Purification And Characteristics Of Bacteriocin From Lactobacillus Plantarum SHY21-2

Microbial safety in food production,processing and storage has been the focus of food industry for a long time.At present,chemical preservatives are commonly used as preservatives,but with the increasing demand of consumers for high-quality food,more and more attention has been paid to the potential harm of chemical preservatives.Therefore,the research and development of biological control technology for food microbial safety is an important development direction in the future.Bacteriocin,as a natural and safe bacteriostatic factor,is one of the development directions.At present,only nisin is used in industry,but nisin has a narrow antibacterial spectrum and can not inhibit Gram-negative bacteria and yeast.Therefore,it is of great significance to find an efficient and broad-spectrum bacteriocin.In this study,lactic acid bacteria isolated from traditional fermented foods such as kimchi,kola,and yak yogurt were used as research objects.A high-yielding broad-spectrum bacteriocin strain was selected as the objective to optimize the bacteriocin purification process and a new bacteriocin plantaricin LP21-2.Some characteristics of this bacteriocin were discussed.The experimental methods and main conclusions are as follows:(1)Screening and identification of bacteriocin-producing lactic acid bacteriaFrom the 28 strains deposited in this experiment,the best antibacterial effect on S.aureus ATCC25923 was screened by the agar diffusion method.The growth curve and bacteriocin production over time of strains were measured within 48 h.Ammonium sulfate was added to the supernatant of the selected strains for precipitation and the antibacterial activity of the precipitates was tested.The strains identified were identified by morphological identification,API lab plus system and 16S r DNA sequence.The results showed that the strain SHY21-2 had the strongest antibacterial effect on Staphylococcus aureus.The bacteriocin production peaked at 26 h of culture,and most of the bacteriocin was precipitated when 80%ammonium sulfate was added.The strain SHY21-2 was identified as Lactobacillus plantarum.The bacteriocin produced was named plantaricin LP21-2 and abbreviated as LP21-2.(2)Purification and physicochemical properties of bacteriocinThe culture supernatant of strain SHY21-2 was separated and purified,and its amino acid sequence was analyzed by LC-MS/MS,and identified by APD database.Purified bacteriocins were treated at 60?,80?and 100?for 10 and 30 min,respectively,and the antibacterial activity was measured after 15 min at 121?.The antibacterial activity was analyzed in different acid-base environments and after protease hydrolysis.It was found that LP21-2 can be purified from the fermentation supernatant of strain SHY21-2 by crude extraction with ammonium sulfate,dextran gel G-10 desalting,cation exchange chromatography,gel column superdex-20010/300 GL purification;and finally obtained by R-HPLC analysis and separation.LP21-2 has a molecular weight of 1362.84 Da,an amino acid sequence of RPNEPRLLLPR,a hydrophobic amino acid content of 27%,and a molecular formula of C₆₀H₁₀₆N₂₀O₁₅.Plantaricin LP 21-2 is a new type of bacteriocin.Its antibacterial activity has not decreased significantly after high temperature treatment,and the optimum p H range is 2-5.Plantaricin LP 21-2 can be hydrolyzed by pepsin,trypsin,and papain,and the antibacterial activity disappears completely.(3)Bacteriostatic mechanism of bacteriocin and sequence analysis of its whole geneThe common contaminating bacteria in food were selected as indicator bacteria to study the bacteriostatic spectrum of LP21-2.The bacteriostatic mechanism of LP21-2was initially analyzed by scanning electron microscope and transmission electron microscope.The whole gene sequence of strain SHY21-2 was determined,and genes related to bacteriocin synthesis were analyzed based on the annotation results of the COG database.The BLASTP program was used to infer the genes encoding the bacteriocin basic structure.It was found that LP21-2 is a broad-spectrum antibacterial peptide,which can inhibit Listeria monocytogenes,Bacillus cereus,Bacillus subtilis,Escherichia coli and Saccharomyces cerevisiae and food-borne pathogens in food.LP21-2 is a type IId bacteriocin that acts on S.aureus by bactericidal rather than bacteriostatic;the mechanism of action is to form holes in the cell membrane,leak the cytoplasm,and finally die.The total gene size of strain SHY21-2 is 3,333,809bp,including 1 chromosome and 4 plasmids,3187 complete coding regions(CDS)were obtained.In the annotation results,Lux S,argc and lytt protein molecules related to quorum sensing were found.The coding gene of LP21-2 infrastructure is located on chromosome.In conclusion,the bacteriocin has the characteristics of high temperature resistance,can be degraded by enzymes in the digestive tract of the human body,and will not affect the human body.In addition,the bacteriocin can not only inhibit Gram-positive bacteria,but also Gram-negative bacteria,even the Saccharomyces cerevisiae.Therefore,bacteriocin plantaricin LP 21-2 can be used as a non-toxic,efficient and broad-spectrum preservative in the food industry.