| The Golden Snub-nosed Monkey in Shennongjia is the subspecies of Rhinopithecus roxellana,the special endangered species of our country.The health and breeding of Golden Snub-nosed Monkeys are seriously effected by various types of gastrointestinal problems,which caused by the imbalance of intestinal flora.In recent years,to achieve the purpose of preventing and treating some gastrointestinal diseases,it has been recognized as an effective method by adding probiotics to adjust intestinal micro-ecological balance of the host.However,there are few studies on micro-ecological researches of Golden Snub-nosed Monkeys at home and abroad.In the early stage of this study,E.faecalis dlt7 a was isolated from the feces of healthy and wild Golden Snub-nosed Monkey residing within the Shennongjia Natural Reserve,and it had been identified as owing good safety and manufacturability.In the study,the fermentation technology of E.faecalis dlt7 a was optimized to prepare the probiotic preparation for Golden Snub-nosed Monkeys in Shennongjia;And to research the effects of E.faecalis dlt7 a on gut microbiome of Golden Snub-nosed Monkeys in Shennongjia,the writer supplemented the diet of monkeys with the probiotics of E.faecalis dlt7 a,and analyzed the results of metagenomic sequencing.Hope to provide a theoretical basis for developing the health regulation technology of Golden Snub-nosed Monkeys in Shennongjia.Exp.1 The fermentation technological optimization of E.faecalis dlt7aIn this experiment,the viable count of dlt7 a in different seed mediums and the growth curve of dlt7 a in seed medium were studied,and the effects of different loading volume,inoculation amount and initial p H value on the proliferation of dlt7 a in fermentation broth were researched.The results showed that the optimum medium for dlt7 a was MRS broth,the best seed broth was inoculated and cultured for 10 h,the best loading volume was 20%,the best inoculation amount was 2% and the optimum initial p H was 6.5.It showed that sucrose,fish meal and KH2PO4 were the main factors affecting the viable count of fermentation broth by PB test.The optimum concentrations of these three factors in fermentation broth were optimized by the steepest climbing test and response surface test.The optimized fermentation medium was 2.55% sucrose,2.63% fish meal,2.50% soybean meal,0.40% yeast extract,1.00% Ca CO3,0.10% KCl,0.10% Mg SO4.7H2 O and 0.014% KH2PO4.The viable count of dlt7 a was 58.83×108CFU/m L,which was 46.53% higher than before the optimization,which provided reference for the industrial production of dlt7 a.Exp.2 The effects of E.faecalis dlt7 a on gut microbiome of Golden Snub-nosed Monkeys in ShennongjiaIn this study,four captive Golden Snub-nosed Monkeys were selected from the artificial breeding base of Shennongjia Golden Snub-nosed Monkey,and the feeding test of monkeys was carried out for 28 days.During the test,E.faecalis dlt7 a probiotics were added to the monkeys' diet three times a day,and we collected 12 fecal samples of four monkeys at Day0,Day14 and Day28,which was responding to each monkey.The genomes of fecal samples were extracted for metagenomic sequencing.And the structural and functional information of gut flora at different time points were compared and analyzed in statistics.The main findings were as follows:1.Here,we carried out species annotation of the intestinal flora.The results were that: The diversity of intestinal microflora did not significantly change by supplementing Golden Snub-nosed Monkeys with E.faecalis dlt7a;The dominant phyla of monkeys' intestinal flora were Firmicutes(40.27%)and Bacteroides(33.60%),and after feeding E.faecalis dlt7 a,the relative abundance of Firmicutes reduced and the relative abundance of Bacteroides increased;At different time points,the main genera of monkeys' intestinal flora were always Bacteroides?Prevotella?Treponema and Ruminococcus,but according to E.faecalis dlt7 a,the relative abundance of Parabacteroides significantly increased(P<0.05),the relative abundance of Bacteroides,Fibrobacter and Roseburia increased by 5.35%,1.66% and 1.33%,respectively;At the species level,after supplementing monkeys' diet with E.faecalis dlt7 a,the relative abundance of Prevotella_copri(2.16%),Fibrobacter_succinogenes(1.66%),Bacteroidales_bacterium(1.61%),Eubacterium_rectale(1.11%)and Bacteroides_sp.(0.84%)increased,however the relative abundance of Prevotella_multisaccharivorax(1.25%),Cetobacterium_somerae(0.66%),Chthoniobacter_flavus(0.36%,P<0.05),Verrucomicrobia_bacterium(0.32%)and Pedosphaera_parvula(0.32%)reduced.The results indicated that it had a certain effect on the structure of monkeys' intestinal flora by supplementing Golden Snub-nosed Monkeys with E.faecalis dlt7 a,which could increase the relative abundance of beneficial bacteria such as some flora of breaking down polysaccharide,and it would be beneficial to the growth and immunization of Golden Snub-nosed Monkeys.2.The predicted genes were blasted to the carbohydrate active enzyme database.The results were that: Compared with Day0,the relative abundance of genes related with GH77,GH117,GH55 and GT14 increased significantly at Day28(P<0.05),but the relative abundance of genes related with CBM51 and GH110 were significantly decreased at Day28(P<0.05);At different time points,the relative abundance of GT2,GH13,GH3 and GH43 related genes were all high and they were much higher at Day28 than at Day0.The results showed that it could increase the abundance of some glycoside hydrolysates and enhance the ability of intestinal flora to digest polysaccharides by supplementing Golden Snub-nosed Monkeys with E.faecalis dlt7 a,so as to provide the nutrient for the intestinal epithelial cells of the host.3.The predicted genes were blasted to KEGG pathway database.The results were that: Compared with Day0,there were 12 KEGG subsystems changed significantly at Day28(P<0.05);The pathways which increased the relative abundance of genes included Alanine aspartate and glutamate metabolism,Tyrosine metabolism,Arginine and proline metabolism,Valine leucine and isoleucine biosynthesis,Valine leucine and isoleucine degradation,Tryptophan metabolism,Oxidative phosphorylation,Glyoxylate and dicarboxylate metabolism,Ethylbenzene degradation and Ether lipid metabolism;Other two pathways which reduced related genes' relative abundance were Streptomycin biosynthesis,Butirosin and neomycin biosynthesis.The results showed that it mainly changed the related genes' relative abundance of pathways such as amino acid metabolism,the synthesis of beneficial substances and the degradation of harmful substances by adding E.faecalis dlt7 a to the diet of Golden Snub-nosed Monkeys,which was beneficial to the health of Golden Snub-nosed Monkeys in Shennongjia. |
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