antiestrogenic with tissue-selective, anti-cancer, antiobesity, anti-oxidation, and tyrosine kinase inhibition activities. CONCLUSION: This review summarized different aspects of soy isoflavones and their molecular mechanisms of pharmacological action on xenobiotic, which demonstrated that soy isoflavones can decrease the incidence of many diseases and benefit for human health. However, since the lack of clinical research for evaluation of the proper dosage of intake of soy isoflavones in diet or adjunctive therapy, there is a need for further studies on the selection of doses, biomedical applications and adverse effects of isoflavones for human health.

Resveratrol compares with melatonin in improving in vitro porcine oocyte maturation under heat stress.

BACKGROUND: Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants, has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the current study, we investigated the beneficial effects of resveratrol on in vitro porcine oocyte maturation under heat stress (HS). The effect of resveratrol, melatonin and their combination on alleviating HS was compared according to the maturation rate of oocytes and the development competence of embryos after parthenogenetic activation (PA). RESULTS: Supplementation with resveratrol (2.0 µmol/L) not only improved the nuclear maturation but also raised the blastocyst rate of porcine embryos' PA from oocytes that underwent HS by increasing their glutathione (GSH) level, reducing reactive oxygen species (ROS) and up-regulating the expression of Sirtuin 1 (SIRT1). It was also found that melatonin (10(-7) mol/L) and the combination of resveratrol (2.0 µmol/L) plus melatonin (10(-7) mol/L) exhibited more potent effects than resveratrol alone regarding their protective activities on oocyte maturation under HS. CONCLUSIONS: This study compared the efficiencies of resveratrol, melatonin and their combination for protecting porcine oocytes from heat stress. The mechanisms are attributed to the fact that each treatment may have different ability to regulate the synthesis of steroid hormones and the expression of mature related genes.

Effects of particle size and drying methods of corn on growth performance, digestibility and haematological and immunological characteristics of weaned piglets.

The objective of this study was to evaluate the effects of particle size and drying methods of corn on growth performance of weaned piglets. Crossbreed weaned piglets (n = 192; Duroc × Landrace × Large White) were assigned to one of four treatments (2 × 2 factorial arrangement). All piglets were fed corn-soybean meal diets and treatments were (1) hot air-dried and coarsely ground corn, (2) hot air-dried and finely ground corn, (3) sun-dried and coarsely ground corn and (4) sun-dried and finely ground corn. The results showed that finely ground corn (FGC) improved the performance of piglets. Additionally, the apparent total tract digestibility (ATTD) of gross energy (GE) and ether extract (EE) were increased by FGC, but the drying methods did not affect the performance of piglets or ATTD. Furthermore, smaller particle size significantly decreased the intestinal permeability, which was also not influenced by drying methods. FGC increased the total number of white blood cells, but not other blood parameters. Finally, the level of serum interleukin-1 was decreased by fine grinding and that of serum tumour necrosis factor α was decreased by sun drying. Conversely, these characteristics of weaned piglets can hardly have been affected either by the corn drying method or its interaction with grinding methods.

Overexpression of MzASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants.

Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-O-methyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E. coli converted its substrates to melatonin with an activity of approximately 5.5 pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2-4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.

Leucine stimulates ASCT2 amino acid transporter expression in porcine jejunal epithelial cell line (IPEC-J2) through PI3K/Akt/mTOR and ERK signaling pathways.

Leucine has been shown to influence intestinal protein metabolism, cell proliferation and migration. Furthermore, our previous study demonstrated that branched-chain amino acids could modulate the intestinal amino acid and peptide transporters in vivo. As the possible mechanisms are still largely unknown, in the present work, we studied the transcriptional and translational regulation of leucine on amino acid transporter production in IPEC-J2 cells and the signaling pathways involved. Treatment of IPEC-J2 cells with 7.5 mM leucine enhanced the mRNA expression of the Na(+)-neutral AA exchanger 2 (ASCT2) and 4F2 heavy chain (4F2hc) and caused an increase in ASCT2 protein expression. Leucine also activated phosphorylation of 4E-BP1 and eIF4E through the phosphorylation of mTOR, Akt and ERK signaling pathways in IPEC-J2 cells. Pre-treatment of IPEC-J2 cells with inhibitors of mTOR and Akt (rapamycin and wortmannin) or an inhibitor of ERK (PD098059) for 30 min before leucine treatment attenuated the positive effect of leucine in enhancing the protein abundance of ASCT2. These results demonstrate that leucine could up-regulate the expression of the amino acid transporters (ASCT2) through transcriptional and translational regulation by ERK and PI3K/Akt/mTOR activation.

Development of immunoaffinity chromatographic method for isolating glycinin (11S) from soybean proteins.

A monoclonal antibody (Mab), 4B2, against soybean glycinin was prepared using the preliminary extracted natural glycinin as the immunogen in our previous study. Herein, we established a novel method for the purification of glycinin by Mab 4B2-based immunoaffinity chromatography. The characteristics of the purified glycinin were identified by SDS-PAGE, Western blot, and histamine release assay. Glycinin was successfully isolated from soybeans with a yield of 16.8% and a purity of 93.8%, which were significantly higher than those produced using other traditional procedures. The acidic polypeptides of the purified glycinin can be recognized by the Mab 4B2, but not the basic polypeptides. In addition, the histamine release ratio of the purified glycinin was similar to that of natural glycinin, which indicated that the purified glycinin maintained its biological activities. Further study revealed that the Mab/gel ratios ranging from 6.0 to 12.0 mg/mL were suitable for the isolation of glycinin using immunoaffinity chromatography. Taken together, this new method based on immunoaffinity chromatography could be used for high-yield and high-purity natural glycinin production and would facilitate future study on the mechanism of soybean-induced food allergy.

A chronocoulometric aptamer sensor for adenosine monophosphate.

The selective recognition of adenosine monophosphate by a half-duplex aptamer-modified electrode leads to a simple chronocoulometric aptasensor based on the changes in surface charges.

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions.

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.