Study On Genomics,Nitrite-Degrading Characteristics Of Lactiplantibacillus Plantarum DMDL9010 And Its Exopolysaccharides In Alleviating Depression

Studies have shown that probiotics play a pivotal role in maintaining human health.Previously we separated a potential probiotic Lactiplantibacillus plantarum DMDL9010（LP9010）with satisfying acid tolerance,bile salt tolerance,and cholesterol-reducing properties from traditional Chinese Paocai.It is of great significance to analyze the mechanism of nitrite degradation by LP9010,explore the internal relationship between the structure and function of LP9010 extracellular polysaccharide（EPS）,and evaluate the potential therapeutic effects of LP9010 and EPS on depression induced by colitis.Based on the genome,comparative genome,transcriptome,and other omics techniques,we have clarified the relationship between the phenotypes such as nitrite degradation characteristics and exopolysaccharide production ability of LP9010 and the corresponding genotypes.We also clarified the mechanism of LP9010 and EPS in the treatment of depression from the perspective of intestinal-brain axis balance,which is of great importance for the development of probiotic therapy for depression.In this study,the safety and probiotic functionality were investigated via genomic and in vitro experiments from genotype and phenotype perspectives.LP9010 was sequenced and analyzed through whole genome sequencing,and it was found that it contained a circular chromosome of 3,334,776 bp,with an average G+C content of 44.47%,and a total of 3324 coding genes.The nitrite reductase gene pgl was found in LP9010,indicating that the bacteria might have the ability to degrade nitrite.LP9010 has 281 genes related to carbohydrate transport and metabolism and 98 carbohydrate active enzyme genes,indicating that it might have the ability to produce EPS.Comparative genome analysis showed that the LP9010 genome structure was similar to the reference strain,but there still existed 10 unique gene families.LP9010 was proved high safe due to its lack of nitro reductase and indole,and showed no hemolysis and no resistance to four antibiotics（tetracycline,streptomycin,erythromycin and ampicillin）.LP9010 had gastrointestinal and oxidative stress tolerance,good self-coagulation ability,and antibacterial activity,and might be related to a wide range of stress-related proteins,cell surface/extracellular proteins,and Plantaricin gene clusters.Using RNA-Seq sequencing and molecular docking approach to study the response mechanism of LP9010 under nitrite stress.The result showed that LP9010 had a certain tolerance towards NO₂^-,and nitrite affected the morphology of LP9010 cells.RNA-Seq sequencing found that the response mechanism of LP9010 under nitrite stress might include:LP9010 cell could be prolonged under nitrite stress,their DNA replication would accelerate and more energy substances（NAD+and ATP）were produced,thereby repairing cell membrane damage caused by nitrite stress.It also regulated the balance of metal ions and thioredoxin system in the cell to accelerate detoxification.In addition,a proper concentration of nitrite could promote the production of nitrite reductase to degrade nitrite.NO₂^-could interact with the active site PRO87 and PRO88residues on the key enzyme Cd1Ni R（pgl）for nitrite degradation to form two hydrogen bonds to interact,thereby contributing to the role of Cd1Ni R（pgl）in the degradation of nitrite.According to the whole genome analysis,it was found that LP9010 contained EPS biosynthesis genes.The EPS was further isolated and purified from the fermentation broth for structural study.EPS gene analysis showed that there were 3 EPS biosynthesis gene clusters in LP9010.LP9010 fermentation broth was purified to obtain EPS-LP1 and EPS-LP2,with purity of 99.68%and 99.41%,respectively.The molecular weights of the two EPS were similar.EPS-LP1 had the most glucose content（86.25%）,while EPS-LP2 mainly composed of mannose（62.07%）.The glycosidic linkage of EPS-LP1 included t-Galp,t-Manp,6-Glcp,4-Glcp and 4,6-Galp,and the relative molar ratio was 1.016:9.874:4.355:78.693:6.062.As for EPS-LP2,the glycosidic bonds included t-Manp,t-Glcp,2-Manp,6-Galp,6-Glcp and 4-Glcp,with a relative molar ratio of 48.884:4.667:17.016:7.650:7.197:14.565.The degradation temperature of the two EPSs was higher than that of conventional food processing and sterilization,indicating that they were with good thermal stability.The model of ulcerative colitis depression was established by gavage of C57BL/6N male mice with Dextran sodium sulfate（DSS）,and the intake of LP9010 and two polysaccharides（EPS-LP1 and EPS-LP2）were investigated.The effect on gut microbiota,inflammatory response,neurotransmitter,intestinal barrier,and short-chain fatty acid content were investigated to understand the influence of LP9010 and its EPS on DSS-induced depression-like behavior,further underlying the gut-brain axis mechanism.The intake of LP9010 and its EPS could alleviate the DSS-induced inflammatory response in mice,repair their intestinal barrier damage,and reduce depression-like behavior in mice.Feeding LP9010 and its EPS could also inhibit neuroinflammation by up-regulating the level of neurotransmitters.As for gut microbiota,intake of LP9010and its EPS could increase the relative abundance of Bacteroidetes and Firmicutes,reduce the relative abundance of Proteobacteria and Verrucomicrobia,thereby improving the intestinal flora.The levels of short-chain fatty acids such as butyric acid and propionic acid in the colon of mice were also increased after feeding LP9010 and its EPS.In addition,the high-concentration of neutral polysaccharides（EPS-LP1）had the best effect on recovering the morphology of mouse intestinal tissues and alleviating inflammation.The overall effect was underlying with the sequence of EPS-LP1>EPS-LP2>LP9010.In summary,LP9010 is a safe and non-toxic grade strain with good gastrointestinal tolerance,self-coagulation ability,antibacterial activity and oxidative stress tolerance.It can degrade nitrite through a certain nitrite stress response mechanism.The EPS-LP1 and EPS-LP2 could be separated and purified from LP9010,and mainly composed of glucose and mannose respectively.LP9010 and its EPS could alleviate depression-like behaviors and regulate the intestinal flora.This paper provides theoretical support for LP9010 as a potential probiotic with higher application value,and provides a basis for LP9010 and its EPS in probiotic therapy for depression.