Processing Stability And Signature Peptide Study Of Shrimp (Metapenaeus Ensis) Allergen

In recent years, food hypersensitivity shows a rising trend and becomes one themost important threats to human health, even trace food allergen can cause seriousallergic reactions. Therefore, establishment of food labeling system is necessary forthe protection of patients. However, currently, there is no effective treatment for foodallergen. Methods used for allergen detection often shows high false negative,especially for processing food. In order to find out the influence of the processingtechnology on food allergen, in this research, shrimp of Metapenaeus ensis waschosen as subject, while immunological and proteomics methods were combined tostudy its immunogenicity stability post food processing. By comparing the peptidechanges post processing, signature peptide that has stability in food processing andcan distinguish with the other crustaceans was determined. The main contents were asfollows:1. Four different treatments including lyophilization, drying, frying and gelationwere chosen to investigate the impacts on the immunocompetence of shrimp allergenrespectively. By the four different treatments, the total proteins and content of themajor allergen, immunocompetence and vitro anti-enzymatic digestion were adoptedto detect the changes of the processed shrimps. The results showed that the majorallergen protein was still retained in the processed shrimps, but theimmunocompetence of the allergen was all reduced after the procedures based on theresults of Western Blot and ELISA. Among these four methods, gelation had the bestreducing effect to the ability combined with IgG, which had bought a87%reductionof total, while drying took85%as follow. The fried sample displayed the least proteinbands and its immunocompetence took about56%. Meanwhile, lyophilizing had littleeffect in reducing the immunocompetence. Nevertheless, the results of in-vitroenzymatic digestion showed the ability of anti-enzyme took subtle effects in theprocesses, and the allergen was lost after about60min into digestion. The conclusion is that the processing can impact on the allergen immunocompetence of the shrimp,but the changes on the ability of anti-enzyme are weaker.2. Samples including shrimp muscles, shrimp protein extracts and purifiedfractions of tropomyosin were treated by four different dosages of electron beamirradiation (EBI). After that, the irradiated samples were profiled by SDSelectrophoresis and changes in the conformity of the allergen were tracked by Westernblot and indirect ELISA. The results showed that the allergenicity of the muscleextracts increased, while the allergenicity of allergen extracts and purified extractsreduced at low irradiation dosages. When it came up to10kGy, the allergenicitydecreased for all of these three irradiated samples, and the musle protein is strongerthan the shrimp extrac lipid, and the major allergen of shrimp still remained in thethree post irradiation samples. In-vitro enzymatic digestion showed that the allergenwas lost after about60min into digestion.3. The shrimp protein were separated by two-dimensional electrophoresis(2DE),and analyzed the protein spots using Melanie7.0. The software detects106proteinspots most of which distributed in the area of pI4.5-7MW20-90kD on the2DE map.However, the protein spots were changes significant after the processing. The proteinspots in pI5-6MW20-60region were missing after fried, meanwhile, the gelationsample produced some new protein spots near the acidic segment.The post EBIsamples missed the protein on the neutral segment at the20kD, nevertheless, thelyophilizationmatched68protein spots to the contrast, signified that the lyophilizationcan keep the protein quality in the shrimp.The2DE techonolgy combined with the western blot analyzed the theimmunization of the Metapenaeus ensis. The results showed that there were elevenpositive spots combined with the serum IgE. Therefore, the No.1, No.4and No.5protein were selected to do the MS analyzation.4. The protein derived from the2DE map was identified using MALDI-MS andMALDI-MS/MS after enzymolysis by trypsin, and the mass spectra results weresubmited and matched with the protein database on the internet. The match resultsshowed that the No.1protein spot is the Met e1allergen of Metapenaeus ensis, which has high identify with other crustaceans and the another arthropod, giving rise to thesevere cross-reaction. However, the peptide of124Ser-Lys139was special to thetropomyosin of shrimp. The No.4protein spot matches with the phosphopyruvatehydratase of Penaeus monodon and enolase, which are99%identity. The peptideswere analyzed by the BLAST tool, and the consequence displayed that198Gly-Lys221can match more than forty kinds of enolase, and273Ile-Arg279can specially match withphosphopyruvate hydratase. No.5protein spot is the outer membrane efflux protein ofRunella slithyformis, which peptides are signature.Through the processing stability study found that the allergen of shrimp（Met e1）showed strong processing tolerance and stability of enzyme digestion. The peptide of124Ser-Lys139derived from tropomyosin can be used as signature peptide for thequantitative detection of the shrimp products.