Isolation Of Pseudomonas Fluorescens W3 And Analysis Of Its Extracellular Protein Products

The development of cold chain storage and transportation has made the growth of most of the foreign microorganisms in raw milk effectively suppressed.Under low temperature conditions,psychrophilic bacteria can continue to grow and gradually become the dominant microorganism in raw milk.Part of the psychrophilic bacteria represented by Pseudomonas can secrete some heat-resistant proteases and lipases,which can still retain some activity after the conventional heat sterilization of raw milk.Such nutrients are decomposed,which leads to a decrease in the quality of dairy products.In this paper,a strain of psychrophilic bacteria with high production of extracellular protease and extracellular lipase was selected from raw milk in Heilongjiang,and the heat resistance of the protease and lipase secreted was studied.The proteomic technique was used to identify and bioinformatics analysis of the extracellular protein of this strain.The raw milk collected from Heilongjiang area was divided into 20 psychrotrophic bacteria using the flat line drawing method,and the 20 isolated psychrotrophic bacteria were identified by Rhodamine B plate method and skim milk plate method respectively.Finally,a strain with the strongest ability to produce protease and lipase was selected.The strain was identified as Pseudomonas fluorescens by 16 S rDNA and named Pseudomonas fluorescens W3.The extracellular proteases and extracellular lipases secreted by Pseudomonas fluorescens W3 are heat-resistant.After heating at 65? for 30 min,the residual rate of lipase was 45.69%,and the residual rate of protease was 47.76%;after heating at 90? for 5 min,the residual rate of lipase was 26.32%,and the residual rate of protease was 35.91%;After heating at 137? for 5 s,the residual rate of lipase was 17.36% and the residual rate of protease was 30.98%.Using proteomics technology to identify the extracellular protein products of Pseudomonas fluorescens W3,a total of 547 proteins were significantly increased and338 proteins were significantly decreased.Proteins causing potential harm to dairy products mainly include protease?Protease Htp X,Metalloprotease Loi P,Putative protease Ydc P,Apr X,Putative zinc protease and Tail-specific protease?,lipase?Triacylglycerol lipase and Alpha/beta hydrolase?and lysozyme?Lysozyme?.GO analysis showed that the differential proteins were distributed in three parts:cellular components,molecular functions and biological processes,and the distribution ratio was about 13:10:13.The biological process part is mainly distributedin the metabolic process and the cellular process;in the cell component part is mainly distributed in the cell component and membrane part;in the molecular function part is mainly distributed in the catalytic activity and binding activity part.The extracellular proteases secreted by W3 are mainly metalloproteinases,and the metal ions are Ca²⁺ and Zn²⁺,which show metalloproteinase activity;two lipases show hydrolytic activity;lysozyme shows chitinase activity.Pathway enrichment analysis showed that differential proteins were mainly concentrated in metabolism,accounting for about89%.In terms of metabolism,differential proteins involved in multiple metabolic pathways such as carbohydrate metabolism,fat metabolism,and amino acid metabolism.Bioinformatics analysis comprehensively analyzes the extracellular protein products of Pseudomonas fluorescens W3,and clarifies the metabolic pathways involved in the products,which provides guidance for the control of psychrophilic bacteria in the future.