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.

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.

Condensation of Soy Protein Peptides Contributes to Sequester Bile Acids and Mitigate LPS-Induced Inflammation.

Soy protein is widely known to have serum triglyceride (TG) and cholesterol-lowering effects associated with a reduced risk of cardiovascular disease. Recent studies highlighted that the extension region (ER) domain of soy 7S globulin (ß-conglycinin) is a key component responsible for the serum TG-lowering effect via modulation of bile acid (BA) homeostasis. Here, we studied the sequestration of BAs by ER peptides during intestinal digestion in vitro and assessed the anti-inflammatory effects of ER peptides using Caco-2/HT29-MTX/RAW264.7 triple-cell cocultures as an intestine cell model. Results show that ER peptides, which share characteristics of intrinsically disordered regions (IDRs), are capable of forming peptide condensates and exhibit the capability to sequester BA-containing colloidal structures during intestinal digestion in vitro. Moreover, BAs enhance the penetration of peptide condensates within the mucus layer, enabling ER peptides to mitigate lipopolysaccharide (LPS)-induced gut inflammation. These results provide a possible explanation for the molecular mechanisms underlying the modulation of BA homeostasis by soybean proteins.

Extension Region Domain of Soybean 7S Globulin Contributes to Serum Triglyceride-Lowering Effect via Modulation of Bile Acids Homeostasis.

SCOPE: Soybean 7S globulin (ß-conglycinin), a major soybean storage protein, has been demonstrated to exert remarkable triglyceride (TG) and cholesterol-lowering effects, yet the underlying mechanism remains controversial. METHODS AND RESULTS: A comparative investigation is performed to assess the contribution of different structural domains of soybean 7S globulin, including core region (CR) and extension region (ER) domains, to biological effects of soybean 7S globulin using a high-fat diet rat model. The results show that ER domain mainly contributes to the serum TG-lowering effect of soybean 7S globulin, but not for CR domain. Metabolomics analysis reveals that oral administration of ER peptides obviously influences the metabolic profiling of serum bile acids (BAs), as well as significantly increased the fecal excretion of total BAs. Meanwhile, ER peptides supplementation reshapes the composition of gut microbiota and impacts the gut microbiota-dependent biotransformation of BAs which indicate by a significantly increased secondary BAs concentration in fecal samples. These results highlight that TG-lowering effects of ER peptides mainly stem from their modulation of BAs homeostasis. CONCLUSION: Oral administration of ER peptides can effectively lower serum TG level by regulating BAs metabolism. ER peptides have potential to be used as a candidate pharmaceutical for the intervention of dyslipidemia.

Bile Acid Profile Influences Digestion Resistance and Antigenicity of Soybean 7S Protein.

Soybean 7S storage protein (ß-conglycinin) is the most important allergen, exhibits resistance in gastrointestinal (GI) digestion, and causes allergies in humans and animals. A previous study has demonstrated that 7S proteins contained innate amyloid aggregates, but the fate of these specific protein aggregates in intestinal digestion and correlation to allergenicity are unclear. In this study, via a modified INFOGEST static in vitro digestion and IgE binding test, we illustrate that the survived amyloid aggregates of soybean 7S protein in GI digestion might be dominant IgE epitopes of soybean protein in humans. The impact of conjugated primary bile acid salt (BS) profile on digestion resistance and immunogenicity of soybean protein is assessed, regarding the binding affinity of BS to protein aggregates with consideration of the BS composition and the physiologically relevant colloidal structure. The results show that chenodeoxycholate-containing colloidal structures exhibit high affinity and unfolding capacity to protein amyloid aggregates, promoting proteolysis by pancreatic enzymes and thus mitigating the antigenicity of soybean protein. This study presents a novel understanding of bile acid profile and colloidal structure influence on the digestibility and antigenicity of dietary proteins. It should be helpful to design in vitro digestion protocol and accurately replicate physiologically relevant digestion conditions.

Digestion Resistance of Soybean 7S Protein and Its Implications for Reinforcing the Gastric Mucus Barrier.

Previous studies have found that soybean protein, especially soybean 7S protein (ß-conglycinin), exhibits digestion resistance, but the mechanism of digestion resistance and its implications for human health are still unclear. Here, we show that the extracted soybean 7S protein contains both oligomer globulins and amyloid aggregates, while the gastric digested soybean 7S protein only contains amyloid aggregates and thus exhibits digestion resistance. An animal experiment shows that un-digestible soybean 7S protein effectively prevents aspirin-induced acute gastric mucosa damage. The impacts of un-digestible soybean 7S protein on gastric mucus barrier properties are investigated using quartz crystal microbalance with dissipation (QCM-D), Langmuir monolayer, and multiple particle tracking (MPT). Results show that these un-digestible protein aggregates can penetrate into gastric mucus, increase the viscosity and compactness of the mucin layer, and reinforce the gastric mucus barrier properties. The findings are helpful to understand that high consumption of non-fermented soybean foods is associated with a decreased risk of gastric cancer.

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.

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.