Anshu Yang

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.

Fluorescent immunosorbent assay for the detection of alpha lactalbumin in dairy products with monoclonal antibody bioconjugated with CdSe/ZnS quantum dots

In this work, a new method termed competitive fluorescence-linked immunosorbent assay (FLISA) was developed for specifically quantification of bovine α-lactalbumin (α-La) in dairy products. The monoclonal antibodies (mAbs) against α-La were produced through hybridoma technology, and the mAbs were covalently conjugated with the CdSe/ZnS quantum dots (QDs) using the crossing-linking reagents. Moreover, a competitive FLISA based on QD-mAb conjugates was established to detect α-La in dairy products. It was shown that there was a good linear relationship between inhibition efficiency, and logarithm of α-La concentration after the detection parameters were optimised in which the concentration of α-La varied from 0.1 to 1000ng/mL. The value of IC50 was 0.03μg/mL, and the FLISA method exhibited high sensitivity with the LOD at 0.1ng/mL. The developed FLISA has been successfully applied to determine α-La in commercial dairy products, providing more sensitive analysis compared with the ELISA method.

Crosslinking of peanut allergen Ara h 2 by polyphenol oxidase: digestibility and potential allergenicity assessment.

BACKGROUND Peanut is one of the eight major food allergens. Its allergen, Ara h 2, can be recognized by over 90% of serum IgE samples from peanut-allergic patients. Therefore, reducing the allergenicity of Ara h 2 is especially important. RESULTS In the present study, polyphenol oxidase (PPO), a protein cross-linking reaction catalyst that acts on tyrosine residue, was used to modify Ara h 2. After crosslinking, the microstructure, digestibility, IgG binding capability and IgE binding capability of Ara h 2 were analyzed. Cross-linking decreased the potential allergenicity of Ara h 2 by masking the allergen epitope, while the antigenicity of Ara h 2 changed slightly. After crosslinking, the apparent diameter of Ara h 2 was altered from 300 to 1700 nm or 220 nm, indicating that polymerization could either be inter- or intramolecular. Regarding digestibility, crosslinked Ara h 2 was relatively more easily digested by gastric fluid compared with the untreated Ara h 2, but much more difficult in the intestinal fluid. CONCLUSION The crosslinking reaction catalyzed by PPO, as a non-thermal process, may be beneficial for avoiding food allergy. The reaction could mask allergen epitopes, decreasing the allergenicity of Ara h 2.

Enzymatic characterization of the immobilized Alcalase to hydrolyze egg white protein for potential allergenicity reduction

BACKGROUND This study examined technique characteristics of the immobilised Alcalase to hydrolyse egg white protein for potential allergenicity reduction. Alcalase was immobilised covalently on carboxyl-functionalised magnetic beads by carbodiimide activation. The technique characteristics of the immobilised Alcalase were investigated, followed by determining the degrees of hydrolysis (DH), immunoglobulin G (IgG) binding, and IgE binding of the digested egg white protein by immobilised Alcalase. RESULTS Enzymatic activity, enzyme loading, and immobilisation yield of the prepared immobilised Alcalase were 20.55 U mg-1 , 925 mg g-1 , and 45%, respectively. Immobilised Alcalase showed maximum activity at pH 8.0 and 60 °C. Compared with free Alcalase, immobilised Alcalase exhibited better thermal and storage stability. Moreover, immobilised Alcalase can be reused 10 times and still maintained 55% of its initial activity. Partial hydrolysis of egg white protein by immobilised Alcalase can effectively reduce IgG and IgE binding of the hydrolysates. CONCLUSION This study indicates that the immobilised Alcalase can be used to hydrolyse continuously egg white protein for potential allergenicity reduction.

Allergen composition analysis and allergenicity assessment of Chinese peanut cultivars.

Peanut (Arachis hypogaea) is among the eight major food allergens in the world. Several attempts have been made to decrease or eliminate the allergenicity of peanut. Systemic screening of thousands of peanut cultivars may identify peanut with low allergenicity. In this study, the allergen compositions of 53 Chinese peanut cultivars were characterized, and their allergenicity to sera IgE of Chinese patients and in a mouse model was assessed. Contents of total protein and allergens were quantified by SDS-PAGE and densitometry analysis on gel. Although the contents of allergens broadly varied among cultivars, they were related to one another. The IgE binding capacity of cultivars was tested by ELISA, and their allergenicity was further evaluated in a mouse model by oral sensitization. Results showed that the allergenicity of peanut was affected by allergen composition rather than a single allergen. Peanut cultivars with low allergenicity may contain more Ara h 3/4 (24 kDa), Ara h 2 and less Ara h 3/4 (43, 38, and 36 kDa), Ara h 6. Screening based on allergen composition would facilitate the identification of low-allergenic peanut.

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.

Purification and recombinant expression of major peanut allergen Ara h 1.

Reaction to peanut, as one of the major food allergens, has become an increasingly common life-threatening disorder. Although peanut allergens have been extensively identified, Ara h 1 is still too expensive to be applied in food safety or clinical utility. In this study, the purification, expression, and immunological analyses of Ara h 1 are investigated. It was shown that a high purity (>95%) of Ara h 1 could be prepared by either purification or expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and mass spectroscopy were used to identify the Ara h 1, and it was found that natural Ara h 1 (nAra h 1) and expressed Ara h 1 (rAra h 1) have the same properties, including amino acid sequence. In particular, rAra h 1 reacted positively with anti-nAra h 1 serum, showing their similar immunological property. Thus, by either purification or expression, Ara h 1 could be prepared with low cost, as performed in the present work. SDS-PAGE, mag trix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), and immunological analysis confirmed that both forms of Ara h 1 had the same properties.

Sequential extractions: A new way for protein quantification—data from peanut allergens

Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by the Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of them in solution. Extracting all of the contents is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein contents and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If all proteins can be extracted with sufficient extraction times, the protein contents in the peanut matrix can be calculated using a mathematical summation formula. This sum should be all proteins in the matrix. The five-step sequential extraction method can provide a means to quantify certain proteins in the matrix.

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.

Prevalence of Celiac Disease Autoimmunity Among Adolescents and Young Adults in China

BACKGROUND & AIMS: In China, epidemiologic information on celiac disease autoimmunity is scarce and fragmented. We investigated the prevalence of celiac disease autoimmunity in the general Chinese population. METHODS: In a cross‐sectional prospective study, 19,778 undiagnosed Chinese adolescents and young adults (age, 16–25 y) were recruited from consecutive new students who underwent routine physical examinations at 2 universities in Jiangxi, China, from September 2010 through October 2013; the students were from 27 geographic regions in China. All subjects were tested for serum IgG, IgG against deamidated gliadin peptides (IgG anti‐DGP), and IgA anti–tissue transglutaminase antibodies (IgA anti‐tTG). We also analyzed HLA genotypes in subgroups of participants with different results from tests for serum markers of celiac disease. RESULTS: A total of 434 students (2.19%) tested positive for serum markers for celiac disease (95% confidence interval [CI], 1.99%–2.41%), 0.36% of the students tested positive for anti‐tTG IgA (95% CI, 0.28%–0.46%), and 1.88% tested positive for anti‐DGP IgG (95% CI, 1.70%–2.09%). The prevalence of celiac disease autoimmunity (positive results in assays for anti‐tTG IgA and anti‐DGP‐IgG) was 0.06% (95% CI, 0.03%–0.10%). Celiac disease autoimmunity was associated with the consumption of wheat and female sex. The prevalence in the Shandong province in north China, where wheat is a staple in the diet, was 0.76% (95% CI, 0.21%–1.95%). The frequencies of the HLA‐DQ2/‐DQ8 genotypes associated with celiac disease were higher in subjects with celiac disease autoimmunity, based on detection of both serum markers, than in subjects with positive results from a single test (P < .01). All subjects with positive results from both assays carried the HLA‐DQ2 genotype. CONCLUSIONS: Approximately 2% of adolescents or young adults in China had positive results from assays for serum markers for celiac disease. The prevalence of celiac disease autoimmunity in the Shandong province in north China, where wheat is a staple in the diet, was 0.76%.