Heat/non-heat treatment alleviates ß-conglycinin-triggered food allergy reactions by modulating the Th1/Th2 immune balance in a BALB/c mouse model.

BACKGROUND: With the increasing popularity of plant protein-based diets, soy proteins are favored as the most important source of plant protein worldwide. However, potential food allergy risks limit their use in the food industry. This work aims to reveal the mechanism of ß-conglycinin-induced food allergy, and to explore the regulatory mechanism of heat treatment and high hydrostatic pressure (HHP) treatment in a BALB/c mouse model. RESULTS: Our results showed that oral administration of ß-conglycinin induced severe allergic symptoms in BALB/c mice, but these symptoms were effectively alleviated through heat treatment and HHP treatment. Moreover, ß-conglycinin stimulated lymphocyte proliferation and differentiation; a large number of cytokines interleukin (IL)-4, IL-5, IL-10, IL-12 and IL-13 were released and interferon γ secretion was inhibited, which disrupted the Th1/Th2 immune balance and promoted the differentiation and proliferation of naive T cells into Th2-type cells. CONCLUSION: Heat/non-heat treatment altered the conformation of soybean protein, which significantly reduced allergic reactions in mice. This regulatory mechanism may be associated with Th1/Th2 immune balance. Our results provide data support for understanding the changes in allergenicity of soybean protein within the food industry. © 2024 Society of Chemical Industry.

Identification of calcium chelating peptides from peanut protein hydrolysate and absorption activity of peptide-calcium complex.

BACKGROUND: Peanut peptides have good chelating ability with metal ions. However, there are few studies on the chelation mechanism of peanut peptides with calcium and absorption properties of peptide-calcium complex. RESULTS: Peptides with high calcium chelating rate were isolated and purified from peanut protein hydrolysate (PPH), and the chelation rate of component F21 was higher (81.4 ± 0.8%). Six peptides were identified from component F21 by liquid chromatography-tandem mass spectrometry, and the frequency of acidic amino acids and arginine in the amino acid sequence was higher in all six peptides. Peanut peptide-calcium complex (PPH21-Ca) was prepared by selecting component F21 (PPH21). Ultraviolet analysis indicated that the chelate reaction occurred between peanut peptide and calcium ions. Fourier transform infrared analysis showed that the chelating sites were carboxyl and amino groups on the amino acid residues of peptides. Scanning electron microscopy revealed that the surface of peanut peptide had a smooth block structure, but the surface of the complex had a granular morphology. Caco-2 cell model tests revealed that the bioavailability of PPH21-Ca was 58.4 ± 0.5%, which was significantly higher than that of inorganic calcium at 37.0 ± 0.4%. CONCLUSION: Peanut peptides can chelate calcium ions by carboxyl and amino groups, and the peptide-calcium complex had higher bioavailability. This study provides a theoretical basis for the development of new calcium supplement products that are absorbed easily. © 2024 Society of Chemical Industry.

Identification and molecular interactions of novel ACE inhibitory peptides from rapeseed protein.

Plant-derived bioactive peptides have drawn much attention because of their physiological functions. This study aimed to evaluate bioactive peptides in rapeseed protein and identify novel angiotensin Ⅰ-converting enzyme (ACE) inhibitory peptides using bioinformatics methods. A total of 24 kinds of bioactive peptides were encrypted in the 12 selected rapeseed proteins by analysis in BIOPEP-UWM, with higher occurrence frequency of dipeptidyl peptidase Ⅳ (DPP-Ⅳ) inhibitory peptides (0.5727-0.7487) and ACE inhibitory peptides (0.3500-0.5364). Novel ACE inhibitory peptides FQW, FRW and CPF were identified by in silico proteolysis, and they had strong inhibitory effects on ACE in vitro, showing IC50 values of 44.84 ± 1.48 µM, 46.30 ± 1.39 µM and 131.35 ± 3.87 µM, respectively. Molecular docking results displayed that these three peptides were able to interact with ACE active site via hydrogen bonds and hydrophobic interactions, and coordinate with Zn2+. It suggested that rapeseed protein could be a good source for the production of ACE inhibitory peptides.

High hydrostatic pressure treatment reduces the potential antigenicity of ß-conglycinin by changing the protein structure during in vitro digestion.

BACKGROUND: High hydrostatic pressure (HHP) treatment has been used to alleviate the allergenicity of soybeans, but there are little data about the potential antigenicity of ß-conglycinin after HHP treatment. RESULTS: We examined the effects of HHP treatment on the antigenicity and structure of ß-conglycinin. When the pressure was 300 and 400 MPa, HHP treatment reduced the immunoglobulin (Ig)G binding capacity of ß-conglycinin, while its IgE binding capacity did not change significantly. After in vitro digestion, both the IgE and IgG binding of ß-conglycinin was obviously inhibited after HHP treatment at 400 MPa and 60 °C, although its binding capacity with linear epitope antibodies increased. Moreover, HHP treatment changed the secondary structure of ß-conglycinin, the content of α-helix and random coils increased, while the ß-sheet and ß-turn decreased. After HHP treatment, the conformational structure was unfolded so that a large number of hydrophobic regions were exposed. CONCLUSION: HHP treatment alleviated the potential antigenicity of ß-conglycinin by modifying its structure, which facilitated in vitro digestion and destroyed epitopes. This research provides a new insight into the mechanism of HHP treatment that affects the sensitization of soy protein allergens. © 2022 Society of Chemical Industry.

Effects of heat treatment on the antigenicity, antigen epitopes, and structural properties of ß-conglycinin.

In this study, the effects of heat treatment on antigenicity, antigen epitopes, and structural changes in ß-conglycinin were investigated. Results showed that the IgG (Immunoglobulin G) binding capacity of heated protein was inhibited with increased temperature, although IgE (Immunoglobulin E) binding capacity increased. Linear antigen epitopes generally remained intact during heat treatment. After heat treatment, ß-conglycinin was more easily hydrolyzed by digestive enzymes, and a large number of linear epitopes was destroyed. In addition, heat denaturation of ß-conglycinin led to the formation of protein aggregates and reduction of disulfide bonds. The contents of random coils and ß-sheet of heated ß-conglycinin decreased, but the contents of ß-turn and α-helix increased. Moreover, the protein structure of heated ß-conglycinin unfolded, more hydrophobic regions were exposed, and the tertiary structure of ß-conglycinin was destroyed. Heat treatment affected the antigenicity and potential sensitization of ß-conglycinin by changing its structure.

Distribution and degradation of DNA from non-genetically and genetically modified soybean (Roundup Ready): Impact of soybean protein concentrate and soybean protein isolate preparation.

To improve genetically modified product labelling legislation and promote the development of genetically modified foods, the mass variations of genomic DNA and length distributions of DNA fragments in non-genetically and genetically modified soybean (Roundup Ready) and the variations in transgenic contents during soybean protein concentrate (SPC) and soybean protein isolate (SPI) preparation were monitored. The material masses throughout the process conformed to the law of mass conservation, and amounts of DNA recovered decreased with SPC and SPI preparation. The successive steps of ethanol extraction led to a decrease in the size distribution of the recovered DNA. For the LECTIN and CP4 EPSPS targets investigated, longer fragments (>800 bp) were more affected than smaller fragments (<200 bp), and both targets degraded slowly upon degradation into small fragments. DNA distribution and degradation thereby affected GMO quantification. After preparation procedures, the transgenic contents of SPC and SPI products were higher than that of raw soybean.

Identification of the linear immunodominant epitopes in the ß subunit of ß-conglycinin and preparation of epitope antibodies.

ß-conglycinin is one of the major allergens in soybean protein. The purpose of this study was to predict and to identify the major linear epitopes of the ß subunit of ß-conglycinin. Potential linear epitopes were predicted and confirmed by three immunoinformatics tools combined with the Immune Epitope Database (IEDB). Ten potential epitope peptides were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis and were validated by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using sera from soybean allergic patients. Polyclonal antibodies, which were prepared by immunizing rabbits with synthesized peptides, were used to confirm their binding ability with ß-conglycinin through western blot and dot blot assays. The results showed that 10 peptides were screened as the main epitopes for the ß subunit of ß-conglycinin. All 10 peptides (P1-P10) presented IgG binding activity, and P2 and P6 were also validated as IgE binding peptides. Moreover, the results of dot blot showed that P5 and P8 might be located inside the protein molecule. Western blot indicated that most of polyclonal antibodies were bound effectively to the ß subunit of ß-conglycinin. In addition, few polyclonal antibodies exhibited an immune cross-reaction with the α and α' subunits.

The separation and identification of the residual antigenic fragments in soy protein hydrolysates.

Soybean is one of the major food allergens. In this study, soy protein isolate was hydrolyzed by Neutrase and Flavourzyme. The hydrolysates were separated by ultrafiltration and ion-exchange chromatography. The antigenicity of proteins was determined by indirect competitive ELISA. The molecular weight distribution was characterized by SDS-PAGE. The amino acid sequence of chromatography fractions was analyzed by LC-MS. The results showed that proteins with >50 kDa in hydrolysates had the highest antigenicity and were further separated into F1 -F5 fragments by ion-exchange chromatography. Fragment F4 , which was the most antigenic, was analyzed by LC-MS. The results of mass spectrometry showed that most of the peptides that contained antigen epitopes in chromatography fraction F4 belonged to glycinin subunits. The antigenicity of soy protein was reduced by enzymatic hydrolysis, but glycinin showed resistance to enzymatic hydrolysis. PRACTICAL APPLICATIONS: The identification of residual antigenicity in soy protein hydrolysates by LC-MS provides important information on the resistance mechanism of enzymatic hydrolysis of soybean protein allergens. In addition, the efficient separation of soy protein hydrolysates could be beneficial for developing low-allergenic soybean products.

A modified SDS-based DNA extraction method from raw soybean.

Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020-2444 ng DNA/mg soybean with A260/280 ratios of 1.862-1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

The structural properties and antigenicity of soybean glycinin by glycation with xylose.

BACKGROUND: Soybean glycinin is considered a major allergenic protein, and glycation is widely used to reduce the allergenic potential of present allergens. Glycation of soybean glycinin with xylose at 55 °C for different lengths of time was investigated. The extent of Maillard reaction was reflected through the content changing of free amino groups, color analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Alteration in the structural properties of glycinin was characterized by Fourier transform infrared spectroscopy, and antigenicity was evaluated by indirect competitive enzyme-linked immunosorbent assay. RESULTS: The changes in the color of glycinin-xylose samples and the reduction of free amino group content in proteins indicated that the Maillard reaction occurred. The degree of glycation increased in glycated samples with the increase in reaction time. Glycation induced the changes in the secondary structure of glycinin and the ordered structure of proteins increased during the glycation reaction. The antigenicity of glycinin was reduced with the increase in reaction time. After glycation for 12 h, the antigenicity of glycinin declined about 18% compared with native glycinin. CONCLUSION: The application of glycation may be an efficient method to reduce the antigenicity of soybean glycinin. © 2016 Society of Chemical Industry.

The influence of glycosylation on the antigenicity, allergenicity, and structural properties of 11S-lactose conjugates.

Soybean is nutritious and is an excellent source of high-quality protein for human food and animal feed. However, glycinin (11S) is considered as the major allergenic protein that causes soybean allergies. Glycosylation is widely used to remove food protein allergens. In this study, soybean 11S was isolated and used in a glycation reaction with lactose at a weight ratio of 4:1 at 55°C and 79% relative humidity for different periods of time. The effects of glycosylation on the antigenicity and residual allergenicity of 11S were investigated, using the specific IgG polyclonal antibodies for glycinin and soy-allergic patient sera by indirect competitive, enzyme-linked immunosorbent assay (indirect competitive ELISA). Meanwhile, the degree of glycation was determined by the trinitrobenzene sulfonic acid (TNBS) method. The structural properties of 11S-lactose conjugates were characterized by SDS-PAGE, Fourier transform infrared spectrum (FTIR), and ultraviolet spectroscopy. Glycosylation effectively decreased the antigenicity and allergenicity of 11S if we increased the reaction time. The antigenicity of 11S after glycosylation was reduced by approximately 30% compared with raw 11S, while allergenicity of 11S was reduced by 9%. The changes in secondary structures of glycated 11S may have influenced the allergic epitopes of protein. Therefore, we suggest that introducing lactose in 11S is an effective method to remove the antigenicity and allergenicity of glycinin.

Preparation of organic tofu using organic compatible magnesium chloride incorporated with polysaccharide coagulants.

Organic tofu using organic compatible coagulants of magnesium chloride and three polysaccharides including carrageenan, guar gum and gum Arabic were generated. For MgCl2 coagulated tofu, carrageenan significantly increased the hardness from 969.5 g to 1210.5 g whereas guar gum (0.6g) decreased the hardness to 505.5 g. Interestingly, gypsum and guar gum (0.6g) increased the yield of tofu significantly. These organic compatible coagulants didn't affect most of 7S and 11S protein subunits. Importantly, the overall-acceptability of organic tofu prepared with MgCl2 combined with guar gum or gypsum was almost the same as conventional tofu made with gymsum while having more beany-flavour. Among these organic coagulants, tofu made from 0.6g guar gum and MgCl2 mixture was the most similar to that coagulated by conventional gypsum. Thus this mixture is promising as coagulant for making organic tofu.

Effects of Maillard reaction conditions on the functional properties of WPI chitosan oligosaccharide conjugates.

Whey proteins are widely used as food material, but their functional properties are affected by processing conditions, in which Maillard reaction commonly occurs. The objective of this study was to investigate the effects of reaction conditions (saccharide rate (SR), setting time and setting temperature) on the functional properties of conjugates of whey protein isolates (WPI) - chitosan oligosaccharide (COS), which were produced by Maillard reaction. Response surface methodology (RSM) was used in this study, models for solubility, heat stability (HS), emulsifying activity (EA) and emulsifying stability (ES) of WPI-COS were established. Results indicated that the solubility of WPI-COS was 98.8 % at 29.8 % SR, 17.4 h, 46.7 °C; the heat stability of WPI-COS was 92.95 % at 27.2 % SR, 18.1 h, 48.7 °C; the EA of WPI-COS was 1.87 at 25.3 % SR, 20.0 h, 46.1 °C; the ES of WPI-COS was 13.28 min at 32.5 % SR, 12.0 h, 35.0 °C and setting temperature was the major factor affecting EA and ES while SR was the major factor affecting solubility and heat stability. Conditions were optimized for the preparation of WPI-COS to obtain improved functional properties.

Milk processing as a tool to reduce cow's milk allergenicity: a mini-review.

Milk processing technologies for the control of cow's milk protein allergens are reviewed in this paper. Cow's milk is a high nutritious food; however, it is also one of the most common food allergens. The major allergens from cow's milk have been found to be ß-lactoglobulin, α-lactalbumin and caseins. Strategies for destroying or modifying these allergens to eliminate milk allergy are being sought by scientists all over the world. In this paper, the main processing technologies used to prevent and eliminate cow's milk allergy are presented and discussed, including heat treatment, glycation reaction, high pressure, enzymatic hydrolysis and lactic acid fermentation. Additionally, how regulating and optimizing the processing conditions can help reduce cow's milk protein allergenicity is being investigated. These strategies should provide valuable support for the development of hypoallergenic milk products in the future.

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins.

BACKGROUND: The main whey proteins alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of alpha-LA and beta-LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS-PAGE electrophoresis. RESULTS: Fermentation by lactic acid bacteria could significantly reduce the antigenicity of alpha-LA and beta-LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, alpha-LA and beta-LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS-PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. CONCLUSION: The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity.