| The bacteriocin secreted by lactic acid bacteria is safe,non-toxic and has strong antibacterial activity,can be degraded by the body,does not produce drug resistance and other advantages.It widely regarded as an effective alternative to antibiotics.Yak is a unique species of the Qinghai-Tibet Plateau.It is survival mportant for the local herdsmen.However,there were no reports about the isolation and identification bacteriocin secreted by lactic acid bacteria from the gastrointestinal tract.The purpose of this experiment is to isolate and identify the bacteriocin secreted by lactic acid bacteria from yak.So we can study its biological characteristics and to interpret the prebiotics in the molecular level.For the further development of yak-specific micro-ecological agents to lay the foundation.Achieved the following results: 1 Isolation and identification of Lactobacillus plantarum from a yak-producing bacteriocin91 strains of lactic acid bacteria were isolated from 19 samples from yak feces,by containing solid medium containing 0.5% calcium carbonate(MRS).The way of filter lactic acid bacteria by Oxford Cup and identified by 16 S rRNA.The results show:After eliminating the interference factors such as organic acid,hydrogen peroxide,9 strains of lactic acid bacteria still inhibit the Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 and sensitive to protease.Identify by 16 S rRNA,4 strains of Enterococcus faecalis,3 strains of Enterococcus faecium,1 Enterococcus faecalis,1 strain of Lactobacillus plantarum.In view of the best antibacterial effect of Lactobacillus plantarum and is the "feed additive variety directory" and "food additives variety directory" allowed to use the bacteria,so the election object,named Lactobacillus plantarum SWUN5815.Further studies have shown that this strain has a strong broad-spectrum antimicrobial activity against clinically isolated yak diarrhea pathogens.The survival rate of this strain was 58.2% at pH = 2,86.9% at pH = 3,66.8% at bile salt concentration of 0.3%.It has a good resistance to acid and salt tolerance.The adhesion rate to Caco-2 cells was 42%.Sensitive to commonly used antibiotics,not resistant.Therefore,the strain is a potential candidate probiotic strain.2 Effects of Lactobacillus plantarum SWUN5815 on growth performance and intestinal mucosal immunity in mice40 male mice of about 18 g were divided into groups 4 of 10 rats each.group 1-3 were treated with 5 × 106 CFU,5 × 107 CFU and 5 × 108 CFU Lactobacillus plantarum SWUN5815 respectively for 21 days.Groups 4 were the control group,and the same volume of MRS broth was fed with control.Each group was randomized to receive 3 experimental groups and 3 control mice.Scrape the small intestine mucosa by adding an equal volume of PBS buffer(pH=7.4)and mix with centrifugal centrifugation at 6000 × g for 15 min.The supernatant was stored at-20 and SIgA ?was measured by double anti-sandwich method.The results showed that the mean weight gain of each dose group was higher than that of the control group(P<0.01).The weight gain of the dose group was significantly higher than that of the control group(P<0.01).And SIgA significantly increased(P<0.01).Therefore,the Lactobacillus plantarum SWUN5815 can promote the growth and development of mice,improve the intestinal mucosal immunity,can be used as a candidate strain of yak probiotics.Effects of Lactobacillus plantarum SWUN5815 on antagonism of Salmonella in mice 3 Effects of Lactobacillus plantarum SWUN5815 on antagonism of Salmonella in mice20 male mice of about 18 g were divided into two groups.The experimental group was fed with 5 108 CFU SWUN5815 per day,while the control group was fed with the same volume MRS broth.After 1 week of continuous administration of 5 × 106 CFU Salmonella typhimurium.The rats were sacrificed at 48 h and 96 h,and the lungs,spleen,liver,duodenum,jejunum,ileum,colon and cecum.4% paraformaldehyde was fixed after slicing,H.E staining,observed histopathological changes.Simultaneous determination of TNF-?,IFN-?,1L-1? and 1L-6 inflammatory cytokines in the tissues of duodenum,jejunum,ileum,colon and cecum by ?-actin internal reference and fluorescence quantitative PCR The change.The results showed that the pathological changes of tissues and organs in the experimental group were significantly lighter than those in the control group at 48 h and 96 h after infection.Compared with the control group,the TNF-?,IFN-?,1L-1? and 1L-6 cytokines were significantly inhibited 48 hours after infection,and the difference between the experimental group and the control group was decreased after 96 h infection.Therefore,SWUN5815 has the effect of inhibiting the expression of inflammatory cytokines and reducing the intestinal pathological damage caused by Salmonella.4 Study on genome and extracellular proteome of Lactobacillus plantarum SWUN5815 strainIn order to further analyze the probiotic properties from the molecular level,the high quality genome of SWUN5815 strain was obtained by 3 generations gene sequencing,and the extracellular protein of the strain was identified by mass spectrometry.The results showed that the genome of the strain was 3345067 bp in length,encoding 3258 genes,Which contains one can be synthesized I class of sulfur bacteriocin and 18 synthetic class II bacteriocin-related genes,So it has broad-spectrum antibacterial activity.While the strain has a gene cluster encoding Atg22 and DUF1440 family proteins.So it has resistance to bile salt and acid resistance.The strain has a gene cluster encoding the Dps family of proteins,which promotes the innate immunity of the host.The extracellular protein was identified by 85 proteins,which involved antimicrobial peptide synthesis,transport,starch and sucrose metabolism,two-component systems,and other metabolic pathways.The results showed that the probiotic characteristics of Lactobacillus plantarum SWUN5815 were explained at the molecular level,which provided a reference for the further development and utilization of the strain. |
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