Study On Rapid Identification And Classification Of Allergens In Crustaceans And Shellfish Based On Infrared Spectroscopy

Aquatic products taste delicious,with rich nutrients,favored by consumers.However,food allergy caused by aquatic products has been increasing in recent years,affecting people’s safety and health and attracting wide attention.Tropomyosin（TM）has been identified as a major allergen in crustaceans and shellfish,and is a myofibrin with a protein weight of about 36kDa and composed of two identical subunits.Currently,detection technologies for TM includeimmunoassay techniques represented by enzyme-linked immunosorbent assays and polymerase chain Reaction（PolymeraseChain Reaction,PCR）technology,and physical and chemical detection methods represented by mass spectrometry.However,immunological detection technology relies on antibodies that are difficult to be prepared.PCR technology does not have tissue specificity and is prone to false negative results.The high cost of the new detection technology limits its application in food analysis.In addition,there is currently no method for the classification of TM from different aquatic products.Therefore,it is necessary to develop a rapid and efficient method for the identification and classification of TM without antibody and with strong specificity.Infrared spectroscopy can detect samples rapidly,nondestructively and multi-component at the same time,and can provide rich sample structure information and overall chemical fingerprint information.Based on the above advantages,infrared spectroscopy technology has been widely used in food,traditional Chinese medicine,materials,environment and other fields.Using infrared spectrum technology,TM and other proteins,samples of different levels of TM andTM from different aquatic were tested.Through the analysis of spectra,a rapid identification and classification methods of allergen TMis established based on the infrared characteristic peaks of TM and infrared characteristic peaks of TM in shrimp,crab and shellfish.The main contents and results of this study are as follows:1.TM was extracted,enriched and purified from aquatic muscle sample.TM was subtracted separately by simulated gastric fluidand Simulated intestinal fluid.As the extension of time,the TM content gradually reduce.Samples of different levels of TM were detected by infrared spectrum.TheFT-IR in The change of the spectral peak in the FT-IR was found in 1700-1000cm^-1wave number range of protein amide bands.Then using infrared spectroscopy to detect the TM and other different protein（including the same type and different types of protein）.It was found that the allergen proteins including TM and Parvalbumin（Parvalbumin,Pa）had the same amideⅠband peak 1645±1 cm^-1and amideⅡband 1540cm^-1.It indicated that FT-IR was unable to distinguish similar proteins.So the second order derivative（SD-IR）for FT-IR was gotten.The results showed that the amideⅠband in SD-IR spectrum had 5 common peaks of proteins in,.In the amideⅡbelt andⅢ,a set of infrared characteristic peaks of TM(1579,1569,1551,1547,1387,1364,1315,1241cm^-1)was found.By comparing the amino acid sequences of TM,it was found that the amino acids corresponding to the characteristic peaks（glutamate,arginine and aspartic acid）were all amino acids with higher content of TM,among which glutamate and arginine were the sensitive amino acids of TM.Therefore,based on the infrared characteristic peaks information of this group of TM and the common peaks information of proteins,this study established a rapid identification method of TM,a major shrimp allergen,based on infrared spectroscopy.2.11 common shrimp,crab and shellfish aquatic products were selected.TMs were extracted and enriched.TM was then detected by infrared spectroscopy.In the FT-IR spectrum,it was found that amideⅠandⅡpeaks of TMs in the 11 kinds of crustaceans and shellfish were similar,respectively within 1644±5cm^-11 and1553±5cm^-1.Further analysis of the 11 kinds of aquatic products SD-IR spectrum,it was found that amideⅠandⅡbands of TMs not only had common peaks of proteins and the similar ratio between the common peaks,which was consistent with the result of the fast identification method.In addition,it was found that 3 TM’s characteristic peaks in shrimp(1241±1cm^-1,1072±3cm^-11 and 1077±3cm^-1),8 TM’s characteristic peaks in crab(1349cm^-1,1299±2cm^-1,1245±2cm^-1,1176±1cm^-1,1145±1cm^-1,1097±3cm^-1,1067±2cm^-11 and 1035±2cm^-1);3 TM’s characteristic peaks in shellfish(1145±5cm^-1,1080cm^-11 and 1024±5cm^-1).Among them,shrimp and crab belong to crustacean,and their characteristic peaks are partially similar(1241±1cm^-1与1245±2cm^-1,1072±3cm^-1与1067±2cm^-1).The cluster analysis based on principal component analysis（PCA）showed that TM of shrimp,crab and shellfish had an obvious clustering trend,and several peaks that contributed a lot to its clustering were all characteristic peaks of various types of TM.Therefore,the infrared spectroscopy established in this study can effectively distinguish TM from shrimp,crab and shellfish,and can be used for the classification of TM from these three species.To sum up,this study established a fast,non-destructive and simple method for the rapid identification of allergen TM based on the 6 common infrared spectral peaks of a group of proteins and 8 characteristic peaks of TM.On this basis,the classification method of TM for shrimp,crab and shellfish was established according to the characteristic peaks of the infrared spectrum of TM in shrimp,crab and shellfish.The method established in this study has the advantages of nondestructive sample,no antibody,rapid and efficient,and can be applied to the large-scale screening of TM,the major allergen in aquatic products,or the rapid determination of the source of food allergy.