Physical And Chemical Properties,Molecular Modification Of BTI And Its Application In Preservation Of Bass(Lateolabrax Japonicas)

Buckwheat trypsin inhibitor(BTI)is a competitive inhibitor of trypsin.The research shows that BTI has practical significance for the development of new clinical drugs and the research of insect-resistant transgenic crops.Based on the first order sequence and three-dimensional structure of BTI,the physical and chemical properties of BTI were further studied,and mutants of BTI were prepared to obtain a new inhibitor.Finally,the application of trypsin inhibitor in the preservation of sea bass was studied.The main contents of this study are as follows:The first part: The inhibitory activities of buckwheat trypsin inhibitor(SBTI)and soybean were compared,and the effects of temperature,p H,salt ions and organic matter on the activities of the two inhibitors were further determined.The experimental results showed that the inhibitory activity of both BTI and SBTI was decreased after heating in water bath at 100?.After heating for 80 min,the residual inhibitory rate of BTI was 75%,while SBTI was completely deactivated.The p H 2-12 buffer had no effect on the activity of BTI and SBTI.Zn2+ significantly decreased the activity of BTI.When the concentration was 2-10 m M,Mg2+,Cu2+ and H2O2 had no effect on either inhibitor.Urea and KSCN treatment had a certain inactivation effect on BTI,but had no effect on SBTI activity.Dimethyl acetamide can enhance the inhibitory activity of BTI and SBTI.Isopropanol and methanol showed a strong inactivation effect on BTI and SBTI.Dixon plot was used to calculate the inhibitory constants Ki of BTI and SBTI as 0.741�10-8mol/L and 0.652�10-8mol/L,respectively.The second part: Study the action site of recombinant buckwheat trypsin inhibitor(BTI)by site-directed mutation technique and construct its mutants.Buckwheat chymotrypsin inhibitor(BCI)was obtained by purification of mutant expression.The purity of BCI was confirmed by SDS-polyacrylamide gel electrophoresis.The activity test showed that BCI had higher inhibitory activity on chymotrypsin.Stability experiments showed that BCI had good temperature stability and p H stability.After heating in water bath at 50?,60?,70?,80?,90? and 100? for 1 hour,the inhibitory activity of BCI did not change significantly.The inhibitory activity of BCI was not affected by p H 2-12 buffer treatment for 1 hour.When the concentration was 2-10 m M,H2O2,KSCN and Mg2+ had no effect on the inhibitory activity of BCI,while Zn2+ had inhibitory effect on BCI.When the concentration was 10%-50%,isopropanol,methanol and dimethyl acetamide all inhibited BCI.The third part: With gelatin and chitosan as raw materials,by adding lactic streptococcus element(Nisin),and can inhibit the endogenous enzyme decomposition of Nisin in food BTI,preparation of fresh-keeping film,with fresh sea bass as the research object,and determine the Total number of colonies each fresh the next day(Total aerobiccount,TAC),volatile base nitrogen(the Total volatile basic nitrogen,TVB-N)content,Thiobarbituric acid reactive substance(TBARS)content,p H value,cooking loss and other quality indicators.The results showed that the application of the coating significantly inhibited the growth of microorganisms in bass,slowed down the growth rate of TVB-N,TBARS and p H,and reduced the cooking loss.Chitosan and Nisin can be used together to improve the antibacterial and antioxidant functions of the coating film,so as to play a better effect on the preservation of sea bass.