Properties And Application Of Antimicrobial Proteins Derived From Rahnella Aquatilis

Antibacterial proteins are usually produced by innate immune systems of different species,such as animals,plants or fungi.These proteins form specific immunity against microbial invasion,and have biological activities such as antifungal,bacterial,and viral.This kind of bioactive protein can play a variety of physiological functions,for example,it can be used in medicine,food and feed processing,and has a broad application prospect.As a natural preservative,antibacterial protein has the characteristics of high safety and broad antibacterial spectrum,which has attracted researchers’interest.This paper mainly provides development prospects for the application of antibacterial proteins.The antibacterial protein obtained from the strains with the effect of inhibiting bacteria screened by the laboratory,combined with 16S r DNA molecular identification and physiological and biochemical identification,was used to identify the strains of the fermentation strains,and the fermentation process conditions of the strains were optimized to achieve the best inhibition.Bacteria effect,study the basic properties of protein On this basis,the anti-bacterial protein’s freshness-preserving effect on beef grains is studied,and attempts to broaden the application range of antibacterial protein to provide a theoretical basis and an effective way for the deep development and utilization of the antibacterial protein.At the same time,it provides powerful measures to solve the abuse of chemical preservatives and food safety crises on the market,which has certain economic and social significance.Finally,the biologically active protein is separated and purified,and finally the biologically active antibacterial protein is obtained,so as to continue to study the deeper level of antibacterial mechanism and the promotion and application of antibacterial protein.The main research contents and results are as follows:1.Identification and fermentation optimization of strains producing antibacterial proteinsA strain producing antibacterial protein was isolated from the lateritic soil of Dongchuan,Yunnan Province.The strain was identified by morphological observation,Gram staining,physiological and biochemical identification and 16S r DNA molecular biology as an aquatic Rahnella Aquatilis),on the basis of single factor experiment,select the significant factors,and then take the bacteriostatic rate as the evaluation index,use the box Behnken experiment to optimize the fermentation conditions to improve the bacteriostatic rate.The optimal conditions were:fermentation time 72 hours,lactose content 2.0%,fermentation temperature 16℃.Under the optimized conditions,the theoretical value of the bacteriostatic rate of E.coli was 63.59%,and the experimental results showed that the bacteriostatic rate was close to the predicted value of 61.3%.2.Study on the properties of crude proteinThe crude protein was obtained by centrifugation,ammonium sulfate salting out,dialysis and desalination.the antibacterial ability of crude protein to 7 different bacterias was compared by 96hole plate method.The results showed that the highest inhibition rate of protein to Escherichia coli was 71.65±0.6%.The antioxidant capacity was determined by DPPH·clearance rate,ABTS^+·clearance rate and hydroxyl clearance rate.ABTS^+·has the best clearance rate and certain antioxidant capacity,and carries out MTT cell acute toxicity test to determine the sterile concentration of the protein（150μg/m L）.3.Application of antibacterial protein in beef preservationUse ultrafiltration technology to ultrafiltration and sterilize the crude protein,and perform MTT cell acute toxicity test to determine the sterile concentration of the protein（150μg/m L）.Select the processed crude protein to keep the beef fresh,4℃Under the conditions of storage,the sensory changes of beef,p H value,volatile basic nitrogen（TVB-N）,thiobarbituric acid（TBA）,methemoglobin content,texture and total number of colonies were used as freshness evaluation indicators.Biofilm made from crude protein can extend shelf life by 2 days compared to sterile water-treated beef.4.Separation and purification of antibacterial proteinsThe obtained crude protein was separated by DEAE weak anion column and S-100 gel column to obtain one main antibacterial active component,and then subjected to SDS polyacrylamide gel electrophoresis detection to obtain pure protein,and the protein was subjected to the optimal temperature,The determination of p H and its stability,and finally the preliminary study of the leakage of K⁺and Na⁺in its bacteriostatic mechanism,in order to classify and identify the unknown antimicrobial protein obtained later.