Preparation Of Surface Mucous And Study On Autolysis Of Urechis Unicinctus

Urechis unicinctus is an economic Marine invertebrate living in the sediment bottom of shallow sea.Previous studies show that the extracorporeal secretions of Urechis unicinctus have certain antibacterial activity,which can avoid the accumulation of pollutants and the growth of harmful microorganisms around the hole.At the same time,it is also found that there is autolysis of Urechis unicinctus,that is,when the body wall is damaged,it is easy to produce body wall perforation,visceral leakage,and eventually lead to the phenomenon of overall liquefaction.[Objective]In view of these two phenomena,this study establishes a preparation method of Urechis unicinctus surface mucus,and analyzes its antibacterial activity.Purification of protease that has the function of dissolving body wall in unicinctus viscera,analysis of its action conditions and main composition,study of its autolysis mechanism,and then explore the application prospect of unicinctus surface mucus and autolysis in medical materials,food processing and other aspects.[Methods]Using microcurrent stimulation method,combined with a simple collection device established,the surface mucus of Urechis unicinctus was collected.After freeze-drying,the antibacterial activity of the mucus against Escherichia coli,Streptococcus hemolyticus,Acinetobacter baumannii and Propionibacterium acnes was tested by filter paper and bacteriostatic zone method.The hemolytic activity was analyzed.After cleaning unicinctus viscera homogenate and extraction,the supernatant is deposited with saturated ammonium sulfate solution at low temperature,and the crude enzyme solution is obtained by dialysis.Using Urechis unicinctus body wall as substrate,the optimal enzymatic hydrolysis conditions and stability were analyzed.Fluorescent casein was used as substrate to demonstrate its serine protease activity.The crude extract was separated by Sephadex G-100,and its molecular weight was determined by SDS-PAGE.The wall breaking effect of these enzymes on Chlorella cells was investigated by blood cell plate counting method.[results](1)The optimal stimulus voltage for Urechis unicinctus surface mucus collection is 6V,and the optimal stimulus temperature is 4?;The maximum preparation amount of mucus was 3.291g,and the yield was 1.533%.The mucus had certain antibacterial activity.At 0.4g/m L,it had certain inhibitory effect on Escherichia coli,Hemolytic Streptococcus and Acinetobacter baumannii.The mucus has certain anticoagulant effect.(2)After saturated salt solution precipitation and dialysis filtration,the visceral protein solution derived from unicinctus has certain autolytic enzyme activity;The average molecular weight measured by SDS-PAGE was about 8k Da.The enzyme activity analysis showed that it had good thermal stability,and the enzyme activity could still maintain about80%after heat treatment at 80-100?for 2h.It has the ability to degrade the unicinctus wall,and the enzymolysis effect is best at 70?and p H 9.Metal ion Pb²⁺could inhibit the activity of the enzyme,while Ca²⁺and Zn²⁺could improve the activity of the enzyme.Histological sections show that such enzymes can destroy the normal structure of unicinctus wall.The cell wall of Chlorella could be degraded in vitro.It is speculated that in the autolysis process of unicinctus,there is at least a phenomenon of structural instability caused by endogenous leakage of this enzyme.[conclusion](1)The preparation method of Urechis unicinctus surface mucus is established and optimized;(2)Unicinctus surface mucus has certain bacteriostatic and anticoagulant functions;(3)The extraction and purification process of Urechis unicinctus autolysase system is established;(4)The autolytic enzyme system contains serine protease,which dissolves and destroys the fibrous structure of the body wall and causes the body wall to lose its homeostasis;(5)One of the main mechanisms of unicinctus autolysis is identified:damage leads to the release of visceral enzymes,dissolution of the body wall causes perforation of the body wall,intensification of visceral leakage,and overall liquefaction;(6)The autolytic enzyme system has a good effect on the dissolution and wall breaking of some algae cells.