| Food allergy is a global food safety and public health problem identified by FAO/WHO,which seriously affects people’s dietary needs and physical health.As one of the most common allergenic fruits worldwide,peach can cause complex skin and mucosal symptoms and digestive system symptoms,and even life-threatening in severe cases.However,the phenomenon of peach allergy has not received widespread attention and high priority worldwide at first,making the proportion of people with peach allergy increasing in recent years.At present,the detection methods for peach allergens are not comprehensive,the interpretation of the allergenic mechanism is still unclear,the allergenicity reduction methods are not sufficient,and the prevention and control system is not yet completed.Therefore,it is very important to develop accurate peach allergen detection methods,to analyze the mechanism of peach allergy,and to investigate effective peach desensitization methods to prevent peach allergy.In this study,we developed an accurate qualitative and quantitative method for the detection of peach allergen proteins,screened the B-cell linear epitopes of the major allergen proteins,and analyzed the structure and allergenicity of peach allergen proteins under ultrahigh pressure and heat treatment from multiple perspectives,In order to provide a theoretical basis for the implementation of peach allergen labeling,as well as to provide a theoretical foundation for comprehensive and in-depth understanding of peach allergens,and the reducing the potential allergenicity of peach foods.The main results are as follows:(1)A precise qualitative and quantitative method for the detection of three major peach allergenic proteins(Pru p 1,Pru p 3 and Pru p 7)was established using shotgun proteomics combined with LC-MS/MS-MRM technique.Eight characteristic peptides with high mass spectrometric response intensity,high specificity and good stability were screened for the qualitative detection of peach allergenic proteins;three peptides with the highest response intensity and best linear fit were further selected as quantitative peptides.A quantitative method based on isotope internal standard method was established for the quantitative detection of peach allergen protein.The LOD and LOQ of the method were 0.4-200 fmol/μL and 1.6-800 fmol/μL,respectively,with the recoveries of 90%-105% for different concentrations of QC samples,and the inter-day and intra-day coefficients of variation were less than 5%,which are accurate,sensitive and stable.(2)Highly pure and immunologically active natural Pru p 3 and recombinant Pru p 7 were obtained by microchromatography and prokaryotic expression,respectively,and their amino acid sequences,protein characteristics,structural features and homology were analyzed,finally,the main B-cell linear epitopes were screened.The results showed that both Pru p 3 and Pru p 7 are rich in cysteine content,and the disulfide bond formed is the main reason for their thermal and digestive stability;their amino acid sequences are highly conserved in ns LTPs and GRPs,respectively,which make them have some possibility of clinical cross-allergic reactions;the secondary structures of Pru p 3 and Pru p 7 are mainly α-helices and random coils,and do not contain β-sheets and β-turns;three major B-cell linear epitopes were screened from each of the two proteins by slot-blot immune microarray assay combined with immune score matrix,and gastric digestion simulation combined with competitive inhibition ELISA results showed that different epitopes have different digestive stability;amino acid mutations combined with immunological validation were used to further screen the key amino acid,cysteine and lysine were the most frequent amino acids in the Pru p 3 and Pru p 7 epitopes,respectively,and had a key role in the Ig E binding ability of these two proteins.(3)In-depth analysis of the effects of ultrahigh-pressure(UHP)treatment on soluble protein content,protein structure and allergenicity of peach.UHP treatment could significantly reduce the soluble protein content of peach,and the effect on the content of different allergen proteins was different;for the rigid protein Pru p 3with small molecular weight,the soluble protein content was not significantly reduced;UHP treatment could cause significant changes in the secondary and tertiary structures of peach protein,mainly manifested by the conversion of α-helices to β-sheets in the secondary structure,and some amino acid residues in the tertiary structure were masked or the conjugated double bond was destroyed The effects of UHP treatment on the Ig E binding ability of peach major allergen proteins were different,among which the Ig E binding ability of Pru p 2 was more sensitive to UHP treatment,while that of Pru p 1 was less sensitive.In addition,UHP treatment significantly down-regulated the release of histamine,TNF-α and IFN-γ after degranulation of KU812,with the former two contributing to allergenicity reduction and the latter detrimental to allergenicity reduction.In terms of epitopes,UHP treatment altered the conformation near the linear epitope of Pru p 3 B cells,leading to partial masking of the enzymatic cleavage site and increasing the risk of allergenicity.(4)In-depth analysis of the effects of heat treatment on soluble protein content,protein structure and allergenicity of peach.Heat treatment at 60℃ had no significant effect on the content of peach soluble protein,and over 80℃ could significantly reduce the content of peach soluble protein.The effects of different temperature treatments on the relative contents of different peach allergenic proteins were also different.Pru p 3 had no significant changes in its content due to its rigid protein structure;the effects of heat treatment on the secondary and tertiary structures of peach proteins were mainly manifested by the disappearance of β-sheets,and the increase of random coils in the secondary structure,and the protein structure trend to be loosened.The rearrangement of the tertiary structure caused changes in the microenvironment near some amino acids,resulting in different degrees of blue and red shifts in the UV and fluorescence spectra and changes in the intensity of the absorption peaks;different temperatures had different effects on the Ig E binding ability of different peach allergic proteins,among which the Ig E binding ability of Pru p 2 and Pru p 7 was more sensitive to temperature.The total Ig E binding capacity of peach proteins decreased significantly above 80°C,while the cytokine release levels of histamine,β-HEX,TNF-α and IL-4 were significantly down-regulated and the release level of IFN-γ was up-regulated after degranulation of KU812 cells,both of which contributed to the abatement of peach allergenicity.In contrast,the trend of IL-6 changes was not significant. |
PDF Full Text Request