Effect Of Surface Layer Proteins From Lactobacillus On The Strains’ Probiotic Properties And Their Antibacterial Function

Lactobacillus surface layer proteins（Slps）,as a special structure outside the cell wall of Lactobacillus,have been proved to have the the function of protecting the integrity and probiotics of the cells.In recent years,more and more studies have begun to pay attention to the biological functions of Slps,such as antioxidant,immune function and antimicrobial effect,but the mechanism of their action on the biological function of Slps and the scope of research is still relatively limited.In this study,the effects of Slps on the probiotic properties of Lactobacillus were studied,and the adhesion,anti-inflammatory and antibacterial functions of the Slps after purification were further explored,which aimed to discover more biological functions of Lactobacillus Slps and provide a theoretical basis for its further research and application.First,seven strains of Lactobacillus carrying Slp were screened by extraction and SDS-PAGE identification（Lactobacillus.acidophilus NCFM,L.acidophilus ATCC 4356,L.acidophilus CGMCC 1.1878,L.fermentum CCFM 421,L.gohnsnil JCM 1101,L.crispatus JCM 2009,L.fermentum CCFM Y40）.After using LiCl to remove the Slps of seven strains of Lactobacillus,the strains’autoaggregation rate,surface charge and Salmonella coaggregation rate were significantly lower than that of the complete strain（P<0.05）.The surface hydrophobicity of the strain was significantly lower than that of the intact strain except L.acidophilus NCFM（P<0.05）,indicating that the presence of the Slp has a positive effect on the surface properties of Lactobacillus.Simulated gastrointestinal tolerance experiments showed that the survival rate of LiCl-treated strains in the simulated gastrointestinal environment was significantly lower than that of the intact strains（P<0.05）.The results showed that Slps had protective effect on lactobacillus in gastrointestinal environment.The results of Lactobacillus adhesion to HT-29 cells showed that after the removal of Slps,the adhesion rate of 7 Lactobacillus was significantly lower than before,and the decrease of L.acidophilus NCFM was the highest,reaching 73.6%（P<0.05）.The Slp of four strains of Lactobacillus was purified（L.acidophilus NCFM,L.acidophilus CGMCC 1.1878,L.fermentum CCFM 421,L.crispatus 2009）,and it was found that four kinds of Slps could significantly inhibit the adhesion of Escherichia coli and salmonella to HT-29 cells（P<0.05）under the exclusion,competition and replacement of three kinds of treatment methods,but the effect of different methods was different.The results showed that the Slps could antagonize the adhesion of the pathogenic bacteria to the HT-29cells by competing with the pathogenic point of the pathogen or covering up and interfering with its recognition adhesion point.By measuring the cell viability and IL-8 secretion of HT-29 cells infected by E.coli,it was found that L.acidophilus NCFM Slp could inhibit the up-regulation of IL-8 in HT-29 cells induced by E.coli,the results showed that the Slp had the effect of regulating inflammation and immune response,and could regulate the IL-8inflammatory factors associated with inflammatory response.To further explore the antimicrobial effect of Slps on pathogenic bacteria,growth curve and viable count confirmed that surface proteins of L.acidophilus NCFM and L.acidophilus CGMCC 1.1878 could enhance the antimicrobial activity of Nisin against Staphylococcus aureus.When Nisin was treated alone or in combination with surface protein,the membrane permeability and release of A_260nm absorbent substance were significantly increased（P<0.05）.In addition,the results of flow cytometry showed that the combined bacteriostasis mechanism of Nisin and Slps involved in the decrease of intracellular enzyme activity,cell membrane damage and the decrease of cell membrane potential.In the future,it is possible to solve the problem of Nisin resistance by means of the combined use.In conclusion,this study found that carrying Slp has a positive effect on the surface properties,gastrointestinal tolerance and adhesion function of bacteria.Purified Slps can antagonize the adhesion of pathogens and regulate the inflammatory response of cells induced by pathogens.In addition,it can be combined with Nisin to inhibit S.aureus.The study showed the contribution of Lactobacillus Slps to the probiotic function of the strain and its potential for bacteriostatic and immunomodulatory,which provided the basic data for its further development and utilization.