Isolation, purification and properties of Antarctic krill (Euphausia superba) major allergens

Antarctic krill (Euphausia superba) is a type of crustacean living in Antarctic waters and is an important strategic marine living resource. Currently, the research on the safety of Antarctic krill is mainly focused on fluorine and arsenic, while the study on Antarctic krill sensitization is rarely reported. How to prevent and control the food allergy caused by Antarctic krill has become urgent work. In order to explore the sensitization problem of Antarctic krill, the main allergens were screened, identified, isolated and purified from Antarctic krill, and the properties of major allergens were studied. Antarctic krill proteins were extracted by salt buffer; Allergens were screened by SDS-PAGE and Western blot (WB); The primary structure of these allergic proteins were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); Major allergens were separated and purified by isoelectric precipitation, ammonium sulfate salting out and anion exchange column chromatography. The heat resistance and the digestive stability of Antarctic krill major allergens to simulated gastrointestinal fluid were analyzed by SDS-PAGE and WB. Results showed that the protein bands of sarcoplasmic protein (SP) and myofibrillar protein (MF) extracted from Antarctic krill were rich and the range of the molecular weight (MW) was wide. Antarctic krill proteins could react with the serum IgE of shrimps/crabs allergic patients, and at least four proteins had positive reactions. The protein with the strongest immune reaction had a MW of about 35 ku and could be recognized by the serum of all allergic patients. This allergic protein was identified as Antarctic krill tropomyosin (TM) by LC-MS/MS. The optimum saturation of ammonium sulfate was 50% for the salting out Antarctic krill TM. After separation and purification, TM with high purity was obtained, which could have a strong immunoreactivity with the serum IgE of shrimps/crabs allergic patients. The measured isoelectric point of Antarctic krill TM was 4.4, and it had good thermal stability. With the extension of digestion time of simulated gastric fluid (SGF), MW of TM gradually decreased and finally stabilized at about 33 ku, and the abundance of degradation fragments with MW of 15 ku and 12 ku gradually increased and existed stably. These degradation bands could still have strong immunoreactivity with the serum IgE of allergic patients. With the extension of digestion time of simulated intestinal fluid (SIF), the original band of TM gradually decreased until disappeared, and finally completely degraded into smaller polypeptide. The immunoreactivity of TM degradation products was greatly reduced after digestion by SIF. The results obtained above indicate that there are allergens in Antarctic krill, and TM is the main allergen with immunoreactivity. TM also has the general characteristics of allergens, such as high temperature resistance, pepsin digestion resistance and partial stability of trypsin digestion.