Comparative Analysis On The Characteristic Of Phenol Oxidase In Different Shrimps And Preliminary Study On The Melanosis Mechanism

Melanosis is a prominent problem in the transportation and storage of shrimps.The production of melanin greatly affects the appearance of shrimps,reduces the value of commodities,and causes huge losses to the fishery economy.Different types of shrimp have very different blackening processes.At present,the process of shrimp melanosis has not yet been fully ascertained,and the differential mechanism of shrimp melanosis remains to be further studied.In order to explore the reasons for the differences in the blackening process between different types of shrimp,this study selected five common species of Litopenaeus vannamei,Metapenaeusensis,Fenneropenaeus chinensis,Penaeus japonicus and Trachypenaeus curvirostris as the research objects,and observed their blackening process,compare the biochemical properties of phenoloxidase(PO),explore the law of proPO activation,analyze the different mechanisms of blackening at different types of prawns.On this basis,select Penaeus japonicus to investigate the effects of grape seed extracts(GTE),pomegranate peels(PPE)and green tea extracts(GTE)on its PO activity and blackening.The main findings are as follows:1.Study on the differences in the blackening process and PO tissue distribution of different species of prawns under 4?cold storage conditions.Observation showed that Litopenaeus vannamei,Metapenaeusensis,Penaeus japonicus and Trachypenaeus curvirostris had obvious blackening on the 3rd day of storage,while Fenneropenaeus chinensis had obvious blackening on the 5th day.Variations are mainly concentrated in the cephalothorax.The PO activity of different parts of five species of prawns was tested,and it was found that all five species of prawns had the main PO activity in the cephalothorax,but there were differences in the PO activity.The PO activity of Trachypenaeus curvirostris was the highest,and that of Fenneropenaeus chinensis was the lowest.The molecular weight of PO in different parts of the five different shrimps is about 270 kDa.2.Comparative analysis of PO biochemical properties of different species of prawns.The crude PO enzymes from the cephalothorax of five species of shrimps were extracted and purified.The final purification folds of the five species of shrimps of Litopenaeus vannamei,Metapenaeusensis,Fenneropenaeus chinensis,Penaeus japonicus and Trachypenaeus curvirostris reached 12.08,10.64,29.94,33.22 and 14.32,respectively.The optimal p H of the five different prawns is between 5.5-7.5,and the optimal temperature is between 35?-55?.Among them,the PO from Trachypenaeus curvirostris has stronger acid-base and high temperature tolerance.Among the five species of shrimp,the Km of Penaeus japonicus PO is the smallest,indicating that it has a stronger affinity for substrates,while the Km of Fenneropenaeus chinensis is the largest,which is consistent with its relatively slow melanization process.3.Study on the activation law of proPO in different species of prawns.After extracting the proPO solution of the five different shrimps,the proPO gradually showed PO activity under storage conditions of 4?,and the activity gradually increased to a relatively stable state with time,but there were obvious differences in the proPO activation process of the five different shrimps.Among them,the activation speed of Fenneropenaeus chinensis is the slowest and the enzyme activity is the lowest when it reaches a stable state,whlie the enzyme activity of Trachypenaeus curvirostris is the highest.Temperature has a significant effect on the proPO activation process.Increasing temperature can accelerate the activation of proPO,high temperature can promote the activation of proPO in a short time,and prolonged high temperature can cause enzyme inactivation.SDS,Ca²⁺,Mg²⁺,methanol,acetone and so on all have a certain promotion effect on the proPO activation of five different shrimps.4.Research the effects of plant-derived enzyme activity inhibitors on delaying the blackening of Penaeus japonicus.The inhibitory effects of grape seed extract(GSE),pomegranate peel extract(PPE)and green tea extract(GTE)on PO enzyme activity had been verified.GTE has a better inhibitory effect at the same concentration.The results of inhibition kinetics experiments showed that GSE and PPE are non-competitive inhibitors,and GTE is a mixed inhibitor.The inhibition constants of the three inhibitors are 10.53 mg/m L,21.68 mg/m L and 1.96 mg/m L,respectively,indicating that GTE has a higher affinity for binding PO.In addition,three inhibitors all have effects in Cu²⁺reduction ability and chelating ability,which can delay and reduce the blackening of Penaeus japonicus,and have a potential to be developed as an anti-blackening and fresh-keeping agent.