| Lactobacillus is one of the most common genera of probiotics widely distributed in nature,with a range of physiological activities,such as immune system modulation,obesity suppression,preservation of intestinal homeostasis,anti-inflammatory,anti-cancer,and antioxidant.The current studies of Lactobacillus have mostly focused on the screening of active strains and exploration of functional mechanisms,and there are few comparative studies on the same species of Lactobacillus from different origins.In this study,a strain of endogenous Lactobacillus plantarum(L.plantarum)was isolated from the feces of C57BL/6 mice(LP-M),and four strains of exogenous L.plantarum were chosen from the isolates from the feces of healthy adults(LP-H),sourdough(LP-S),fermented milk(LP-F),and pickles(LP-P).Based on the genome and anti-inflammatory activity,the study of endogenous and exogenous Lactobacillus was conducted to examine the differences in the effects of endogenous and exogenous Lactobacillus on gut health,as well as underlying mechanisms contributing to the differences.The aim of this study is to provide a reference for the rational application of Lactobacillus.The main findings are as follows:1.The genomes of five strains of L.plantarum were sequenced using Illumina Hiseq+Pac Bio and functional genes were annotated through the gene information databases.The results showed that the genome size,GC content,and the number of coding genes of the five L.plantarum strains were comparable with fundamental information on the genome of known L.plantarum.The gene annotation revealed that the five L.plantarum strains’ genes were primarily involved in metabolic and molecular processes.The comparative analysis of genomes indicated the genes encoded information of five L.plantarum were not related to their isolated origin.However,LP-M and LP-S differed from the other three L.plantarum strains in terms of cell wall/membrane/envelope biogenesis,replication,recombination,and repair in information storage processing,carbohydrate transport and metabolism,and overall membrane components.2.The anti-inflammatory activity of five strains of L.plantarum was investigated using dextran sulfate sodium(DSS)-induced colitis mice,and the mechanism of L.plantarum was analyzed based on intestinal metabolism,intestinal immunity,intestinal mucosa,and gut microbiota.The outcomes demonstrated that each L.plantarum effectively prevented weight loss,diarrhea and bleeding,colonic shortening,splenomegaly,thymic atrophy,and colonic mucosal damage in mice with DSS-induced colitis.Among them,the effect of endogenous Lactobacillus LP-M was the most pronounced,which was proved by the insignificant differences(p≥0.05)in colon length and histopathology score between LP-M administrated mice and normal mice.Five different strains of L.plantarum increased the amount of short-chain fatty acids(SCFAs),inhibited the activity of myeloperoxidase(MPO)significantly(p<0.05),and restored the diversity of the gut microbiota.LP-M and LP-H significantly reduced(p<0.05)the amount of pro-inflammatory cytokines,and endogenous Lactobacillus inhibited the activation of the NF-κB pathway,raised the expression of the tight junction proteins ZO-1,Occludin,and Claudin,and brought the gut microbiota’s fundamental makeup closer to that of healthy mice.Although all of the L.plantarum strains were able to treat the gut microbiota problems seen in colitis animals,endogenous L.plantarum was superior at preventing the loss of beneficial bacteria and the growth of pathogenic bacteria.The above results suggested that endogenous L.plantarum alleviated mice colitis more effectively by restoring intestinal flora,repairing intestinal mucosa,and improving intestinal immunity,which was possibly related to the interaction between endogenous L plantarum and gut microbiota.3.in vitro probiotic potential of Lactobacillus isolated from five origins was compared by their tolerance,cell surface properties,bacterial inhibition,and safety,to analyze the reasons for the differences in anti-inflammatory activity between endogenous and exogenous Lactobacillus based on the basic properties of bacteria.The results indicated that five strains of L.plantarum were safe and had adequate bile salt tolerance,but other performance metrics differed.Overall,LP-P isolated from pickles demonstrated the best probiotic potential among tested L.plantarum primarily in terms of improved low pH tolerance,superior cell surface properties,and bacterial suppression.This result indicated that there was no significant correlation between the basic probiotic properties of Lactobacillus and its anti-inflammatory activity in vivo.4.The intestinal colonization,and the impact on gut microbiota of Lactobacillus were examined to explore the potential reasons for the difference in the effects of endogenous and exogenous Lactobacillus on colitis mice.Meanwhile,the in vitro simulated colon fermentation was conducted to study the synergistic effect of five origins of L.plantarum and fecal microbiota.Firstly,L.plantarum was labeled using the fluorescent probe 5-(6-)carboxyfluorescein diacetate succinimidyl ester(c FDA-SE),and the labeled bacteria in the intestinal contents of mice were found by flow cytometric analysis to study the intestinal colonization.The results showed that L.plantarum colonized primarily the cecum and colon of mice,showing an increasing first,followed by a decreasing trend in the first 7 days.A higher level of colonization detected on day 14 indicated that five L.plantarum strains were able to colonize at the intestinal for a long period.The dominance of endogenous Lactobacillus was not associated with their capacity to colonize the intestinal tract,since no significant differences in labeled bacteria were seen in the same gut tissue on the same day.Secondly,the effects of a single gavage of L.plantarum on intestinal metabolism,organism immunity,and gut microbiota in mice were studied.The results were described as follows: on day 7 after gavage,the acetic acids content in cecum contents increased,the content of pro-inflammatory cytokine and MPO were upregulated significantly(p<0.05),the level of anti-inflammatory cytokines decreased significantly(p<0.05),while there were no obvious changes observed in gut microbiota.On day 14 after gavage,mice in LP-treated groups had significantly higher acetic acids(p<0.05)than that in NC group mice,while cytokines and MPO levels were normalized.In addition,the basic composition of gut microbiota in mice intervened by exogenous L.plantarum was different from normal mice,and the abundance of beneficial genera such as Lactobacillus and Muribaculum was decreased.Endogenous L.plantarum did not change the basic composition of the gut microbiota and significantly increased the relative abundance of the beneficial genus Romboutsia(p<0.0.5)and decreased the relative abundance of the harmful genus Erysipelatoclostridium(p<0.0.5).Finally,the results of in vitro simulated colonic fermentation showed that LP-M and fecal flora had a more effective interaction to promote sugar metabolism and the buildup of the primary metabolite SCFA.The above results revealed that there was no significant correlation between the probiotic properties and intestinal colonization ability of L.plantarum and their isolated origins,while the gut microbiota was the key factor related to the difference in activity between endogenous and exogenous L.plantarum.Gut microbiota and LP-M had a certain synergistic effect,which may encourage the accumulation of advantageous metabolites in the intestine,stimulate the proliferation of advantageous genera,inhibit the growth of detrimental genera,and improve intestinal health protection.5.Based on the aforementioned study,LP-P was chosen as the exogenous Lactobacillus,which showed better adhesion ability,but relatively poor anti-inflammatory activity.The anti-inflammatory activity of endogenous and exogenous Lactobacillus+chitooligosaccharide(COS)synbiotics was examined separately using DSS-induced colitis mice.LP-M,LP-P,COS,and synbiotics all alleviated the symptoms of colitis in mice.The effects of LP-M and endogenous synbiotics on intestinal immunity and metabolism were not statistically different(p≥0.05),and the relative abundance of the advantageous bacterial genera Lactobacillus and Blautia increased in mice of both groups.However,exogenous synbiotics were more significant in promoting the accumulation of SCFA,inhibiting pro-inflammatory cytokines and MPO activity(p<0.0.5),and converging the composition of the gut microbiota to that of normal mice when compared to LP-P.This indicated that the anti-inflammatory activity of exogenous LP-P can be increased by combining it with COS as a synbiotic. |
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