| With the change of people's living environment and dietary habits,many new diseases begin to appear and pose a serious threat to human health.Under the background of the concept of "big health" and its industrialization,people's health awareness has been gradually improved.Compared with various problems caused by the use of antibiotics,probiotic therapy has attracted extensive attention.With the continuous research of probiotic therapy in the application of diseases,the new functions of probiotics in the field of new and important disease prevention and treatment are constantly refreshing people's cognition.China has a large population and significant difference in dietary culture,resulting in high diversity and richness of intestinal flora,which provides a good source of samples for the study of probiotics.Lactic acid bacteria are representative probiotics,and as the dominant flora in human intestinal tract,they can regulate the balance of human intestinal flora and improve host health,which is particularly important for disease prevention.The aim of this study was to isolate lactic acid bacteria strains from intestinal tract of healthy Chinese population and select lactic acid bacteria strains with bacteriostatic activity.Preliminary study on potential probiotics of isolated strains were carried out through simulated gastric juice,intestinal juice test and surface hydrophobicity test,,and drug resistance of strains was analyzed by antibiotic disk diffusion method,.The main bacteriostatic substances in fermentation broth of selected potential probiotic and safe strains were analyzed,in order to obtain strains were more in line with the intestinal characteristics of Chinese population,besides they possesshigh bacteriostasis and good probiotics,which could be expected to be applied in industry production in the future.The main results are as follows.Eighty lactic acid bacteria strains were isolated and purified from the feces of 10 healthy adults.According to the 16 Sr RNA gene similarity analysis,it was confirmed that they belong to12 species of Lactobacillus,Lactococcus,Leuconostoc,Enterococcus and Streptococcusrespectively.To save moneyand material resources,eleven representative strains of Enterococcus,Lactococcus garvieaeand Streptococcus salivarius with low application potential were selected according to phylogenetic relationships.They were strain 2(Lactococcus garvieae),strain 11(Lactococcus garvieae),strain 17(Enterococcus thailandicus),strain 28(Enterococcus gallinarum),strain 29(Streptococcus salivarius),strain 30(Enterococcus faecium),strain 32(Enterococcus gallinarum),strain 33(Enterococcus hirae),strain 59(Enterococcus durans),strain 63(Enterococcus faecalis),strain 78(Enterococcus faecium),and Lactobacillus strains 1,6,8,20,60,65(Lactobacillus salivarius),strain 9(Lactobacillus ghanensis),strain 24(Lactobacillus paracasei),and strain 12(Leuconostoc lactis)respectively,a total of 20 strains for subsequent bacteriostatic screening test.Using Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 as indicator bacteria strains,twenty strains of lactic acid bacteria were tested by Oxford cup double-layer AGAR diffusion method.And 5 strains of lactic acid bacteria with obvious antibacterial activity against both indicator bacteria strains were selected,which was strains 1(Lactobacillus salivarius),strains 9(Lactobacillus ghanensis),strains 12(Leuconostoc lactis),strains 24(Lactobacillus paracasei)and strains 63(Enterococcus faecalis)seperatedly.Probiotic traits and drug resistance studies were carried out on 5strains of lactic acid bacteria with antibacterial activity.Three strains including strain 1,strain 12 and strain 24 were selected,which had strong viability in simulated gastric juice at p H 3.0 and intestinal fluid containing 0.3% bovine bile salts.Meanwhile they also possess certain adhesion ability and didn't have multi-drug resistant.The bacteriostatic substance and properties studies of these 3 strains showed that the bacteriostatic substances in the fermentation broth had good thermal stability,and hydrogen peroxide were not the effective ingredient which exerting antibacterial activity.The main antibacterial component of strain 12 was organic acid;the main antibacterial components of strain 1 and strain 24 were organic acids and a kind of protein material.Ammonium sulfate precipitation method,organic solvent extraction method and aqueous two-phase extraction method were used to extract the protein bacteriostatic substances which may be present in the fermentation broth,and the extraction conditions were optimized.Results showed that the extraction effect of sample by ammonium sulfate precipitation method was unsatisfied.It was speculated that they were small molecular peptides which existed in the fermentation broth of the two strains.The organic solvent method and the two-phase aqueous method both had good extraction effects on the samples,and the extraction effect of the aqueous two-phase system was the best.After the aqueous two-phase extraction,the antibacterial activity of the crude fermentation solution of the strain 1 was determined,and the specific activity of that was 260.16,which was 6.41 times higher than that of the original fermentation broth.The specific activity of the bacteriostatic substance obtained from the crude fermentation of strain 24 was 161.61,which was 4.35 times higher than that of the original fermentation broth.The electrophoresis analysis of the sample indicated that the bacteriocin were indeed present in the fermentation broth of strain 1 and strain 24,and it was confirmed again that the aqueous two-phase system had better extraction effect.The bacteriostatic spectrum of crude bacteriocins test showed that bacteriocin produced by the two strains had relative broad-spectrum antibacterial effect.In summary,three probiotic lactic acid bacteria with high antibacterial activity were screened in this study,which might provide strain resources and application basis for the development of probiotic preparations for the prevention and treatment of gut diseases caused by intestinal pathogens. |
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