Characterization Of Lactic Acid Bacteria Strains From Yogurts Produced By Herders In Qinghai Province And Their Antimicrobial Activity Against Escherichia Coli From Ready To Eat Food

Lactic acid bacteria(LAB)are industrially used in fermented dairy products and are also widely used in food preservation and in health promoting supplements.Commercial LAB strains are used as starter cultures to improve texture,flavor and nutrition value of fermented products such as yogurt in food industry.LAB strains such as Lactobacilli and Bifidobacteria,both found in the human gut,could beneficially affect human health by improving balance of microbiota.Nomadic people living in Qinghai province have a long tradition of making and consuming yogurt made from yak or buffalo milk.Those yogurts are rich in LAB but these wild LAB strains are not well studied.They may represent a good source of potentially commercial starter culture or be developed as biopreservative to control microbial contamination in food products.In this study,yogurt samples were collected in nomadic herders in Qinghai province and used for isolation of LAB strains.LAB strains were isolated by conventional methods and identified by 16 s RNA gene sequencing.Then the isolates were characterized by acid tolerance and bile salts tolerance.Antimicrobial activities against common bacterial foodborne pathogens and antibiotic susceptibility profiles were further investigated.Moreover,their capability to adhere to human intestinal Caco-2 cells and antioxidant activity were determined.To test their potential application in ready to(RTE)food to control E.coli contamination,E.coli were first isolated from RTE foods collected from different cities and characterized by phylogenetic grouping,antimicrobial susceptibility testing,and screened for the presence of STEC and class 1 integron.Then antimicrobial activities of selected LAB strains were determined against E.coli strains from RTE food.The major findings from this study are as follows:1.A total of two hundred and nine presumptive LAB were isolated from sixty-eight yogurt samples including 38 yak yogurt and 30 yellow buffalo yogurt,which were collected in nomadic herders in Qinghai province,China.One isolate was chosen from each sample and tested by Gram staining and catalase assay.Of these,fifteen isolates were confirmed as LAB by using 16 S r RNA sequence analysis.BLAST search identified the LAB isolates as follows,Lactobacillus delbrueckii subsp.(6 isolates),Streptococcus thermophilus(1 isolate),Enterococcus durans(5 isolates),Enterococcus hirae(2 isolates),and Enterococcus faecium(1 isolate).Phylogenetic tree was constructed by including certain LAB isolates in NCBI database.Phylogenetic analysis revealed a high phylogenetic diversity at the genus level.2.Antimicrobial activity of LAB strains against four bacterial foodborne pathogens(one Escherichia coli,two Staphylococcus aureus,and one Listeria monocytogenes)were tested.The results indicated that antimicrobial activity of LAB differed among different pathogens and 15 selected LAB isolates showed different level of inhibitory action against the pathogens.Antimicrobial activity of most LAB isolates was affected by neutralization of p H and catalase treatment,indicating that organic acid,hydrogen peroxide were main antimicrobial compounds produced by these selected LAB isolates.However,LAB isolates 14 and 16 kept their antimicrobial activity after p H neutralization and catalase treatment,which suggests that bacteriocin-like inhibitory substance(BLIS)may also play role in their antimicrobial activity.3.Selected LAB isolates were further characterized by antimicrobial susceptibility test,acid,and bile salt tolerance,antioxidant capacity and adherence to Caco-2 cells.LAB isolates showed high sensitivity to clindamycin and ampicillin.Vancomycin resistance was frequently detected.LAB isolate 14(Enterococcus durans),LAB isolate 16(Enterococcus faecium)showed tolerance to low p H and bile salt,which indicated their probiotic potential.Moreover,nine selected isolates(10,14,16,33,34,40,63,and 67)were further investigated to evaluate their capability to adhere to human intestinal Caco-2 cell.The strongest in vitro adherence was detected in LAB isolate 14(20.0±6.22%),followed by LAB isolate 34(8.42±3.50%)and isolate 40(7.13±3.15%).Various antioxidant compounds have reduction capacity of the DPPH' radical as a result of antioxidant radical scavenging activity through the hydrogen donation to form stable DPPH.LAB isolate 12 had the minimum DPPH percentage scavenging activity(30.79 ± 0.24%)while LAB isolate 16 showed maximum DPPH scavenging activity(76.20 ± 0.52 %).The antioxidant activity of remaining isolates was decreased in the order as follows 14>67>68>34>40.4.There is an increasing concern in the microbiological safety of ready-to-eat(RTE)food.RTE could serve as vehicle for transmission of pathogenic E.coli.The present study also test the prevalence of E.coli in RTE foods and the potential to use LAB strains as a biopreservative to inhibit growth of E.coli from RTE food.For the isolation and characterization of E.coli,a total of 300 RTE foods samples were collected in Shaanxi province,China.Two hundred and sixty-seven(89.0%)RTE foods were positive for E.coli.Among E.coli isolates,73.0% belong to phylogenic A,12.4% to phylogenic B1,6.4% to phylogenic B2,and 8.2% to D.All isolates were negative for Shiga toxin genes.These results indicate that RTE foods in Shaanxi were commonly contaminated with antibiotic resistant E.coli,which may pose potential risk for consumer health and for transmission of antibiotic resistance.Furthermore,selected LAB isolates were tested for their antimicrobial activity against some E.coli isolates from RTE foods.Two LAB isolates were demonstrated to be effective against most E.coli isolates and were potential candidate as a biopreservative for controlling E.coli in RTE food.In summary,a variety of LAB genera were isolated from yogurt samples collected from nomadic herders in Qinghai province,some of which were found to exhibit broad-spectrum antimicrobial activity.Certain LAB isolates also showed good survival under acidic and bile salt environment,and exhibited good adherence to intestinal Caco-2 cells in vitro,all indicated their probiotic potential.In addition,LAB isolates showed good antimicrobial activity against E.coli from RTE food,which suggests their potential application in RTE food to control microbial contamination.