Antioxidant Properties Of Lactic Acid Bacteria Isolated From The Qinghai-Tibetan Plateau And Their Impact On Gut Microbiota

Objective:Lactic acid bacteria（LAB）with potent antioxidant properties have demonstrated efficacy in mitigating oxidative damage in humans,making them valuable in the prevention and treatment of oxidative stress-related diseases and the modulation of intestinal microecology.The unique climate and geographical environment of the Tibetan plateau,also known as the"third pole of the earth,"may endow LAB with distinct biological properties.In this study,we isolated and screened LAB strains with robust antioxidant capacity from the Qinghai-Tibet Plateau and evaluated their probiotic potential and modulation of intestinal microecology.Our aim was to provide new data and strategic insights to support health interventions targeting the regulation of intestinal microorganisms.The results of our investigation suggest that LAB strains from the Qinghai-Tibet Plateau exhibit notable antioxidant properties and show potential as probiotics for the modulation of gut microbiota.These findings may provide a foundation for further exploration of LAB as a natural and effective approach to promote gut health.Methods:Biological samples from different yak and cow taxa in the Tibet Autonomous Region,including milk ducts,milk,birth canal,and rumen,were collected and used to isolate and culture lactic acid bacteria.Phylogenetic relationships were predicted based on phenotypic characteristics,colony size,Gram staining,and 16S r DNA sequencing.To identify lactic acid bacteria with high antioxidant activity,various tests were conducted,including hydrogen peroxide（H₂O₂）tolerance,2,2-diphenyl-1-trinitrophenylhydrazine（DPPH）radical scavenging capacity,superoxide anion(O^2-)scavenging capacity,hydroxide ion（HO·）scavenging capacity,reducing capacity,and total antioxidant（T-AOC）capacity.Lactobacillus rhamnosus CICC 6001was selected as the high antioxidant strain.The probiotic function and safety of the selected strains were evaluated in vitro based on hydrophobicity,self-aggregation,acid resistance,bile salt resistance,survival rate under in vitro simulated gastrointestinal conditions,and antibiotic resistance.To simulate and explore the effects of the selected strains on human intestinal biological structure,diversity,function,and short-chain fatty acids,in vitro fermentation experiments,high-throughput sequencing,and Gas Chromatography-Mass Spectrometry（GC-MS）were performed.Our findings provide valuable insights into the potential health benefits of the selected strains and highlight the need for further research to explore their potential as natural agents for promoting gut health.Results:（1）A total of 95 strains were obtained through microscopic observation of colony size and Gram staining.Further sequencing of the 16S r DNA gene revealed that 90 of these strains were lactic acid bacteria.Among the nine lactic acid bacterial species identified,Pediococcus pentosaceus was the most abundant（44.44%）,followed by Weissella pararmesenteriodes（28.89%）and Lacticasebacillus sakei（8.89%）.These findings provide valuable insights into the diversity of lactic acid bacteria present in the biological samples collected from yak and cow taxa in the Tibet Autonomous Region.（2）Six strains were selected as having good antioxidant capacity in the initial screening tests for H₂O₂ tolerance and DPPH scavenging capacity:L.paracasei TDM-2,P.pentosaceus TCM-3,W.paramesenteroides TYC-53,L.paracasei TDM-68,P.pentosaceus TDM-101,and W.paramesenteroides TDM-116.Further evaluation of the antioxidant capacity of these strains demonstrated significant differences in their effects on different assays,indicating that each strain may mitigate the effects of reactive oxygen species through distinct cellular mechanisms.W.paramesenteroides TDM-116exhibited the highest antioxidant capacity under 2.0 mmol/L H₂O₂ incubation（74.69%,P<0.05）,followed by P.pentosaceus TCM-3 and L.paracasei TDM-2（71.79%and63.65%,respectively）.P.pentosaceus TCM-3 showed the highest DPPH scavenging capacity（87.99%,P<0.05）,while L.paracasei TDM-2 exhibited the highest reducing capacity（29.48%,P<0.05）.In addition,L.paracasei TDM-2 showed a total antioxidant capacity of 4.10μmo L/m L（P<0.05）.Notably,L.paracasei TDM-2 and P.pentosaceus TCM-3 demonstrated the most stable performance among the various antioxidant assays measured（P<0.05）.（3）L.paracasei TDM-2 and P.pentosaceus TCM-3 exhibited noteworthy resilience in acid and bile salt tolerance assays.Specifically,L.paracasei TDM-2demonstrated the highest tolerance in the bile salt assay（P<0.05）,and both strains displayed exceptional acid tolerance.Further,under simulated in vitro gastrointestinal conditions,L.paracasei TDM-2 exhibited the highest survival rate（96.20%,P<0.05）.In terms of physicochemical properties,L.paracasei TDM-2 and P.pentosaceus TCM-3 exhibited over 85%hydrophobicity and self-aggregation capacity at 6 h.Regarding antibiotic susceptibility,all strains were sensitive to vancomycin,and L.paracasei TDM-2 and P.pentosaceus TCM-3 were generally more sensitive to most antibiotics.（4）The two selected strains,L.paracasei TDM-2 and P.pentosaceus TCM-3,were subjected to simulated in vitro colonic fermentation experiments,based on their antioxidant assay and probiotic characteristics.The results of the 24-hour fermentation showed a total of 123 operational taxonomic units（OTUs）detected in the three groups.The species richness（Chao 1）of the L.paracasei TDM-2 group was significantly lower than that of the P.pentosaceus TCM-3 group or the control group（P<0.05）.The principal coordinate analysis（PCo A）based on the weighted Unifrac distance matrix indicated a significant dispersion between groups and respective aggregation within groups.The addition of P.pentosaceus TCM-3 showed significantly higher content of acetic acid,propionic acid,and total short-chain fatty acids（SCFAs）than that of L.paracasei TDM-2 or the control group（P<0.05）.Conclusion:This study has successfully identified two promising probiotic strains,Lactobacillus paracasei TDM-2 and P.pentosaceus TCM-3,with high antioxidant capacity,which were isolated and purified from diverse biological samples collected from the Qinghai-Tibet Plateau.The results of this investigation show that these strains can effectively regulate the intestinal microecology and promote the overall health of an organism.These findings provide a strong basis for further research and development of probiotics that target antioxidant and intestinal microecological regulation as a means of improving health in the unique high-altitude environment of the Qinghai-Tibet Plateau.