Identification And Screening Of Intestinal Probiotics From Golden Monkey In Shennongjia

Rhinopithecus roxellanae is the first class national protected animals, a rare and endemic species in China. Shennongjia is the most eastern end where the Rhinopithecus roxellanae is found. Digestive tract disease is one of golden monkey recurrent disease, which is one of the leading causes of death among infant monkeys. Maintenance of intestinal microecological balance and reduce the incidence of gastrointestinal diseases golden monkey, for monkey has important implications on the health of individuals and growth of population.In this study, the Rhinopithecus roxellanae as the study object.Collection of wild and health Rhinopithecus roxellanae300feces samples.By using MRS medium,92lactic acid bacteria isolates were isolated from monkey feces. The fermentative supernatant fluid of these lactic acid bacteria showed inhibitory activity to the pathogens bacteria, but there is a big difference. Eight isolates dlt6a, dlt6b, dlt6c, dlt7a, dlt7b, dlt9, dlt10a and dlt10b were showed obvious inhibitory activity. Eight isolates were identified as Enterococcus faecalis. Analysis of16S rRNA gene sequences and species-specific fragments ddlE.faecalis,ddlE.faecium, further confirmation isolated strains were Enterococcus faecalis.To ensure the safety of eight Enterococcus faecalis, isolated strains were tested for the presence of virulence factors and antibiotic resistance. The virulence factors cylA^esp、gelE、agg、efaAfS、ace、efaAfm were evaluated by PCR. Test confirmed dlt6a was agg+esp+efaAfs+, dlt6b was agg+esp+efaAfs+, dlt6c was agg+esp+efaAfs+dlt7a was esp+efaAfs+, dlt7b was esp+efaAfS+, dlt9was agg+cylA+esp+efaAfs+, dlt1Oa was agg+cylA+efaAfs+, dlt10b was agg+esp+efaAfs+. Basing on antimicrobial activity and virulence factors, three Enterococcus faecalis isolates dlt7a, dlt7b and dltlOa for the further experiments. Antibiotic susceptibility test showed three Enterococcus faecalis resistance to penicillin, tetracycline, and sensitivity to ampicillin, vancomycin, erythromycin, ciprofloxacin, norfloxacin, nitrofurantoin, Chloromycetin, doxycycline, gentamicin and spectinomycin. Animal acute toxicity experiment evaluation of bacteria on the safety of animal body.These results suggested that Enterococcus faecalis dlt7a and dlt7b are safe to be used. But dlt10a has potential dangerous, therefore discarded.In order to screen the dominant strains, experiment evaluated strains isolated from the growth curve, tolerance and resistance to high temperature capability and so on. These results suggested that Enterococcus faecalis dlt7a and dlt7b have the following characteristics:1. Able to tolerate the acidic environment.2. Able to tolerate bile salt at concentration of0.5%.3. Highly proliferative capacity and no lag phase,1%bacterial inoculation4h after entering logarithmic phase,9h after entering stationary phase.4.Able to survive at the temperature above80℃,2min. These results suggested that Enterococcus faecalis was able to successfully go through the gastrointestinal.They providing a valuable foundation for the production and applications.