Research On Screening And Probiotic Effects Of Probiotics Derived From Breast Milk

Breast milk,as the main food for infants,contains nutrients that can meet all the growth needs of infants.Antibodies in breast milk provide passive immunity for infants to protect and regulate the development of the infant's immune system.Breast milk probiotics promote the development of infant gut microbiota,immune and metabolic functions.In this study,by analyzing the geographic specificity of the culturable microbiota of breast milk,a potential probiotic unique to rural breast milk(Lactobacillus reuteri FN041)was isolated.Then,FN041 was investigated for its alleviation effect on dyslipidemia and intestinal mucosal barrier damage caused by hyperlipidemia,and its effect on diurnal changes of intestinal microbiota was also investigated.Finally,the effects of maternal intake of FN041 during gestation and weaning,or offspring intake after weaning,on the intestinal immune function of offspring mice were investigated.The main findings are as follows:1.Based on the 42-day mature milk samples of 89 healthy lactating mothers collected by the National Center for Disease Control and Prevention from 11 cities in my country,MRS and colonic bacterial anaerobic medium were used for enrichment culture of viable bacteria,and 16 S r RNA gene(v3-v4 region)amplicon sequencing was used to analyze the composition of the breast milk microbiota.The results showed that the bacterial composition of breast milk was geographically specific,and the alpha diversity and the relative content of Lactobacillus in the northwestern and northern samples were higher than those in other regions.The core species of the Lintan sample in Gansu included Lactobacillus reuteri and Lactobacillus gasseri.Postpartum BMI was negatively and positively correlated with the relative content of Lactobacillus lactis and Staphylococcus,respectively.The relative content of Lactobacillus lactis and Streptococcus was negatively correlated with Staphylococcus.In this study,the dominant genera of breast milk culturable microbiota were Staphylococcus,Streptococcus,Enterococcus and Lactobacillus.Brevibacterium,Bifidobacterium,Propionibacterium,Yersinia,and other bacteria were also detected in breast milk(at an abundance of less than1%).2.Rabbit anti-human Ig A serum combined with streptavidin-modified magnetic beads was used to enrich the Ig A-binding microbiota of breast milk in Lintan,Gansu.Eleven strains of Lactobacillus reuteri were isolated using selective medium.Among them,the survival rate of strain FN041 was 85% in p H=2.5 3h and 86% in 0.3 bile salt medium for 3h.The ability of FN041 to adhere to mucin was 4.25 times that of Lactobacillus rhamnosus GG.It is sensitive to commonly used antibiotics and is not resistant.Therefore,FN041 is a potential probiotic strain.Whole genome sequencing and analysis of FN041 were performed.The results showed that the assembled FN041 genome was 2.4 Mb in size,containing 2 plasmids,and the GC content was 38%.It was predicted that 2442 genes,18 r RNAs,and 75 t RNAs were obtained.16 S r RNA gene sequence phylogenetic tree analysis showed that FN041 shared a common ancestor with the type strain Lactobacillus reuteri JCM 1112.3.To induce hyperlipidemia in mice fed with high-fat diet,and to evaluate the alleviating effect of FN041 on dyslipidemia and intestinal mucosal barrier damage.The results showed that FN041 intervention significantly reduced high-fat diet-induced weight gain and peritesticular fat accumulation in mice,and decreased motor locomotor activity during active periods.FN041 intervention could significantly reduce high-fat diet-induced hypercholesterolemia,up-regulated expression of hepatic Fas gene m RNA and downregulated expression of Srebp1 c gene m RNA.FN041 partially treated high-fat diet-induced endotoxemia and mucosal barrier damage.FN041 significantly affected the high-fat dietinduced changes in the composition of gut microbiota and intestinal short-chain fatty acids in mice,and the changes were significantly correlated with circadian rhythms.4.The beneficial bacteria vertically transmitted to the infant gut will affect the gut immune development of offspring.We investigated the role of FN041 in regulating immune function in young mice.After the female mice and male mice were co-co-mated in a 3:1 ratio,the lactating female mice were divided into two groups,namely the normal group and the FN041 gavage group.The pups were divided into groups according to the normal group,only the mothers,only the offspring,and both the mothers and offsprings,and the offsprings were intervened after weaning,and the intervention lasted until 5 weeks.The results showed that all interventions resulted in enhanced gut barrier function in 5-week-old offspring,especially female offspring.Colon tissue immunoglobulin A plasma cells and colonic fecal secretory Ig A(s Ig A)increased in the offspring of the co-intervention group.The m RNA expression of 17 intestinal mucosal barrier-related genes was activated in the mouse ileum,especially in 5-week-old female offspring;the effect of co-intervention on the Shannon index of intestinal microbes in 5-week-old offspring was related to sex.The Shannon index of s Ig A-coated microbiota showed an upward trend.The ?-diversity of s Ig A-coated microbiota showed differences between groups.Actinobacteria and Bifidobacterium were increased in female mice.s Ig A-coated Bifidobacterium was positively correlated with Tlr9 m RNA expression,and s Ig A-encapsulated Lactobacillus was negatively correlated with Cldn2 m RNA expression in male offspring.In conclusion,the detection rate of Lactobacillus reuteri in breast milk was higher in Lintan,Gansu than in other cities.The typical probiotic strain Lactobacillus reuteri FN041 isolated in this study could alleviate hyperlipidemia and regulate lipid metabolism rhythm in high-fat-induced mice.Ingestion of FN041 in maternal mice promoted the development of intestinal immunity in offspring.This study provides a theoretical basis for the industrial application of FN041.