Characterization Of The Allergens From Mud Crab (Scylla Paramamosain)

Recently, people around the world have paid much more attention on food allergy. Each year, events of people consuming allergic food triggered allergic reaction still increase, some of the allergic food threaten people's life. According to the research, the number of people allergic to crabs increased day by day. There are two kinds of allergens in crabs, one is tropomyosin (TM), the other is arginine kinase (AK). Right now, there were lots of researches on TM, mainly focus on its purification and immunology, while the studies of AK were most on its enzymatic properties and molecular cloning. Therefore, it's significant to study the allergic properties of AK and TM.This study used mud crab (Scylla paramamosain) as material, the extraction and purification of AK and TM from mud crab were studied, and the ELISA detecting standard curves of AK and TM were established, which could be used to detect the concentration of AK and TM in aquatic products. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and inhibition ELISA were adopted to analyze part of the physicochemical property and digestibility of AK, then the digestibility and allergenicity of crab TM after processed were analyzed. In addition, in vitro simulated gastrointestinal two phase digestion was adopted to compare the digestibility of AK and TM, and the tertiary structure of AK and TM were predicted.AK was purified by ammonium sulfate fractionation, column chromatography, etc. Firstly, the researches on the thermal and pH stability, digestibility of AK were conducted, the results showed that AK was thermal unstable, the protein structure of AK was variable during temperature rising, especially when the temperature higher than 46℃. As to the pH stability, AK started to degrade in acid buffers (pH 1.0) when incubation for 1.5 h. Referring to simulated gastrointestinal digestive experiment in the United States Pharmacopoeia, results of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) reaction indicated that AK was completely digested at 1 h digestive time, to the opposite, it was stable under the digestion of trypsin or chymotrypsin. In addition, other proteins in myosin would affect the digestibility of AK. Secondly, the purified AK was used to establish AK ELISA detecting standard curve and have been used to detect the content of AK in eight aquatic products. The results demonstrated that the content of AK of crustaceans, such as grass prawn, white-leg shrimp and mantis shrimp were higher, as to the mollusca and bivalvia such as short necked clam, oyster, squid et al., the content were however lower. This standard curve can mainly used to detect the content of AK especially in crustaceans.TM was purified by isoelectric precipitation, ammonium sulfate fractionation, etc. And different processing methods (such as boiling, ultrasonic assist boiling, high temperature and pressure, simulated crab can treatments, cooking treatment et al) were adopted to treat mud crab meat, and the analysis of the digestibility of treated crab TM was done according to the experiments of SGF and SIF based on the United States Pharmacopeia. The results showed that different processing methods could affect the digestibility of TM, among the processing methods, high temperature and pressure treatment could enhance the degradation of TM in the digestion, the allergenicity of TM and its degraded fragments were reduced either. In the process of simulated crab can treatment, the addition of sodium phytate could to some extent accelerate the degradation of TM in SIF or SGF. Otherwise, proper quantity of vinegar could also boost the digestion of TM in SGF. Above all, the high temperature and pressure treatment could significantly reduce the allerginicity of TM. Similarly, the purified TM was used to establish TM ELISA detecting standard curve and have been used to detect the content of TM in aquatic products. The results indicated that the content of TM of crustaceans, such as grass prawn, white-leg shrimp and mantis shrimp were higher, while the content in the mollusca and bivalvia such as short necked clam, oyster, squid et al., were however lower.Finally, the in vitro simulated gastrointestinal two phase digestion was used to compare the digestibility of AK and TM, and the tertiary structure of AK and TM were predicted. The results displayed that AK was easily degraded by pepsin than TM, while TM was not resistant to trypsin and chymotrypsin digestion. Otherwise, the tertiary structure of TM was much simple than AK, the tertiary structure of TM was mainly superhelix, while AK contendα-helix,β-sheet andβ-loops. The difference of the tertiary structure of AK and TM may lead to the difference of their physicochemical properties and digestibility.To sum up, in this study, the physicochemical and digestibility characters of crab AK were studied, showing that AK was thermal and acid unstable for the first time. Meanwhile, the processing properties of crab TM were mainly probed, indicating that high temperature and pressure, sodium phytate assist crab can treatment and vinegar addition during cooking could accelerate the digestive degradation of TM, and reduce its allergenicity.In addition, the in vitro simulated gastrointestinal digestive experiment was used to compare the digestibility of AK and TM, demonstrated that TM was a much stable allergen. These results are expected to provide a necessary justifies for the further researches on properties and desensitization of crab allergens.