| Due to the endogenous proteases from postmortem Litopenaeus vannamei, the shrimp muscle becomes soft, leading to the decline of freshness and commercial value of shrimp. Trypsin from the head of shrimp, having high content, strong activity and powerful degradation, may be connected with the softening of postmortem shrimp muscle. In order to confirm whether trypsin is the key factor of the softening of shrimp muscle, trypsin was extracted and labeled with FITC, the migration of trypsin from shrimp head to muscle was studied, and the degradation of muscle and protein by trypsin was investigated, which could improve the shrimp quality and develop new preservation technology of shrimp. The main tasks and conclusions are as follows:Trypsin from the shrimp head was purified by 30%-80% of ammonium sulfate fractions, Q-Sepharose F.F. anion chromatography and Sephacryl S-100 gel chromatography with 30 kDa of molecular weight, 885.07 U/mg activity, 13.77% yield and 21.77-fold increase in purity.Trypsin was labeled with FITC to get FITC-trypsin. Base on the single factor experiments, the effects of FITC/trypsin, time, temperature and pH on FITC-trypsin with the F/P, F and trypsin activity as indicators, L9(34)orthogonal experiments were investigated to gain the best labeling conditions. The best factors containing FITC/trypsin, time, temperature and pH were 1:50, 24 h, 8℃, and 8.0, respectively, under which the F/P, F and trypsin activity were 3.57, 1025.93, 94.90%, respectively. Therefore, FITC was suitable for trypsin labeling, but the labeling effect of FITC-trypsin affected by FITC/trypsin, time, temperature and pH.Different endogenous proteases activities from shrimp were compared, the distribution and changes of total proteolytic and trypsin activities in shrimp during ice storage were detected, and the migration of FITC-trypsin in shrimp muscle and myofiber was researched with the purpose to investigate the migration of trypsin from shrimp head to muscle. Results showed proteolytic activity in the shrimp head was 90.17 U/mg, which is much higher than that of muscle. Trypsin activity, the highest one of six endogenous proteases activities, was up to 13.59 U/mg, 100 times higher than that of cathepsins in muscle. Trypsin from the shrimp head was leaked from head and penetrated into the shrimp muscle during the ice storage. Total proteolytic and trypsin activities in shrimp muscle were detected during extended storage of 4 days. Total proteolytic activity in shrimp muscle was up to 1.66 U/mg after 8 days storage, while trypsin activities were 0.97 U/mg and 1.84 U/mg when BApNA and TAME were used as substrate, respectively. The fluorescence results clarified that shrimp head had a 42.66% drop in F value while the first segment of muscle had a 55.77% rise in F value during 4 days of storage. The second segment of muscle raised just 12.60% after 8 days of storage. In addition, FITC-trypsin could pass through the myolemma and sarcoplasm of myofiber. Therefore, trypsin possibly penetrated into muscle from head during storage and moved into myofiber of shrimp.The comparison of the proteolytic degradation of myofibrillar protein by main endogenous proteases and the observation of the dissolution of muscle by trypsin were studied in order to demonstrate that trypsin played a main role in muscle degradation. The proteases from shrimp head is the main factor of the degradation of myofibrillar protein, but not the proteases from shrimp muscle. The proteases from shrimp head had an obvious destruction on MHC and Actin, and the degradation rate increased with the storage temperature. The degradation of myofibrillar protein by trypsin from shrimp head was more remarkable than by pepsin. The break of MHC was stronger, but the dissolution of Actin was little slower by trypsin, and the degradation rate also increased with the storage temperature. Microscopic results indicated that trypsin could dissolute the muscle tissue of shrimp. As a consequence, trypsin was the main destroyer of muscle and protein from shrimp. |
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