The Research On The Probiotic Potential Of Yak-Derived Probiotics

Yak is a rare cattle species mainly distributed in high altitude areas of China,and yak breeding is one of the important pillar industries in alpine Tibetan areas,but the traditional rough breeding mode is prone to many diseases.Antibiotics can improve the growth performance and disease resistance of animals in the short term,but long-term use is prone to various problems such as antibiotic residues,intestinal flora disorders and drug resistance.With the implementation of the antibiotic ban in 2020,it is urgent to find alternatives to antibiotics,and probiotics,as a natural alternative to antibiotics,can effectively avoid the various problems brought about by antibiotics.At present,the domestic probiotic industry is still in its infancy,and research on probiotics for ruminants is rare.China is the world’s largest livestock industry,and the livestock industry has a great demand for probiotics,so the screening and application of good probiotics is particularly important.Therefore,in this paper,the in vitro probiotic properties and safety of four strains of yak enteric-derived probiotics were analyzed,including enzyme production capacity,tolerance capacity,bacterial inhibition capacity,bacterial surface properties and antibiotic susceptibility;and the safety to mice was studied by in vivo tests;in addition,the effects of two strains with better in vitro probiotic properties on the intestinal flora of mice were investigated.The results of the study are as follow:1.Identification and evaluation of probiotic properties of yak-derived probioticsThe four strains of Yak-derived probiotics were identified by morphology and molecular biology,one strain was Lactobacillus rhamnosus,named L1;two strains were Bacillus subtilis,named B2 and B4 respectively;one strain was Bacillus atrophaeu atrophaeu,named B3.In the tolerance test,all four strains were able to tolerate high temperature,acidic environment,artificial gastrointestinal fluid and high bile salt environment,L1 and B4 had the strongest acid tolerance.In the inhibition test,L1 inhibited all five pathogenic bacteria,B2 and B4 inhibited three pathogenic bacteria,and the diameter of the inhibition circle of B4 was significantly higher than the other groups.In the enzyme production test,L1 hydrolysed protein but not starch;B2,B3 and B4 hydrolysed protein and starch,and B4 hydrolysed protein the most.In the surface property test,L1 showed the highest rate of self-agglutination and coagglutination with pathogenic bacteria,while B4 showed the highest rate of hydrophobicity.In the drug sensitivity test,L1 was completely resistant to 10 antibiotics;B2 and B4 were completely resistant to penicillin G and ceftazidime;B3 was completely resistant to penicillin G.Four probiotics were administered to Kunbai mice for 14 d.No abnormalities such as death or diarrhoea occurred in the mice;the anatomical structures of all organs were normal and no lesions were visible to the naked eye;there were no significant changes in the organ indexes of the organs;no bacterial translocation occurred in the organs;the weight gain and food utilisation of the mice in the L1-fed group were significantly higher than those in the other groups.3.Effect of L1 and B4 on intestinal flora of miceIn vitro probiotic characteristics of strains L1 and B4 were fed to Kunbai mice for 30 d.The effects of both strains on the intestinal flora of mice were studied using high-throughput sequencing.At the phylum level,L1 increased the relative abundance of Bacteroidota and decreased the relative abundance of Firmicutes in the mouse intestinal flora;B4 decreased the relative abundance of Bacteroidota and increased the relative abundance of Firmicutes in the mouse intestinal flora.At the genus level,the relative abundance of Lactobacillus increased significantly in group B4;The relative abundance of Unclassified＿f＿Lachnospiraceae,Turicibacter,Desulfovibrio,Eubacterium＿brachy＿group,Romboutsia increased significantly and Escherichia-Shigella decreased significantly in group L1.In conclusion,all the four strains of yak-derived probiotics were safe and non-toxic to mice,and L1 and B4 had good in vitro probiotic properties.Feeding them to mice altered the diversity and abundance of intestinal flora.Increasing beneficial intestinal bacteria and reducing pathogenic bacteria,and can be used as safe and effective yak-derived probiotics for further study.