Isolation Of Halophilic Microorganisms And Identification Of New Specie In Panjin Grasshopper Sub Shrimp Paste

Shrimp paste is a traditional fermented aquatic product in coastal areas of China and southeast Asia.Panjin shrimp paste is made by mixing the shrimps of liaohe river and bohai sea in panjin area with 15%²0%sodium chloride for several months.As a kind of fermented aquatic products,the microbial community and its role in shrimp paste have been widely concerned.In this paper,the halophilic microorganisms in panjinshopper shrimp paste were identified by colony morphology and 16S rRNA analysis.A strain was identified as a new species by cell morphology,physiology,biochemistry and molecular biology,and named Lentibacillus panjinensis.The main contents and conclusions are as follows:1.Experimental methods:The microorganisms were separated and purified from panjin grasshopper shrimp paste.Single colonies were selected from the culture dishes for gram staining to identify the pure bacteria,and then the Tiangen bacterial genome extraction kit was used to extract DNA from bacteria with uniform color and single morphology observed within the scope of the microscope field.If the bacterial colonies with inconsistent color and different morphology were not purified,the DNA was extracted.After DNA extraction,the concentration of bacteria was tested by agarose gel electrophoresis and enzyme labeling apparatus.General primers 27F and 1492R were used for PCR amplification of DNA,and the obtained PCR products were handed over to sangon biotech for sequencing.Halophiles were screened by concentration gradients of 5%,10%,15%,20%and 25%,respectively.Strains are obtained by NCBI BLAST analysis.Strain G56^T morphology analysis（gram staining,scanning electron microscope bacteria,flagella）,physiological and biochemical experiments（sugar alcohol fermentation experiment,enzymes,the determination of fatty acids,polar lipid testing）,molecular biology identification（DNA extraction,PCR and sequencing,the establishment of the phylogenetic relationship）.2.Through to set up a file in the shrimp paste,the separation and identification of halophilic microorganisms found ten strains of bacteria resistant to salinity is as high as 25%,after the extraction of DNA and PCR amplification of sequencing results,they respectively belong to Kouria,Allobacillus,Paenibacillus,Salinicoccus,Bcillus and Lentibacillus.3.A Gram-positive,aerobic,motile,short rod-shaped bacteria,strain G56^T,was isolated from panjin grasshopper shrimp paste.The optimum growth temperature,NaCl concertration and pH of strain G56^T is 37℃,10%and 7.0.G56^T was positive for catalase and oxidase,but had no ability to reduce nitrate.D-ribose,D-xylose,D-galactose,glycerin,arbutin,salicylic acid,D-mannose,D-fibrous disaccharide,N-acetylglucosamine,glycogen,D-trehalose can be used as substrate to produce acid.But xylitol,L-sorbitol,D-tagose,erythritol cannot be used as substrate to produce acid.The main fatty acids were anteiso-C_15:0（69.7%）and anteiso-C_17:0（23.3%）.The DNA G+C content of G56^T strain was 44.7%mol%.Through polyphasic classification and analysis,we reported that strain G56^T（CGMCC 1.17100^T）is a new type of bacteria,which named Lentibacillus panjinensis.Conclusion:in this study,a new strain named Lentibacillus panjinensis was obtained through the isolation and screening of halophilic microorganisms in grasshopper shrimp paste from panjin area of liaoning province.Which can provide reference for the analysis of the diversity of microbial community and food safety of shrimp paste.