Screening Of Probiotic Lactic Acid Bacteria From Fruit And Vegetable Ferments And Investigation Of Their Potential Application

The probiotic lactic acid bacteria screened from the fruit and vegetable ferments can be used for silage preparation of perishable fruit and vegetable,reducing environmental pollution and alleviating the shortage of forage.Probiotics,an important boost for the efficient and green development of animal husbandry,can enhance the natural defense mechanism of livestock and poultry against diseases and improve their health status and productivity through regulating the structure of intestinal flora,nutrition metabolism and intestinal immune homeostasis,etc.1.Screening of probiotic lactic acid bacteria from fruit and vegetable fermentsIn this study,96 strains of lactic acid bacteria were isolated and purified from naturally fermented pineapple fruit waste silage and pickled vegetable from Hubei region using MRS medium.Three novel probiotic lactic acid bacteria,Lactobacillus plantarum A1,C1 and C10,were finally screened through safety and probiotic potential assessment.All three strains were peroxidase-negative,non-hemolytic,non-virulence genes(virulence genes tested included adherent virulence factors efaAfs,asa,ace,secretory virulence factors gelE,cylA,hyl and biogenic amine production factors hdc,tdc and odc),sensitive to rifampicin,gentamicin,erythromycin,amoxicillin,ampicillin,tetracycline and chloramphenicol,and tolerant to vancomycin,streptomycin and kanamycin.All three strains were able to survive at the acidic pH of 2 and the 1%bile salt supplementation.After 3 h of incubation at pH 3 and transfered to 1%bile salt environment for another 3 h,the final survival rates of three strains were 27.1%,91.7%,and 96.6%,respectively.All three strains were able to inhibit the activity of E.coli K99 and Salmonella Dublin in vitro by producing organic acid and lowering the environmental pH.They were all able to completely inhibit the activity of the pathogenic bacteria within 8 h when co-cultured with two strains of pathogenic bacteria in equal amounts(1×108 CFU/mL)in MRS×LB mixed medium.The cell surface hydrophobicity of the three strains was 71.92%,45.50%and 66.9%,respectively.They were able to adhere significantly to the cell surface after co-culture with bovine small intestine epithelial cells for 2 h and were difficult to be rinsed off with PBS solution.2.Genomic sequence analysis of probiotic lactic acid bacteriaThe genomes of the three strains were not matched for virulence genes and acquired antibiotic resistance genes.The genes encoding vancomycin resistance,bacitracin resistance and aminoglycoside modifying enzymes(acetyltransferase,nucleotidyltransferase and phosphotransferase)were matched,which clarifying the resistance mechanisms of the strains to vancomycin,streptomycin and kanamycin,and presuming that the three strains might be able to tolerate bacitracin.A series of genes encoding Na+/H+ antiporter,cation transport ATPase,amino acid permease,acidresistance membrane protein,alkaline shock protein and dltD manipulator were identified as being related to the acid tolerance ability.MES-type transporter-encoding gene,which is closely related to bile salt tolerance,was identified.The genes encoding fibronectin-binding domain and sortase,which are related to cell adhesion,were identified.Bacteriocin gene cluster are present in both L.plantarum A1 and C1 genomes,encoding plantaricin EF and two putative two-peptide bacteriocins in L.plantarum A1,and plantaricin EF,plantaricin JK,plantaricin NC8 in L.plantarum C1,respectively.3.Assessment of antioxidant potential of probiotic strains and investigation of their mechanismL.plantarum A1,C1 and C10 were all able to tolerate high concentrations of hydrogen peroxide and proliferated up to 3.5,3.5 and 4.0 mmol/L of hydrogen peroxide environment,respectively.The antioxidant capacity was mainly achieved through glutathione peroxidase,peptide-methionine sulfoxide reductase and alkylhydroperoxidase,as hypothesized by whole genome sequence analysis.Compared to bovine small intestinal epithelial cells without additional treatment,the addition of three strains in vitro significantly reduced the accumulation of reactive oxygen species in bovine small intestinal epithelial cells under both conventional conditions and hydrogen peroxide stimulation,and enhanced the total antioxidation capacity activities under hydrogen peroxide stimulationL.plantarum Al,C1 and C10 are novel probiotic lactic acid bacteria with high safety and probiotic potential,which can be developed into probiotic additives for animals.The three strains show higher hydrogen peroxide resistance than the reported L.plantarum MA2 and AR113,which can be developed into antioxidants with probiotic properties and have broad application prospect.