Effects of pH and salt concentration on freeze-thaw fractionation of soymilk protein.

BACKGROUND: The two major storage proteins of soymilk are the globulins 7S and 11S. Freeze-thaw fractionation is a simple method for separating these proteins in raw soymilk. In this study, we assessed the freeze-thaw fractionation ability of raw soymilk under various pH (4.3-11.6) conditions and added salt (sodium chloride) concentrations (0.00-0.67 mol L-1). RESULTS: We successfully achieved fractionation within a pH range of 5.8-6.7 and when the salt concentration was 0.22 mol L-1 or lower. Analysis of particle size distribution and microscopic examination of soymilk revealed no direct correlation between particle size and freeze-thaw fractionation ability. Interestingly, it was confirmed that the ranges of zeta potential values associated with successful freeze-thaw fractionation in raw soymilk remained consistent across different pH and salt concentration conditions. These ranges were between -23 and -28 mV at pH levels ranging from 5.8 to 6.7 and between -18 and -29 mV at added salt concentrations ranging from 0 to 0.22 mol L-1. CONCLUSION: The pH and salt concentration in raw soymilk markedly influence the freeze-thaw fractionation process. We confirmed that the range of zeta potential values where fractionation was possible remained consistent under various pH and salt concentration conditions. These findings suggest that the zeta potential value might serve as an indicator for evaluating the freeze-thaw fractionation ability of raw soymilk. © 2024 Society of Chemical Industry.

Transport of glycinin, the major soybean allergen, across intestinal epithelial IPEC-J2 cell monolayers.

Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysate transport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-ß-cyclodextrin, significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

Potential protective effects of sodium butyrate on glycinin-induced oxidative stress, inflammatory response, and growth inhibition in Cyprinus carpio.

Investigated mitigating effects of sodium butyrate (SB) on the inflammatory response, oxidative stress, and growth inhibition of common carp (Cyprinus carpio) (2.94 ± 0.2 g) are caused by glycinin. Six isonitrogenous and isoenergetic diets were prepared, in which the basal diet was the control diet and the Gly group diet contained 80 g/kg glycinin, while the remaining 4 diets were supplemented with 0.75, 1.50, 2.25, and 3.00 g/kg SB, respectively. The feeding trial lasted for 8 weeks, and the results indicated that supplementing the diet with 1.50-2.25 g/kg of SB significantly improved feed efficiency and alleviated the growth inhibition induced by glycinin. Hepatopancreas and intestinal protease activities and the content of muscle crude protein were significantly decreased by dietary glycinin, but supplement 1.50-2.25 g/kg SB partially reversed this result. SB (1.50-2.25 g/kg) increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hepatopancreas and reduced the activities of AST and ALT in the serum. Glycinin significantly reduced immune and antioxidant enzyme activities, whereas 1.50-2.25 g/kg SB reversed these adverse effects. Furthermore, compared with the Gly group, supplement 1.50-2.25 g/kg SB eminently up-regulated the TGF-ß and IL-10 mRNA, and down-regulated the IL-1ß, TNF-α, and NF-κB mRNA in hepatopancreas, mid-intestine (MI), and distal intestine (DI). Meanwhile, supplement 1.50-2.25 g/kg SB activated the Keap1-Nrf2-ARE signaling pathway and upregulate CAT, SOD, and HO-1 mRNA expression in hepatopancreas, MI, and DI. Summarily, glycinin induced inflammatory response, and oxidative stress of common carp ultimately decreased the digestive function and growth performance. SB partially mitigated these adverse effects by activating the Keap1-Nrf2-ARE signaling pathway and inhibiting the NF-κB signaling pathway.

The protective role of vitamin C on intestinal damage induced by high-dose glycinin in juvenile Rhynchocypris lagowskii Dybowski.

This study was to evaluate the mitigative effects of vitamin C (VC) on growth inhibition and intestinal damage induced by glycinin in juvenile Rhynchocypris lagowskii Dybowski. 270 healthy juvenile Rhynchocypris lagowskii Dybowski (4.65 ± 0.04 g) were randomly divided into 3 treatments, and fed with control diet, 80 g/kg glycinin diet and 80 g/kg glycinin+200 mg/kg VC diet respectively for 8 weeks. The results showed that glycinin significantly decreased the weight gain rate, specific growth rate, protein efficiency rate, feed efficiency rate and feeding rate of fish compared with the control group (P < 0.05), while VC supplementation improved the growth performance and feed utilization efficiency, and reached a level similar to the control group. Similarly, VC significantly increased the crude protein content of muscle and whole-body, and hepatopancreas and intestinal protease activities of fish fed with glycinin diet (P < 0.05). The distal intestine of fish in glycinin group showed typical damage characteristics, including breakage and atrophy of intestinal mucosal fold, and increased intestinal mucosal permeability. However, fish fed the glycinin + VC diet showed an unimpaired normal intestinal morphology. Usefully, VC supplementation could also restore impaired immune function and antioxidant capacity. VC down-regulated the mRNA levels of pro-inflammatory cytokines TNF-α and IL-1ß, and up-regulated the mRNA levels of anti-inflammatory cytokines IL-10 and TGF-ß in the distal intestine of fish fed with glycinin. Furthermore, glycinin exposure could reduce the mRNA levels of HO-1, CAT and GPx by inhibiting the activation of Nrf2-Keap1 signaling pathway, while VC supplementation reversed this phenomenon and maintained the homeostasis of antioxidant defense system. Concluded, glycinin causes growth inhibition, digestive dysfunction and intestinal damage of Rhynchocypris lagowskii Dybowski, while sufficient VC intake is beneficial for fish to resist the adverse effects of glycinin.

Commensal Bacillus siamensis LF4 ameliorates ß-conglycinin induced inflammation in intestinal epithelial cells of Lateolabrax maculatus.

ß-conglycinin and glycinin, two major heat-stable anti-nutritional factors in soybean meal (SM), have been suggested as the key inducers of intestinal inflammation in aquatic animals. In the present study, a spotted seabass intestinal epithelial cells (IECs) were used to compare the inflammation-inducing effects of ß-conglycinin and glycinin. The results showed that IECs co-cultured with 1.0 mg/mL ß-conglycinin for 12 h or 1.5 mg/mL glycinin for 24 h significantly decreased the cell viability (P < 0.05), and overstimulated inflammation and apoptosis response by significantly down-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and significantly up-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). Subsequently, a ß-conglycinin based inflammation IECs model was established and used for demonstrating whether commensal probiotic B. siamensis LF4 can ameliorate the adverse effects of ß-conglycinin. The results showed ß-conglycinin-induced cell viability damage was completely repaired by treated with 109 cells/mL heat-killed B. siamensis LF4 for ≥12 h. At the same time, IECs co-cultured with 109 cells/mL heat-killed B. siamensis LF4 for 24 h significantly ameliorated ß-conglycinin-induced inflammation and apoptosis by up-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and down-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). In summary, both ß-conglycinin and glycinin can lead to inflammation and apoptosis in spotted seabass IECs, and ß-conglycinin is more effective; commensal B. siamensis LF4 can efficiently ameliorate ß-conglycinin induced inflammation and apoptosis in IECs.

Soybean glycinin and ß-conglycinin damage the intestinal barrier by triggering oxidative stress and inflammatory response in weaned piglets.

PURPOSE: Soybean glycinin (11S) and ß-conglycinin (7S) are major antigenic proteins in soybean and can induce a variety of allergic reactions in the young animals. This study aimed to investigate the effect of 7S and 11S allergens on the intestine of piglets. METHODS: Thirty healthy 21-day-old weaned "Duroc × Long White × Yorkshire" piglets were randomly divided into three groups fed with the basic diet, the 7S supplemented basic diet, or the 11S supplemented basic diet for 1 week. Allergy markers, intestinal permeability, oxidative stress, and inflammatory reactions were detected, and we observed different sections of intestinal tissue. The expressions of genes and proteins related to NOD-like receptor thermal protein domain associated protein 3 (NLRP-3) signaling pathway were detected by IHC, RT-qPCR, and WB. RESULTS: Severe diarrhea and decreased growth rate were observed in the 7S and 11S groups. Typical allergy markers include IgE production and significant elevations of histamine and 5-hydroxytryptamine (5-HT). More aggressive intestinal inflammation and barrier dysfunction were observed in the experimental weaned piglets. In addition, 7S and 11S supplementation increased the levels of 8-hydroxy-2 deoxyguanosine (8-OHdG) and nitrotyrosine, triggering oxidative stress. Furthermore, higher expression levels of NLRP-3 inflammasome ASC, caspase-1, IL-1ß, and IL-18 were observed in the duodenum, jejunum, and ileum. CONCLUSION: We confirmed that 7S and 11S damaged the intestinal barrier of weaned piglets and may be associated with the onset of oxidative stress and inflammatory response. However, the molecular mechanism underlying these reactions deserves further study.

Amino acids induce high seed-specific expression driven by a soybean (Glycine max) glycinin seed storage protein promoter.

KEY MESSAGE: We characterize GFP expression driven by a soybean glycinin promoter in transgenic soybean. We demonstrate specific amino acid-mediated induction of this promoter in developing soybean seeds in vitro. In plants, gene expression is primarily regulated by promoter regions which are located upstream of gene coding sequences. Promoters allow transcription in certain tissues and respond to environmental stimuli as well as other inductive phenomena. In soybean, seed storage proteins (SSPs) accumulate during seed development and account for most of the monetary and nutritional value of this crop. To better study the regulatory functions of a SSP promoter, we developed a cotyledon culture system where media and media addenda were evaluated for their effects on cotyledon development and promoter activity. Stably transformed soybean events containing a glycinin SSP promoter regulating the green fluorescent protein (GFP) were generated. Promoter activity, as visualized by GFP expression, was only observed in developing in planta seeds and in vitro-cultured isolated embryos and cotyledons from developing seeds when specific media addenda were included. Asparagine, proline, and especially glutamine induced glycinin promoter activity in cultured cotyledons from developing seeds. Other amino acids did not induce the glycinin promoter. Here, we report, for the first time, induction of a reintroduced glycinin SSP promoter by specific amino acids in cotyledon tissues during seed development.

Soybean Antigen Protein-Induced Intestinal Barrier Damage by Trigging Endoplasmic Reticulum Stress and Disordering Gut Microbiota in Weaned Piglets.

Endoplasmic reticulum (ER) stress is a crucial factor in the pathogenesis of intestinal diseases. Soybean antigenic proteins (ß-conglycinin and soy glycinin) induce hypersensitivity reactions and intestinal barrier damage. However, whether this damage is associated with ER stress, autophagy, and the gut microbiome is largely unclear. Therefore, in this study, we aimed to investigate the effect of dietary supplementation with soy glycinin (11S glycinin) and ß-conglycinin (7S glycinin) on intestinal ER stress, autophagy, and flora in weaned piglets. Thirty healthy 21-day-old weaned "Duroc × Long White × Yorkshire" piglets were randomly divided into three groups and fed a basic, 7S-supplemented, or 11S-supplemented diet for one week. The results indicated that 7S/11S glycinin disrupted growth performance, damaged intestinal barrier integrity, and impaired goblet cell function in piglets (p < 0.05). Moreover, 7S/11S glycinin induced ER stress and blocked autophagic flux in the jejunum (p < 0.05) and increased the relative abundance of pathogenic flora (p < 0.01) and decreased that of beneficial flora (p < 0.05). In conclusion, 7S/11S glycinin induces intestinal ER stress, autophagic flux blockage, microbiota imbalance, and intestinal barrier damage in piglets.

The preparation of soy glycinin/sugar beet pectin complex network gels catalyzed by laccase under weakly acidic conditions.

BACKGROUND: Traditional soy protein gel products such as tofu, formed from calcium sulfate or magnesium chloride, have poor textural properties and water retention capacity. Soy glycinin (SG) is the main component affecting the gelation of soy protein and can be cross-linked with polysaccharides, such as sugar beet pectin (SBP), and can be modified by changing system factors (e.g., pH) to improve the gel's properties. Soy glycinin/sugar beet pectin (SG/SBP) complex double network gels were prepared under weakly acidic conditions using laccase cross-linking and heat treatment. The structural changes in SG and the properties of complex gels were investigated. RESULTS: Soy glycinin exposed more hydrophobic groups and free sulfhydryl groups at pH 5.0. Under the action of laccase cross-linking, SBP could promote the unfolding of SG tertiary structures. The SG/SBP complex gels contained 46.77% ß-fold content and had good gelling properties in terms of hardness 290.86 g, adhesiveness 26.87, and springiness 96.70 mm at pH 5.0. The T22 relaxation time had the highest peak, and magnetic resonance imaging (MRI) showed that the gel had even water distribution. Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM) indicated that the SG/SBP complex network structure was uniform, and the pore walls were thicker and contained filamentous structures. CONCLUSION: Soy glycinin/ sugar beet pectin complex network gels have good water-holding, rheological, and textural properties at pH 5.0. The properties of soy protein gels can be improved by binding to polysaccharides, with laccase cross-linked, and adjusting the pH of the solution. © 2022 Society of Chemical Industry.

Identification of allergenic epitopes destroyed by two processing technologies of glycinin A2 from soybean.

BACKGROUND: Glycinin is one of the most highly allergenic proteins in soybeans, and G2 is one of the five allergenic subunits of glycinin. Compared with the alkaline chain, the acidic chain A2 of the G2 subunit has strong allergenicity. However, the precise epitopes of A2 and the epitopes destroyed during processing are still unknown. RESULTS: In the present study, preparation of two specific antibodies damaged by processing and phage display techniques were applied to locate the antigenic epitopes of glycinin A2 polypeptide chains disrupted by two processing techniques (thermal processing and ultra-high pressure combined thermal processing). Bioinformatics methods were used to predict the possible epitopes of the A2 chain. The A2 chain and its overlapping segments were introduced into T7 phages and expressed on phage shell by phage display. An indirect enzyme-linked immunosorbent assay was used to screen for antigenic epitopes that had been disrupted by the two processing technologies. The results showed that the dominant antigenic region disrupted by processing was located mainly in the A2-3-B fragment. The reacting experiment with the serum of allergic patients showed that the A2-3-B fragment protein was not only an antigenic region, but also an allergenic region. The two processing technologies destroyed the allergenic epitopes of A2 chain, thereby reducing the allergenicity of protein. The amino acids where the dominant allergenic region disrupted by processing was located were: 233 AIVTVKGGLRVTAPAMRKPQQEEDDDDEEEQPQCVE268 . CONCLUSION: Precise epitopes of the acidic chain A2 in glycinin were identified and epitopes destroyed in two common processing methods were also obtained. The application products of rapid detection of de-allergenicity effect of processed food can be developed according to the location of processed destruction allergenic region, which is of great significance with respect to preventing the occurrence of soybean allergenic diseases. © 2022 Society of Chemical Industry.

Taurine can improve intestinal function and integrity in juvenile Rhynchocypris lagowskii Dybowski fed high-dose glycinin.

The present study evaluated the protective effect and the regulatory mechanism of taurine on growth inhibition and intestinal damage induced by glycinin in juvenile Rhynchocypris lagowskii Dybowski. The control diets had no glycinin and taurine, the glycinin diets contained only 80 g/kg glycinin, and the glycinin + taurine diets contained 80 g/kg glycinin+10 g/kg taurine. Juvenile Rhynchocypris lagowskii Dybowski (4.65 ± 0.03 g/tail) were respectively fed with these 3 diets for 8 weeks. The results showed that glycinin significantly decreased the final body weight, weight gain rate, specific growth rate, protein efficiency rate, feed efficiency rate and feeding rate of fish compared with the control group (P < 0.05). While taurine supplementation improved the growth performance and feed efficiency, but final body weight, weight gain rate, specific growth rate of the glycinin + taurine group were still significantly lower than the control group (P < 0.05). Compared with the glycinin group, taurine supplementation significantly increased whole-body and muscle crude protein content, and hepatopancreas and intestinal protease activities (P < 0.05). Distal intestinal villous dysplasia and mucosal damage, and increased intestinal mucosal permeability were observed in the glycinin group, while taurine supplementation alleviated these adverse effects. Usefully, taurine supplementation could also partially restore the impaired immune function and antioxidant capacity of fish fed glycinin diets. Compared with the glycinin group, taurine supplementation down-regulated pro-inflammatory cytokines TNF-α and IL-1ß mRNA levels, and up-regulated anti-inflammatory cytokines IL-10 and TGF-ß mRNA levels. Furthermore, taurine partially reversed the reduction of antioxidant genes Nrf2、HO-1, CAT and GPx mRNA levels in distal intestine induced by glycinin. Concluded, 80 g/kg glycinin led to intestinal damage, digestive dysfunction and increased intestinal mucosal permeability in juvenile Rhynchocypris lagowskii Dybowski, and these adverse effects were ultimately manifested in growth inhibition. But taurine supplementation could partially mitigate the negative effects induced by glycinin.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

A Multi-Year, Multi-Cultivar Approach to Differential Expression Analysis of High- and Low-Protein Soybean (Glycine max).

Soybean (Glycine max (L.) Merr.) is among the most valuable crops based on its nutritious seed protein and oil. Protein quality, evaluated as the ratio of glycinin (11S) to ß-conglycinin (7S), can play a role in food and feed quality. To help uncover the underlying differences between high and low protein soybean varieties, we performed differential expression analysis on high and low total protein soybean varieties and high and low 11S soybean varieties grown in four locations across Eastern and Western Canada over three years (2018-2020). Simultaneously, ten individual differential expression datasets for high vs. low total protein soybeans and ten individual differential expression datasets for high vs. low 11S soybeans were assessed, for a total of 20 datasets. The top 15 most upregulated and the 15 most downregulated genes were extracted from each differential expression dataset and cross-examination was conducted to create shortlists of the most consistently differentially expressed genes. Shortlisted genes were assessed for gene ontology to gain a global appreciation of the commonly differentially expressed genes. Genes with roles in the lipid metabolic pathway and carbohydrate metabolic pathway were differentially expressed in high total protein and high 11S soybeans in comparison to their low total protein and low 11S counterparts. Expression differences were consistent between East and West locations with the exception of one, Glyma.03G054100. These data are important for uncovering the genes and biological pathways responsible for the difference in seed protein between high and low total protein or 11S cultivars.

Interactions between Mannosylerythritol Lipid-A and Heat-Induced Soy Glycinin Aggregates: Physical and Chemical Characteristics, Functional Properties, and Structural Effects.

Protein-surfactant interactions have a significant influence on food functionality, which has attracted increasing attention. Herein, the effect of glycolipid mannosylerythritol lipid-A (MEL-A) on the heat-induced soy glycinin (11S) aggregates was investigated by measuring the structure, binding properties, interfacial behaviors, and emulsification characteristics of the aggregates. The results showed that MEL-A led to a decrease in the surface tension, viscoelasticity, and foaming ability of the 11S aggregates. In addition, MEL-A with a concentration above critical micelle concentration (CMC) reduced the random aggregation of 11S protein after heat treatment, thus facilitating the formation of self-assembling core-shell particles composed of a core of 11S aggregates covered by MEL-A shells. Infrared spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry also confirmed that the interaction forces between MEL-A and 11S were driven by hydrophobic interactions between the exposed hydrophobic groups of the protein and the fatty acid chains or acetyl groups of MEL-A, as well as the hydrogen bonding between mannosyl-D-erythritol groups of MEL-A and amino acids of 11S. The findings of this study indicated that such molecular interactions are responsible for the change in surface behavior and the enhancement of foaming stability and emulsifying property of 11S aggregates upon heat treatment.

Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

The cell structure damage and embden-meyerhof-parnas pathway inhibition of Listeria monocytogenes induced by glycinin basic peptide.

Glycinin basic peptide (GBP) is a natural antibacterial peptide. This study aimed to explore the antibacterial characteristics of GBP against Listeria monocytogenes (L. monocytogenes) by measuring the membrane potential, membrane permeability, cell damage, morphological changes, respiration metabolism inhibition and DNA content. GBP increased the surface zeta potential and decreased the trans-membrane potential of L. monocytogenes in a dose-dependent manner. Compared with the control, the electrical conductivities of GBP-treated bacterial suspensions were significantly increased. The percentages of bacteria with damaged membrane increased from 6.40% to 70.90% with GBP from 0 to 0.8 mg/mL. Obvious rupture and deform of bacterial cells with GBP were observed by transmission electron microscope (TEM), showing the destructive effect of GBP on L. monocytogenes. GBP also inhibited the embden-meyerhof-parnas pathway of the bacterial respiration metabolism and reduced the activities of its key regulator enzymes. Besides, the content of DNA in GBP-treated L. monocytogenes was lower than that in control.

Soybean glycinin impaired immune function and caused inflammation associated with PKC-ζ/NF-κb and mTORC1 signaling in the intestine of juvenile grass carp (Ctenopharyngodon idella).

Glycinin is a major protein and antinutritional factor of soybean. However, how dietary glycinin affect intestinal immune function of fish were largely unknown. In this study, we used juvenile grass carp as a model to investigate the impacts of glycinin on intestinal immune function of fish and involved mechanisms. We set three treatments including control, glycinin and glycinin + glutamine in this trial. For immune components, results revealed that compared with control group, glycinin group had lower acid phosphatase activities in the foregut, midgut and hindgut, lower C3 and C4 content, and lower mRNA abundances of IgM, IgZ, hepcidin, LEAP-2A, LEAP-2B and ß-defensin-1 in the midgut and hindgut rather than foregut of grass carp. For pro-inflammatory cytokines and relevant signaling, glycinin elevated mRNA abundances of IL-1ß, IL-8, IL-12p35, IL-12p40 and IL-17D in the midgut and IL-1ß, IFN-γ2, IL-6, IL-8, IL-12p35, IL-12p40 and IL-17D in the hindgut, and increased protein abundances of PKC-ζ and nuclear NF-κB p65 in the midgut and hindgut in comparison to control. For anti-inflammatory cytokines and relevant signaling, glycinin reduced mRNA abundances of TGF-ß1, TGF-ß2, IL-4/13B (rather than IL-4/13A), IL-10 and IL-11 in the midgut and hindgut, and reduced p-mTOR (Ser 2448), p-S6K1 (Thr 389) and p-4EBP1 (Thr 37/46) protein abundances in the midgut and hindgut rather than foregut. Co-administration of glutamine with glycinin could partially enhance intestinal function and reduce intestinal inflammation compared with glycinin treatment. Concluded, glycinin decreased intestinal immune components and caused intestinal inflammation associated with PKC-ζ/NF-κB and mTORC1 signaling.

Sodium butyrate can improve intestinal integrity and immunity in juvenile Chinese mitten crab (Eriocheir sinensis) fed glycinin.

Butyrate is a fermentation byproduct of gut microbiota and is susceptible to chronic oxidative stress. This study investigates the mitigative effects of sodium butyrate (SBT) on growth inhibition and intestinal damage induced by glycinin in juvenile Chinese mitten crab (Eriocheir sinensis). All four experimental diets containing 80 g/kg glycinin were formulated with 0, 10, 20 and 40 g/kg SBT respectively. There was no glycinin or SBT in the control diet. Juvenile crabs (0.33 ± 0.01g) were respectively fed with these five diets for eight weeks. The diets with 10 and 20 g/kg SBT significantly improved the survival and weight gain of the crabs compared with those in the 0 g/kg SBT group, and showed no difference with the control group. The crabs fed diets containing glycinin without SBT had lower glutathione and glutathione peroxidase activities but higher malondialdehyde in the intestine than those in the control group. Moreover, dietary glycinin decreased the lysozyme and phenoloxidase activities and improved the level of histamine in the intestine compared with the control group, while the supplementation of SBT counteracted these negative effects. The addition of SBT could also restore the impaired immunity and morphological structure of the intestine. Dietary SBT could increase the mRNA expression of antimicrobial peptides genes (anti-lipopolysaccharide factor 1 and 2) and decrease the content of pro-inflammatory factor TNF-α. The SBT could restore the intestinal microbial community disorganized by glycinin. The abundance of pathogenic bacteria (Aeromonas, Vibrio and Pseudomonas) decreased significantly and the potential probiotic bacteria (Bacillus, Lactobacillus, Chitinibacter and Dysgonomonas) increased significantly in the 10 g/kg SBT group. This study suggests that sodium butyrate supplementation can mitigate the negative effects induced by glycinin such as growth inhibition, intestinal inflammation and reduction of beneficial flora in the gut.

Ultrasound-Assisted Mild Heating Treatment Improves the Emulsifying Properties of 11S Globulins.

Ultrasonic technology is often used to modify proteins. Here, we investigated the effects of ultrasound alone or in combination with other heating methods on emulsifying properties and structure of glycinin (11S globulin). Structural alterations were assessed with Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), intrinsic fluorescence spectroscopy, ultraviolet (UV) absorption spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The size distribution and zeta-potential of 11S globulin were evaluated with a particle size analyzer. An SDS-PAGE analysis showed no remarkable changes in the primary structure of 11S globulin. Ultrasound treatment disrupted the 11S globulin aggregates into small particles with uniform size, narrowed their distribution and increased their surface charge density. Fluorescent spectroscopy and second-derivative UV spectroscopy revealed that ultrasound coupled with heating induced partial unfolding of 11S globulin, increasing its flexibility and hydrophobicity. FTIR further showed that the random coil and α-helix contents were higher while ß-turn and ß-sheet contents were lower in ultrasound combined with heating group compared to the control group. Consequently, the oil-water interface entirely distributed protein and reduced the surface tension. Moreover, ultrasound combined with heating at 60 °C increased the emulsifying activity index and emulsifying stability index of 11S globulins by 6.49-folds and 2.90-folds, respectively. These findings suggest that ultrasound combined with mild heating modifies the emulsification properties of 11S globulin.

Changes to the core and flanking sequences of G-box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters.

Cis-regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G-box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6-bp G-box provided information on the base level of GFP expression while modifications to the 2-4 flanking bases on either side of the G-box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2-bp sequences on either flank of the G-box significantly affected G-box activity, while the distal 2-bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2- to 4-bp flanking sequence of a G-box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G-box variant was first converted into a classical G-box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.