Microbiota Characteristics In Neonatal Feces And Breast Milk And Its Mechanism In Promoting The Development Of Intestinal Barrier

Newborns often suffer from diseases such as diarrhea,feeding intolerance and enteritis,which are caused by incomplete intestinal mucosal barrier function.Breast milk can provide infants with nutrients needed for growth,which also contains a large number of probiotics and prebiotics and plays a crucial role in the development of the infant’s intestinal tract.There are abundant microorganisms in breast milk and infant feces.However,the effects of these microorganisms on the composition of infant intestinal flora and the functional development of infant intestinal mucosal barrier need to be further studied to assist clinical improvement of infant intestinal dysplasia.Herein,this study analyzed the characteristics of breast milk microflora and infant stool microflora,in order to find probiotics promoting the intestinal mucosal barrier function of infants from breast milk and infant stool,and explore the mechanism of action.Feces and corresponding breast milk samples of newborn infants in Harbin were collected.The species diversity and relative abundance of bacteria were analyzed by Illumina high-throughput sequencing method.Illumina high-throughput sequencing analysis showed that the diversity of breast milk flora was higher than that of infant feces.Among them,Enterobacteriaceae,Moraxella,Staphylococcaceae and Streptococcaceae were the main breast milk flora.The fecal flora of infants was mainly Enterobacteriaceae and Bifidobacteriaceae.The Sourse Tracker analysis found that,on average,44.90% of the bacterial flora in babies’ faeces came from breast milk.There is potential transmission between breast milk flora and infant fecal flora.Different modes of delivery,feeding and mother’s use of antibiotics have an effect on the microflora structure of breast milk and infant feces.PICRUSt function prediction analysis showed that the dominant flora in the stool of vaginal delivery and breast milk of mothers who did not use antibiotics was closely related to the development of digestive system.At the same time,Lactobacillus,Bifidobacteria and Enterococcus were the dominant flora in maternal breast milk and feces of babies born naturally without antibiotics.Bifidobacteria,Lactobacilli and Enterococcus were isolated and screened from the feces of spontaneous newborns and breast milk samples of mothers who did not use antibiotics.Finally,three strains with potential to promote intestinal mucosal barrier function were obtained.A total of 480 strains of Bifidobacteria,Lactobacillus and Enterococcus were isolated from feces of babies and breast milk samples of mothers without antibiotics.Among them,176 strains had good surface hydrophobicity,115 strains had good tolerance to gastric juice,and 84 strains had good tolerance to intestinal juice.Furthermore,30 strains with good adhesion ability were obtained by screening the adhesion ability of intestinal epithelial cells IEC-6.Finally,it was found that three strains,m L-329,ML-446 and FL-228.1,had a good ability to promote the proliferation of intestinal epithelial cells IEC-6.By 16 S r RNA,they were identified as Limosilactobacillus oris ML-329,Lacticaseibacillus paracasei ML-446 and Bifidobacterium bifidum FL-228.1.The effects of ML-329,ML-446 and FL-228.1 on intestinal development were investigated by measuring the indexes in rats.The results showed that the three strains had good intestinal colonization ability.Among them,ML-446 had a good promotion effect on intestinal length and weight of rats.ML-329 and FL-228.1 could promote the growth of villus length in colon and ileum of rats after intervention.Meanwhile,FL-228.1,ML-329 and ML-446 significantly increased the intestinal antioxidant capacity,and FL-228.1,ML-329 and ML-446 significantly increased the content of s Ig A in ileum and colon tissues.The effects of ML-329,ML-446 and FL-228.1 on the differentiation and tight junctions of intestinal mucosa epithelial cells were studied in the rats with in utero hypoplasia.The results showed that these three strains could activate Wnt/β-catenin signaling pathway by promoting the expression of target gene r-spondin.Meanwhile,these three strains promoted the proliferation of Paneth cells and goblet cells,and participated in the regulation of the proliferation and differentiation of intestinal epithelial cells through Notch signaling pathway.They balanced the number of secretory cells and absorbent cells.After FL-228.1 and ML-446 treatment,the intestinal permeability of rat pups was significantly reduced.Immunofluorescence staining also proved that the expression of Claudin 3,Occludin and ZO-1 in jejunum,ileum and colon were significantly increased.FL-228.1 and ML-446 significantly affected the expression of F-actin in ileum and colon.FL-228.1 and ML-446 gavage intervention reduced the expression of Rho A/ROCK/MLC pathway related genes and proteins in intestinal cells of rats with IUGR.Meanwhile,after ML-329,ML-446 and FL-228.1 gavage intervention,the intestinal microflora structure of IUGR rats was significantly improved,and the abundance of dominant lactobacillus was significantly increased,which alleviated the retardation of intestinal mucosal barrier development caused by intestinal microflora disorder of IUGR rats,and effectively improved the intestinal mucosal barrier function.To sum up,this study obtained three strains ML-329,ML-446 and FL-228.1which have the potential to promote the intestinal mucosal barrier function through the analysis of the flora characteristics of neonatal breast milk and infant feces,and determined that they can enhance the intestinal mucosal barrier function by promoting the differentiation of Paneth’s cells and goblet cells,and promoting the expression of tight junction proteins.