Jinyan Gao

High‐pressure microfluidisation‐induced changes in the antigenicity and conformation of allergen Ara h 2 purified from Chinese peanut

BACKGROUND Peanut allergy is one of the most serious food allergies, and Ara h 2 is one of the most important peanut allergens as it is recognised by serum immunoglobulin E from more than 90% of peanut-allergic individuals. Dynamic high-pressure microfluidisation has been widely used in food processing as a new technology. The aim of this study was to investigate the effect of high-pressure microfluidisation on the antigenicity and structure of Ara h 2. Extracted peanut allergen Ara h 2 was treated under a continuous pressure array of 60, 90, 120, 150 and 180 MPa. Immunoreactivity was measured by indirect enzyme-linked immunosorbent assay with rabbit polyclonal antibodies. Secondary structure was analysed by circular dichroism. Surface hydrophobicity and sulfhydryl groups were assessed via fluorescence and UV absorption spectra respectively. RESULTS High-pressure microfluidisation treatment decreased the antigenicity of peanut allergen Ara h 2, changed its secondary structure and increased its UV absorption intensity and surface hydrophobicity. CONCLUSION The change in conformation contributed to the decrease in antigenicity of Ara h 2, and the spatial conformation of peanut allergen Ara h 2 plays a critical role in its antigenicity.

The tip of the "celiac iceberg" in China: a systematic review and meta-analysis.

Objective Until recently, celiac disease was considered to be rare in China. We aimed to estimate its true status. Methods By searching the MEDLINE database and four Chinese full-text databases (CNKI, CBM, VIP and WANFANG) (up to August 2012), as well as two HLA allele frequency net databases and the Chinese Statistics Yearbook databases, we systematically reviewed the literature on definite and suspected cases of celiac disease, the predisposing HLA allele frequencies, and on gluten exposure in China. Meta-analysis was performed by analyzing DQ2, DQ8 and DQB1*0201 gene frequencies and heterogeneity in populations from different geographic regions and ethnicities in China. Results At present, the number of reported celiac disease cases is extremely low in China. The frequencies of the HLA-DQ2.5 and HLA-DQ8 haplotypes were 3.4% (95% confidence interval 1.3–5.5%) and 2.1% (0.1–4.1%), respectively. HLA-DQ2 and HLA-DQ8 antigen frequencies were 18.4% (15.0–21.7%) and 8.0% (4.5–11.4%), respectively. The frequency of the DQB1*0201 allele was 10.5% (9.3–11.6%) and it was more common in the northern Chinese than in the southern Chinese populations. The chance of being exposed to gluten is rapidly increasing all over China nowadays. Conclusion The data on HLA haplotyping, in conjunction with increasing wheat consumption, strongly suggests that the occurrence of celiac disease is more common in China than currently reported. Coordinated measures by the Chinese government, medical and agricultural research institutions, and food industries, would be justified to create more awareness about celiac disease and to prevent it becoming a medical and societal burden.

A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation

Peanut allergen Ara h 6 was isolated and irradiated at 1, 3, 5, or 10 kGy, and a whole peanut protein extract (WPPE) was also treated by irradiation. Alteration in structure of Ara h 6 was characterised by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and SDS-PAGE, and antigenicity was evaluated by immunoblotting and indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation induced significant changes in the secondary and tertiary structures of Ara h 6, and the antigenicity of both purified Ara h 6 and WPPE were reduced upon increasing the irradiation doses. Moreover, a good correlation between the loss in α-helix and IgG binding to Ara h 6 was observed. This indicated that irradiation might be an efficient approach to reduce or eliminate peanut allergenicity.

Conformation affects the potential allergenicity of ovalbumin after heating and glycation

Ovalbumin (OVA), one of the major allergens in hen egg white, and has widespread use in experimental models of allergy. The aim of this research was to assess the effect of glycation and heat treatment on the potential allergenicity of OVA prepared from hen egg white. Secondary and tertiary structures of OVA were also characterised to show the relationship between potential allergenicity and the conformation of OVA after heating and glycation. Glycation significantly reduced the potential allergenicity of OVA tested with egg allergy patients’ sera, which was caused by conformation changes. An increased IgG reactivity was measured using rabbit anti-OVA and was supposed to be caused by protein unfolding which exposed hidden epitopes. Heating reduced the potential allergenicity of OVA at the expense of increased IgG reactivity. It is suggested that conformational changes of OVA induced by glycation and controlled heating significantly reduced its potential allergenicity.

Combined effect of glycation and sodium carbonate-bicarbonate buffer concentration on IgG binding, IgE binding and conformation of ovalbumin.

BACKGROUND Ovalbumin (OVA) is a major allergen in hen egg. During thermal processing, reducing sugars contained in the hen egg white might easily undergo glycation with OVA, but few studies have been conducted on its corresponding immunoreactivity changes. The aim of the present study was to assess changes of the antigenicity, potential allergenicity and conformation of OVA after glycation in a wet-thermal processing system under different concentrations of sodium carbonate-bicarbonate buffer. RESULTS IgE binding of the glycated OVA was increased after glycation, and the higher the sodium carbonate-bicarbonate buffer concentration, the higher the IgE binding capacity. The increase in IgE binding of OVA corresponded well with the disruption of the disulfide bond, which exposed the epitopes initially buried. Antigenicity of the glycated OVA was increased, and the amount of the increase varied among samples treated under different buffer concentrations. CONCLUSION Glycation increased the allergenic potential for OVA, with the amount of increase varying with different sodium carbonate-bicarbonate buffer concentrations.

Identification and characterization of the antigenic site (epitope) on bovine β-lactoglobulin: common residues in linear and conformational epitopes

BACKGROUND β-Lactoglobulin is recognised as one of major allergens in milk and its epitopes include linear and conformational epitopes contributed to milk allergy. RESULTS In our work, two types of epitopes have been identified. Linear epitopes identified by using SPOT™ peptide arrays approach and three common peptide sequences AA77-82 (KIPAVF), AA126-131 (PEVDNE) and AA142-147 (ALPMHI) were obtained by reacting with specific sera from two rabbits. At the same time, mimotopes were screened by the panning of a phage display peptide library and the corresponding conformational epitopes were calculated by the web tool of Peptiope server with Mapitope algorithm. Three conformational epitopes against two specific sera were identified, in which there were 15 common residues as well and located in the different position and appeared mainly as an α-helix. CONCLUSION Common residues on the linear and conformational epitopes were identified in the first time, respectively, which could be regarded as informative epitopes for detection of allergen in dairy products.

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The Km and Vmax values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min−1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.

Allergenicity characteristics of germinated soybean proteins in a BALB/c mouse model.

Soybean proteins are widely used in many food products. However they also commonly cause food allergy. This study aimed to characterize the allergenicity of germinated soybean proteins in a BALB/c mouse model. Mice were orally sensitized with germinated soybean proteins or soybean proteins using cholera toxin as adjuvant. Anaphylactic shock reactions as well as changes in body temperature, specific antibody levels, mast cell protease-1 (mMCP-1) concentrations, morphological structure of duodenum, and cytokines were determined after the mice were challenged with germinated soybean proteins or soybean proteins. In contrast to soybean proteins, oral sensitization to germinated soybean proteins did not result in anaphylactic shock symptoms or decreased body temperature in mice. However, a minor damage of the intestinal villus existed after the challenge. A tendency toward decreased allergen-specific IgE, IgG and IgG1 levels, and mMCP-1 concentration was observed, accompanied by a repression of IL-4, IL-5, IL-13 and IFN-γ production in spleen cell cultures. Results indicate that germinated soybean proteins did not provoke remarkable allergic reactions compared to soybean proteins. Germinated soybean proteins have the potential to be a safe dietary formula for humans at risk of soybean allergy. However, additional studies on the underlying mechanisms and clinical trials are necessary.

Characterization of Physicochemical Properties and IgE-Binding of Soybean Proteins Derived from the HHP-Treated Seeds

The aim of this work was to evaluate the characterization of physicochemical properties and IgE-binding of soybean proteins derived from the high hydrostatic pressure (HHP) treated seeds. Soybean seeds were treated by HHP at different pressures, and changes in the physicochemical properties of soybean proteins were characterized by proteins solubility, free sulfhydryl (SH) content, surface hydrophobicity, and secondary structures. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunoabsorbent assay (ELISA) were used to define the proteins patterns and IgE-binding ability. The results showed that HHP treatment in the ranges of 0 to 500 MPa led to a slight but gradual decline in free SH content. The solubility and hydrophobicity of soybean proteins increased sharply from 100 to 200 MPa, and gradually decreased upon the further increase of pressure. The α-helix and β-sheets contents of soybean proteins decreased, while the random coil content increased. The SDS-PAGE showed that HHP treatment of 100 to 200 MPa could dissociate the proteins, breaking the aggregates into smaller units, while the treatment ranging from 300 to 500 MPa could induce the proteins aggregation into larger units. Moreover, the ELISA revealed that the IgE-binding of soybean proteins after HHP treatment at 200 MPa decreased 61.7% compared to the untreated group. Our findings suggested that HHP processing could not only modify the physicochemical properties of soybean proteins, but also significantly reduce its IgE-binding at an appropriate pressure level.

Blocking celiac antigenicity of the glutamine-rich gliadin 33-mer peptide by microbial transglutaminase

Celiac disease (CD) is a T cell-mediated enteropathy of the small intestine and caused by the ingestion of wheat gluten and related prolamins in barley and rye. However, there is no effective therapy to alleviate symptoms of celiac disease except for a life-long gluten-free diet. Recent studies showed that modification by microbial transglutaminase (mTG) could reduce the gliadin-specific immune response. In the present study, different acyl-acceptor substrates in combination with mTG were used to modify the model 33-mer peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), which is a particular celiac toxic α-gliadin peptide. RP-HPLC and LC-ESI-MS were performed to test the extent of the modifications. R5 ELISA and G12 ELISA were used to analyze the antigenicity of the modified peptide. The shifts of retention time and molecular weight showed great modification of 33-mer peptide after 2 h of incubation with mTG. When acyl-acceptor substrates were crosslinked with 33-mer peptide, the antigenicity of modified peptide forms was decreased compared to its initial level. In summary, it is demonstrated that mTG is active on a variety of chemically acyl-acceptor substrates. Transamidation by mTG with an appropriate amine donor can be used to block the antigenicity of gliadin peptide related to celiac disease. These findings highlight a potential strategy to prevent cereal toxicity in celiac disease.