Screening Of Probiotics From Breast-fed Infants Faeces And The Research Of Immune Effects And Related Mechanism On Inflammatory Bowel Disease

Early life is the key period for the correct establishment of intestinal microflora.The establishment of intestinal flora directly affects the development and maturity of the immune system of the body,and thus has a short-,medium-or even long-term consequences for the host health.During such life stage,vertical mother-to-infant microbial transmission usually plays a pivotal role in initial colonization of the neonatal gut,and among the many transmission pathways:amniotic fluid,vagina,cavity,skin,breastfeeding,etc.,especially breastfeeding is the most important.Lactic acid bacteria,as an important probiotic in the human intestinal flora,are closely related to the health of the host:specific strains have been reported to regulate immune,relieve oxidative stress,and alleviate inflammatory bowel disease,etc.Some reports believe that there is a specific mutual selection relationship between the organism and the gut microbe:when the source of the strain and its application to the object are consistent,it will significantly enhance the specificity and effectiveness of the strain on the probiotic function of the body,namely:the ideal probiotic is best derived from the body itself.This study collected fresh feces from exclusively breast-fed infants,isolated a series of lactic acid strains,and selected a lactic acid bacteria with potential probiotic characteristics by using anti-artificial digestive juice ability,bile salt resistance and adhesion ability as indicators.By sequencing 16S rRNA combining with physiological and biochemical test results,it was initially identified as L.plantarum,named BF₁5.At the same time,the safety（antibiotic sensitivity,harmful metabolites,hemolysis,etc.）and probiotic function（immune function,antioxidant capacity）of the strain were initially evaluated in vitro;the colonization ability of the strain in the intestinal tract of mice and the immunomodulatory function in vivo were further analyzed;Finally,with the animal model of ulcerative colitis（UC）induced by dextran sodium sulfate（DSS）,we evaluated it’s function of relieving UC in terms of regulating immunity,alleviating oxidative stress and balancing intestinal flora,even through proteomics to reveal its mechanism of action in the intervention of inflammatory bowel disease.The research contents are as follows:1.Hungate anaerobic culture technique was used to separate lactic acid bacteria in the feces of exclusively breastfed infants,and L.rhamnosus GG was used as a positive control strain,and the strains with potential probiotic properties were screened by biological characteristics such as acid resistance,bile salt tolerance,tolerance to human digestive juice,and adhesion ability.At the same time,it was identified by 16s r RNA sequencing combined with physiological and biochemical tests,and the safety（drug sensitivity,harmful metabolites,hemolysis）and probiotic functions（Immune and antioxidant ability）of the selected strain was further evaluated.The results showed that a strain with excellent probiotic performance（high tolerance of artificial gastrointestinal fluid and high adhesion in vitro）was screened out by various indicators.The strain was identified as L.plantarum by physiological and biochemical index test and 16S rRNA gene sequence analysis,named BF₁5;in vitro safety experiments showed that BF₁5 is resistant to oxacillin and cefotaxime,in addition to its intrinsic resistance to aminoglycosides and glycopeptide antibiotics,but without a resistance plasmid,and the strain does not produce harmful metabolites,has no hemolytic property,and thus has certain safety.The results of in vitro probiotic function test indicated that the activity of live/heat inactived BF₁5 within a certain range of bacteria concentration（1×106～107 CFU/mL）could promote the proliferation of mouse lymphocytes in vitro,and there was no significant difference in the immunomodulatory ability of LGG under the same bacterial concentration conditions（P>0.05）.BF₁5 is able to tolerate high concentration of H2O2（3.5 mmol/L）and has strong free radical（O2-·、·OH and DPPH）clearance and anti-lipid peroxidation capacity.2.With the PCR-DGGE molecular technique based on rpo B gene to analyze the colonization of BF 15 in the intestine of mice.Cyclophosphoramide induced immunosuppression in mice as a model,LGG as a positive control strain,by measuring organ index,transformation of splenic lymphocytes,serum hemolysin level,toe swelling degree,macrophage phagocytic,and the diversity of intestinal flora,the immunomodulatory capacity of BF₁5,which protect mice from cyclophosphamide-induced immunosuppression and intestinal flora disturbance,was evaluated.The results showed that BF₁5 could still be detected in mouse feces on the 14th day after stopping the gavage,with strong colonization ability.And BF₁5 with the early gavage can significantly resist the reduction of immune-related indicators（organ index,spleen lymphocyte transformation value,serum hemolysin level,toe swelling and macrophage phagocytic index）induced by CTX（P<0.01）,and intestinal flora imbalance（the relative abundance of Bacteroidetes is reduced,the relative abundance of the thick-walled bacteria and the Proteobacteria containing a large number of potential pathogenic bacteria（E.coli and Pseudomonas）is increased）,having a certain immunomodulatory ability.3.DSS induced mouse UC as a model,to evaluate the mitigation effect of BF₁5 on UC from multiple aspects such as regulating immune function,relieving oxidative stress,and balancing intestinal flora,further through the proteomics of colon tissue,the mechanism of action of BF₁5 intervention in UC was revealed from the protein level.The results show:through early gavage in vivo,BF₁5 reduced intestinal mucosal damage and inflammatory cell infiltration,relieved intestinal mucosal oxidative stress damage（to increase SOD and GSH-Px activity,reduce MDA content）,improved energy metabolism（improve ultra-micro ATPase activity）,regulated of intestinal mucosal immune function（inhibiting pro-inflammatory factors:IL-6,IL-17A,IFN-y and TNF-α）,promoting anti-inflammatory factors:IL-10 and sIgA,balancing the intestinal flora(alleviating the structural disorder of the intestinal flora induced by DSS:the relative abundance of Bacteroides and Actinobacteria were reduced,and the relative abundance of Firmicutes,Proteobacteria,Verrucomicrobia and Tenericute were increased,the disordered trend of increasing the ratio of F/B（Firmicutes/Bacteroides）,thereby achieving the purpose of effectively interfering with UC.Finally,through bioinformatics analysis of differentially expressed proteins in different groups,revealed BF₁5 mainly by calling back differential proteins（BF₁5 mainly callback GSH-Px,pIgR,AR,ALOX15,MMP-2,ApoC-III and so on,LGG mainly callback pIgR,ALOX15,ApoC-III,TN,MMP-2 and so on）in UC to normal expression level,which in turn regulates antigen processing and antigen presentation system（APC）,to intervenes UC.The research results of this thesis will enrich the development of original human probiotics in China and provide excellent strain resources for functional foods of infants.