Effect Of High Hydrostatic Pressure On The Allergenicity And Structure Of Tropomyosin From Shrimp

High Hydrostatic Pressure (HHP) is often chosen as the preferred processing technology for shrimp at present. At the same time, it is mainly considered as the technology to reduce the allergenicity of shrimp. For further research on the mechanism of HHP to change the shrimp allergenicity,the study was carried out about the effect of HHP on the allergenicity, molecular structure and antigen epitope of tropomyosin (TM) from shrimps (Litopenaeus vannamei). The change rule for HHP treatment on the allergenicity and structure of TM was also discussed. The main conclusions were summarized as follows:We established a method for purification of TM from shrimps based on two isoelectric point (pH 4.5) precipitation, saturated ammonium sulfate (50%) precipitation and anion exchange chromatography (UNO(?)Q1 (C18)). The method had the advantages of simple operation, high purity (above 95%),4% recovery rate, and high allergenicity, and could meet the requirements of subsequent experiments. Bioinformatics prediction results showed that TM had strong hydrophilicity, and its secondary structure was rich in a-helix (above 88%). For special structure of TM, it was made up of two polypeptide chains of a-helix, and no complicated tertiary and quaternary structure were found. Epitopes in TM were mainly linear epitopes.Fluctuation changes were showed in TM allergenicity after the HHP treatment. With the fixed dwell time of 10 min at room temperature, the allergenicity decreased from 200 to 300 MPa, increased from 400 to 500 MPa, and then slightly decreased when up to 600 MPa. At 300 MPa, TM allergenicity was the lowest, while at 500 MPa, its allergenicity was highest; Under the treatment of 300 MPa from 5 to 15 min at room temperature, TM allergenicity gradually decreased following extension of the dwell time. However, no changes were observed with the dwell time more than 15 min. At 300 MPa, with the dwell time of 15 min, the TM allergenicity firstly increased from 35 to 45 ℃, and decreased from 55 to 65 ℃, then slightly increased at 75 ℃.TM conformational structure could be influenced by HHP treatment. The primary structure of TM was not changed by HHP alone, that is, the amino acid chain of TM was not destroyed. Along with the rising of pressure, the a-helix content decreased slightly at 200 MPa but reached the maximum at 300 MPa, and fluorescence intensity decreased gradually. From 400 to 500 MPa, the a-helix content began to decline, the minimum happened at 500 MPa, while fluorescence intensity rose to the highest. At 600 MPa, the a-helix content rose, and fluorescence intensity decreased; with the dwell time extended, from 5 to 15 min, the a-helix content increased, and decrease was found in fluorescence intensity. No changes were happened in both a-helix and fluorescence intensity when the dwell time was much than 15 min. HHP combined with heat treatment could change the primary structure of TM. The a-helix content changed a little from 35 to 45 ℃, and then significantly decreased from 55 to 75 ℃, so that β-sheet, P-turn and random coil structure content increased, while the fluorescence intensity decreased.It was found that Peptide 1、Peptide6、Peptide9, and Peptide3 of TM were the key epitopes, and caused the changes in the allergenicity. TM had the ability of antipepsin enzymatic hydrolysis, which could be little effected by HHP. And the enzymatic product still has allergenicity.The mechanism of reducing the allergenicity of TM by HHP treatment was provided. By means of single HHP treatment, the change of TM allergenicity was effected by both the secondary structure and the tertiary structure. Under the condition of high pressure, the a-helix content increased; the natural conformation of hydrogen bonds rearranged, then the protein structure was further coiled; the surface hydrophobicity decreased; and the protein conformational space become tight. Therefore, the linear epitopes exposed in the protein surface were reduced, especially Peptide 1, Peptide 6 and Peptide 9, and conformational epitope, which reduced the opportunity of the reactivity between TM and epitopes and thus decreasing TM allergenicity. With combination of HHP and heat treatment, the allergenicity of TM changed because degradation of the primary structure caused changing of the secondary and tertiary structures so that destruction of the linear and conformational epitopes occurs, and then the TM allergenicity decreased.