Zhihua Wu

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.

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.

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.

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.

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%.

Changes in the structure, digestibility and immunoreactivities of glycinin induced by the cross-linking of microbial transglutaminase following heat denaturation

Summary This study aimed to evaluate the effects of microbial transglutaminase (MTG)-mediated modification on the structure, digestibility and immunoreactivities of glycinin. Glycinin was separated from soya bean and cross-linked with MTG, and the sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cross-linked glycinin was higher than that of native glycinin. Individual MTG cross-linking could maintain stable secondary structures and spatial structure. Sequential heat denaturation and MTG cross-linking could promote the unfolding of protein structures and reduce their hydrophobicity. The digestibility of glycinin was decreased, and its immunoreactivities were increased because of MTG-induced structural alteration, including primary and spatial structures.

Highly Sensitive Detection of Bovine β-Lactoglobulin with Wide Linear Dynamic Range Based on Platinum Nanoparticles Probe

Cows milk allergy is one of the most frequent and severe IgE-induced food allergies for children, demanding sensitive analytical methods, and β-lactoglobulin (BLG) can be as an important biomarker for detection of milk protein. In this study, a highly sensitive sandwich enzyme-linked immunosorbent assay (sELISA) based on a specific polyclonal antibody against human IgE linear epitopes of BLG and an anti-BLG polyclonal antibody-platinum nanoparticles probe was described for detection of BLG. This sELISA exhibited an ultrawide linear range of 0.49-1.6 × 104 ng/mL, covering more than four orders of magnitude. The limit of detection was 0.12 ng/mL, which was 16-fold lower than that using traditional sELISA with the same antibodies. Furthermore, the proposed approach showed high recoveries (93.53%-111.95%) and low coefficient of variation (1.49%-12.50%) after analysis of samples fortified with BLG. The presence of allergenic BLG residues also could be detected in partially hydrolyzed infant formulas. These results, in comparison with conventional and commercial BLG detection sELISAs, highlight that this proposed sELISA could be a reliable and user-friendly tool to monitor trace amounts of BLG and its potentially allergenic residues in foods.

Structure characterization and IgE-binding of soybean 7S globulin after enzymatic deglycosylation

ABSTRACT This study investigated the effects of enzymatic deglycosylation following ultrasound pretreatment on structure and immunoreactivity of soybean 7S globulin. Soybean 7S globulin was pretreated by ultrasound (40 kHz, 300 W) and enzymatically deglycosylated by peptide-N-glycosidase F (PNGase F). Changes in structure of processed soybean 7S globulin were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, ultraviolet absorption spectrum, circular dichroism spectrum, and surface hydrophobicity analysis. Enzyme-linked immunoabsorbent assay was used to evaluate IgE-binding ability. The results showed that the glycan moieties of soybean 7S globulin were effectively removed by PNGase F, which significantly modified protein structures including the secondary and tertiary structures of 7S globulin. Individual enzymatic deglycosylation could reduce IgE-binding capacity of 7S globulin, whereas enzymatic deglycosylation following ultrasound pretreatment enhanced its IgE-binding capacity. In conclusion, soybean 7S globulin treated by single enzymatic deglycosylation can reduce potential allergenicity and may be employed in hypoallergenic food preparation.

Caffeic acid-assisted cross-linking catalyzed by polyphenol oxidase decreases the allergenicity of ovalbumin in a Balb/c mouse model

Ovalbumin (OVA) is the most abundant egg white protein, but is also a major egg allergen. Desensitization of OVA may be a good way to control an egg allergy. In this study, caffeic acid-assisted cross-linked OVA catalyzed by polyphenol oxidase (PPO) was prepared, the effect of cross-linking on the allergenicity of OVA was tested in a Balb/c mouse model. Mice were orally sensitized with OVA or cross-linked OVA using cholera toxin as adjuvant. Clinical signs of allergy, specific antibody levels, serum histamine levels, mast cell protease-1 (mMCP-1) concentrations, morphological structure of duodenum, and cytokines were determined after mice were challenged with OVA or cross-linked OVA. Both OVA and cross-linked OVA induced allergic diarrhea in Balb/c mice, however, histological symptoms of small intestine were much milder in mice fed with cross-linked OVA than in those fed with OVA. A tendency toward decreased allergen-specific IgE, IgG, IgG1 and IgG2a levels, as well as serum histamine and mMCP-1 concentration were observed in cross-linked OVA group, accompanied by an inhibition of IL-4, IL-5, IL-13 and IFN-γ production in the stimulated spleen cell. It could be concluded that caffeic acid-assisted PPO-catalyzed cross-linking significantly reduced the potential allergenicity of OVA, but may not completely eliminate it.