Study On The Antibacterial Stability Of Nisin Based On Protease Activity Regulation Strategy

Nisin,a natural antibacterial peptide produced by Streptococcus lactis,is the most commonly used food preservative.Nisin is sensitive to application environment,protease and high temperature conditions can affect the antibacterial stability of Nisin,reduce its antiseptic effect.In this study,we screened protease activity regulators from natural polyphenols to protect the antibacterial stability of Nisin in high protease environment by inhibiting protease activity.Secondly,Nisin compound preparation based on protease activity regulation was prepared,and its application effect in low salt emulsified sausage was explored.The main research contents and results are as follows:1.Screening of protease regulators（1）Computer simulation of Nisin hydrolysis was used to analyze and identify the types of proteases that could hydrolyze Nisin.The results showed that only 57.20%of Nisin antibacterial potency was retained after trypsin treatment,and its antibacterial activity was inhibited.The results showed that curcumin,catechin and（-）-epigallocatechin gallate（EGCG）decreased the trypsin activity by 17.50%,23.20%and 26.80%,respectively.These three polyphenols can be used as trypsin regulators.（2）The interaction between the regulator and trypsin was analyzed by molecular docking method.The results showed that there were hydrogen bond interactions between hydroxyl groups of the regulators（curcumin,catechin and EGCG）and amino acid residues Asn157（A）,Asn212（A）,Asn296（C）and Asn304（C）in trypsin.The protective effect of the regulator on Nisin antibacterial stability in protease environment was verified by bacteriostatic experiments.The results showed that catechin had no protective effect on Nisin’s antibacterial stability,while curcumin and EGCG increased Nisin’s antibacterial activity by 37.60%and 33.00%,respectively,and had protective effect on Nisin’s antibacterial stability in protease environment.2.Study on the mechanism of EGCG protecting the antibacterial stability of Nisin（1）The protective effect of EGCG in nisin-EGCG complex on Nisin was investigated by measuring the antibacterial potency of Nisin.The results showed that the titer loss rate of Nisin after EGCG treatment was reduced from 41.73%to 30.52%at 126℃,indicating that EGCG can protect the antibacterial stability of Nisin in high temperature environment to a certain extent.（2）The results of electrophoresis,gel chromatography,particle size and potential analysis and scanning electron microscopy showed that EGCG could form high molecular weight complex with Nisin.The interaction between Nisin and EGCG was further analyzed by using spectroscopy（circular dichroism,infrared and fluorescence spectroscopy）.Circular dichroism spectra showed that the characteristic peak of Nisin appeared red shift after EGCG was added,indicating that EGCG affected the secondary structure of Nisin.Infrared spectra showed that nisin-EGCG complex had characteristic peaks of hydrogen bond vibration at 1235.53 cm^-1,1144.90 cm^-1 and 1035.57 cm^-1,and the thermodynamic reaction parameters of nisin-EGCG complex were calculated asΔG（297K,-19.132 k J/mol）,ΔS（-1.210 k J/mol）andΔH（-380.922 k J/mol）are all negative values,indicating that the binding process of EGCG and Nisin occurs spontaneously,and the hydrogen bond interaction predominates the binding process.Binding molecular docking analysis showed that the polyhydroxy structure of EGCG could form hydrogen bonds with amino acid residues such as Lys45,Met44 and Thr31 of Nisin.（3）The changes of bacterial count during the storage of meat products were measured at 25℃to evaluate the protective effect of EGCG on Nisin.The results showed that the storage period of meat products treated with Nisin-EGCG complex was extended from 48 h to 72 h,indicating that Nisin-EGCG complex can effectively inhibit the growth and reproduction of microorganisms,and EGCG can protect the antibacterial stability of Nisin in meat products.3.Application of Nisin-EGCG complex in low salt emulsified sausage（1）Total colony count（TVC）,volatile base nitrogen content（TVB-N）and thiobarbituric acid active substance content（TBARS）were measured during storage of low-salt emulsified sausage,and the bacteriostasis effect of Nisin-EGCG complex in low-salt emulsified sausage was evaluated.The results showed that Nisin significantly inhibited the growth and reproduction of microorganisms in low-salt emulsified sausage 3 days before storage（P<0.05）,and the total number of bacteria in nisin-EGCG complex group approached the minimum value of 5 log（CFU/g）on the 7th day.The results of TVB-N and TBARS were consistent with the colony number.The nisin-EGCG complex can slow down the corruption of low-salt emulsified sausage,inhibit the growth of microorganisms,and has an outstanding fresh-keeping effect on low-salt emulsified sausage.（2）The texture（elasticity,hardness,cohesiveness and chewiness）and color of low-salt emulsified sausage during storage were measured,and the application effect of nisin-EGCG compound in low-salt emulsified sausage was evaluated.The results showed that nisin-EGCG complex can improve the texture characteristics of low-salt emulsified sausage to a certain extent,and improve the total color differenceδE of low-salt emulsified sausage.In this study,the antimicrobial stability of Nisin was protected by regulating protease activity,trypsin regulators were screened and identified,and nisin-EGCG compound preparation was prepared to explore the protective mechanism of EGCG against Nisin,and the application effect of Nisin-EGCG complex in low-salt emulsified sausage was investigated.The application of Nisin in the field of meat processing was expanded.