Inhibiting Effect Of Trypsin Inhibitor From Soybean On The Tryptase And Protein Degradation Of Litopeneaus Vannamei During Iced Storage

Litopenaeus vannamei is a main shrimp species in our country and very popular among consumers. But the shrimp muscle is apt to softening and autolysis in the process of transportation and sales, which leads to the decline of commercial value and shorten of storage time. Our research group previous study shows the spread of tryptase from hepatopancreas and degradation on muscle protein are the main reason of muscle softening and quality loss during storage. In addition, literature has reported that soybean extract is rich in trypsin inhibitor and it is able to inhibit the protein degradation of the shrimp. In order to explore the role of soybean trypsin inhibitor in the shrimp preservation, the inhibiting mode of soybean trypsin inhibitor on tryptase, effect of crude extracts of soybean trypsin inhibitor on the muscle properties were investigated. The proteomics technologies(2-DE and MS) were also applied to study the changes of muscle protein during iced storage as well as the effect of crude extracts on the protein degradation of Litopeneaus Vannamei. The main studied contents and conclusions are as follow:(1) Tryptase from Litopeneaus Vannamei was purified by 30%-80% of saturated ammonium sulfate precipitation, Q-Sepharose F.F. column chromatography and Sephacryl S-100 chromatography. The tryptase was determined to have a molecular mass of 32.6 kDa. The soybean(Glycine max) powder was extracted by 0.15 M Na Cl, followed by thermal denaturation, ultra-filtration and Sephacryl S-100 chromatography to obtain Kunitz-type trypsin inhibitor. The inhibiting effect of the trypsin inhibitor on the tryptase from Litopeneaus Vannamei was investigated. Results showed that the inhibitor was purified 12.51-fold with a yield of 28.95% and displayed a main band on SDS-PAGE with a molecular mass of 16 k Da. The inhibitor exhibited a significant inhibiting effect on the tryptase and their reaction mode was irreversible.(2) In order to evaluate the effect of crude extracts of trypsin inhibitor from soybean on the muscle properties of Litopeneaus Vannamei during storage(0℃), the dynamic rheometer and differential scanning calorimeter were used to investigate the difference between the experimental group which injected the crude extracts and the blank. Dynamic rheology research showed that two groups of shrimp could be able to form thermal gel. The formation of the gel began at about 47 ℃ and better network occurred at high temperature(about 78 ℃); The gel was further reinforced as the following cooling. Elastic modulus(G’) of injecting group was higher than the blank during storage of 2, 4, 6 days on the whole. The gel elasticity of injecting group was better. DSC results revealed that during storage of 0, 2 days, two groups of shrimp had 3 endothermic peaks and the denaturation temperature were about 48、68、79 ℃,respectively. A new endothermic peak( about 53 ℃) appeared with the time extension. However, the denaturation temperature of 4 endothermic peaks of injecting group was higher than the blank group on the whole. The crude ext racts of trypsin inhibitor had remarkable effect on the denaturation temperature at storage of 2, 4 days(p < 0.05). Therefore, the crude extracts of trypsin inhibitor from soybean could be able to improve the elastic of the thermal gel and protein stability during the early stage of the storage(< 6 days), achieving the effect of delaying the softening and degeneration.(3) The proteomics technologies were used to study changes of muscle protein of Litopeneaus Vannamei during iced storage. Results shown that among the differentially expressed protein spots of A0 down-regulated, myosin heavy chain type 1, adenosine monophosphate deaminase and the natural protein which degraded to I135(not identified) degraded to I19, I14 and I135. The change of I19 gray value had a good linear positive correlation with the iced time. The correlation coefficient R2 was 0.920. Gray value of I135, I14 had a good quadratic polynomial regression with the iced time and their R2 were to be 0.974 and 0.986 respectively. Among the differentially expressed protein spots of A0 up-regulated, myosin heavy chain type 1, myosin heavy chain type 2, troponin I belonged to those proteins that degraded gradually.Their difference fold changes of gray value were on the rise with the iced time going by. Therefore, myosin heavy chain type 1, myosin heavy chain type 2, adenosine monophosphate deaminase and troponin I could be used as freshness indicators of Litopeneaus Vannamei during iced storage.(4) In order to analyze the effect of crude extracts of soybean trypsin inhibitor on protein degradation of Litopeneaus Vannamei, shrimps were divided into two groups, the blank and the experimental group which injected the crude extracts. Differentially expressed protein spots of shrimp were analyzed at the same time of iced storage. Results shown that the content of heavy chain type 1 in the injected group was higher than that in the blank group, indicating that myosin degraded more slowly in the injected group. On the contrary, the content of arginine kinase, enolase and pyruvate kinase in the blank was higher than that in the injected group, showing that the metabolism in the blank group was faster than the injected group. Therefore, the crude extracts of soybean trypsin inhibitor could slow down the degradation of protein during post mortem of Litopeneaus Vannamei.