Screening,Identification And Probiotics Of Lactic Acid Bacteria Derived From Breast Milk

As beneficial bacteria,lactic acid bacteria is widely used in dairy products,infant formula milk powder and other food because of its probiotic functions such as regulating intestinal flora,improving immunity and promoting the absorption of nutrients.And as a source of nutrient for healthy growth of infants,breast milk is also an important screening library for probiotics.Therefore,the purpose of this study is to screen the candidate strains of probiotics from healthy breast milk,and to preliminarily determine their probiotics.It is expected to be applied to baby food after commercialization to ensure the health of babies who cannot be breastfed.In this experiment,lactic acid bacteria were isolated and screened from 10 breast milk samples,and a total of 22 lactic acid bacteria were screened.Then,a strain of Lactobacillus plantarum R9 was obtained by acid and bile salt resistant double screening and further study was carried out.The tolerance?acid,bile salt and antibiotic sensitivity?and probiotics?inhibition of pathogenic bacteria and adhesion to intestinal epithelial cells HT-29?of L.plantarum R9 were studied.The results showed that L.plantarum R9 was cultured at p H2.5 for 24h and bile salt concentration was 4.5%for 6h,the number of viable bacteria was still above 10⁶cfu/m L,That means it had a strong tolerance.The inhibition diameters of the fermentation broth and supernatant on 8 common pathogenic bacteria including Echerichia coli,Listeria monocytogenes,Staphylococcus aureus,Shigella sohn,Bacillus cereus,Salmonella typhi,Enterobacter sakazakii and Salmonella enteritis were all above 20mm.It is sensitive to ampicillin,erythromycin,amikacin,clindamycin,ceftriaxone,ciprofloxacin and azithromycin,mildly sensitive to chloramphenicol.and resistant to penicillin G and tetracycline.The adhesion test results showed that the adhesion ability of L.plantarum R9 to cell HT-29 was affected by the action time,the concentration of the bacterial solution and the concentration of Ca²⁺.When the action time was 1.5 hours,the concentration of the bacterial solution was 1.0×10¹⁰cfu/m L and the concentration of Ca²⁺reached 11mmol/L,the maximum adhesion numbers of the bacterial strains reached were?6.52±0.32?cfu/cell,?8.14±0.26?cfu/cell and?8.14±0.26?cfu/cells,respectively.In the adhesion inhibition test of pathogenic bacteria,L.plantarum R9 showed that the best adhesion inhibition method for escherichia coli and staphylococcus aureus was rejection inhibition,for salmonella was competitive inhibitionThe results of whole gene sequencing showed that the genome size of L.plantarum R9was 3,530,009 bp and the G+C content was 44.67%,which were consistent with the characteristics of L.plantarum.The GO,KEGG and COG databases were used for annotation.The results showed that L.plantarum R9 contained a large number of genes involved in catalysis,carbohydrate transport and metabolism,amino acid transport and metabolism,indicating that L.plantarum R9 had strong catalytic and carbohydrate utilization abilities.Through CARD database comparison,a total of 15 resistance genes and 7 resistant antibiotics were identified,among which rifampicin had the largest number of genes and appeared twice,the Identity value reached 70%.In summary,L.plantarum R9 has good acid resistance,bile salt resistance and adhesion characteristics,has obvious antibacterial effect on 8 common diseases,is sensitive to 7antibiotics,and lays a foundation for probiotic characteristics research at the gene level,which can be used as a candidate strain of probiotics.