南极磷虾主要过敏原的分离纯化及性质研究

为了探究南极磷虾致敏问题,从南极磷虾中筛选、鉴定、分离纯化其主要过敏原,并对过敏原的性质进行研究,实验通过缓冲盐溶液提取南极磷虾蛋白;采用SDS-PAGE和Western blot(WB)筛选南极磷虾过敏原;使用高效液相色谱-串联质谱鉴定过敏蛋白;通过等电点沉淀、硫酸铵盐析、阴离子交换柱层析分离纯化过敏原;采用SDS-...

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. The huge biological resources and good utilization potential give Antarctic krill broad prospects for development and utilization, so its edible safety has also attracted extensive attention, especially the sensitization of Antarctic krill. At present, the research on Antarctic krill at home and abroad is more focused on Antarctic krill protein, lipid, enzyme, astaxanthin, frozen shrimp, shrimp powder, processing and utilization. The research on the safety of Antarctic krill is mainly focused on fluorine and arsenic, while the study on Antarctic krill sensitization problem is rarely reported. With the wide application of Antarctic krill as food raw materials in the food field, its allergenicity and other potential food safety problems have attracted special attention. Because of its excellent nutritional value and high economic value, Antarctic krill has become an important object of pelagic fishery in China. The potential food safety problems of Antarctic krill may have a great impact on China"s Pelagic Fisheries, especially on the fishing and processing of Antarctic krill. How to prevent and control the food allergy caused by Antarctic krill has become an urgent work. In order to study 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 buffer salt solution; The Antarctic krill allergens were screened by SDS-PAGE and Western blot (WB); High performance liquid chromatography-mass spectrometry was used to identify the primary structure of these allergic proteins; 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 sarcoplasmic protein (SP) and myofibrillar protein (MF) were successfully extracted from the Antarctic krill. Protein bands in electrophoresis were rich and the range of molecular weight was wide. After identified by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), the protein with the highest abundance in SP was arginine kinase with a molecular weight of about 43 kDa, and the protein with the highest abundance in MF is tropomyosin with a molecular weight of about 35 kDa. Results of WB showed that 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 molecular weight of about 35 kDa and can 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 fractional 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, molecular weight of TM gradually decreased and finally stabilized at about 33 kDa, and the abundance of degradation fragments with molecular weight of 15 kDa and 12 kDa gradually increased and existed stably. These degradation bands could still have a strong immunoreactivity with the serum IgE of allergic patients. With the extension of digestion time of simulated intestinal fluid, the original fragment of TM gradually decreased until disappeared, and finally completely degraded into peptides with smaller molecular weights. The immunoreactivity of TM degradation products was greatly reduced after digestion by simulated intestinal fluid. The results obtained above indicate that there are allergens in Antarctic krill, and TM is the main allergic protein with immunoreactivity. TM also has the general characteristics of allergens, such as high temperature resistance, pepsin digestion resistance and partial stability of trypsin digestion. The research has practical significance for the early warning, prevention, and control of potential food safety problems of Antarctic krill. It also provides basic information for the processing and utilization of Antarctic krill and the construction of allergen reduction technology.