| ObjectiveThe microorganism quantity of big bone chicken?s cecum bacterial tops the list of each intestines section,which have an effect upon the digest and quality of the host nutrition.The purpose of the topic research includes the aspects as follows:1,the analysis of community composition and the species abundance of cecum microorganism fauna to acquire the advantage bacterial community,which serves the probiotics selection as a theoretical basis.2,the selection of probiotics strain from the cage-free chicken cecum,which provide the premise conditions for the development of the microecologicsMethodsThe DNA of cecum bacterial community was extracted,then the variable region of the cecum contents of V4 16s rDNA was analyzed by the metagenome and high-throughput sequencing and the application of software Qiime and Mothur.Two kinds of probiotics were cultivated and isolate many strains from the cecum contents among which the Clostridium strain was through colony morphology observation,staining microscopy and physiological and biochemical identification.And lactic acid bacteria through colony morphology observation,staining microscopy,catalase test and fermentation liquid measuring the p H value,the resule was screened lactic acid bacteria,both probiotic research using plate count method,MATH method,Oxford cup measured bacteriostatic circle method,selected probiotic better strains for the 16s rDNA molecular identification.Results1,Through the analysis of cage-free group and control group cecum microflora sequencing,it was concluded that the cage-free group and the control group of Chao values were 4127.87/2273.06,Ace value were 5876.50/3100.56,Shannon value were 4.58/3.86,Simpson value were 0.04 /0.09.At the phylum level,there have three significant phyla in cage-free group and the control group(P<0.05).The cecum bacterial community of cage-free group belong to 24 phyla,the percentage of Bacteroidetes,Firmicutes and Proteobacteria was 82.90%,the percentage of Bacteroidetes and Firmicutes were 51.57%,21.56%.While the cecum bacterial community of control group belong to 17 phyla,the percentage of Bacteroidetes,Firmicutes and Proteobacteria was 53.05%,the percentage of Bacteroidetes and Firmicutes were 26.70%,16.49%.In the class level,the cecum bacterial community of cage-free group belong to 42 classes,the percentage of Bacteroidia and Clostridia were 51.56%,20.57%,In the control group,the cecum bacterial community belong to 26 classes,the percentage of Bacteroidia and Clostridia were 26.69%,16.07%.At the level of the genus,there were 60 genera,and there were 10 differences in the cage-free group and control group(P<0.05).After NMDS analysis,the microbial communities in the different groups were different in the cage-free group and the control group.2,Through the colony morphology observation and physiological and biochemical identification,screened of three strains of Clostridium.At the same culture time,when the p H value was 2,the survival rate of D-19 and D-2 was 50.53%,48.60%,that is significant higher than d-8(P<0.05),when p H value was 3,the survival rate of D-19 and D-2 was 62.93%,61.20%,that is significant higher than D-8(P<0.05).When the p H value was 4,the survival rate of D-19 is 81.17%,significantly higher than the other two strains(P<0.05);when the bile salt concentration was 0.2%,D-19 survival rate was 44.47%,when the concentration was 0.3%,41.67% are significantly higher than those in the other two groups(P<0.05).In the aspect of hydrophobic rate of D-19 was 19.07%,significantly higher than the other two groups(P<0.05).In the antibacterial activity against Escherichia coli and Staphylococcus aureus,three strain of bacteriostatic ring between 11.40mm-15.97 mm.Comprehensive experimental results,selected D-19 as 16 S rDNA molecular identification.3,Through the colony morphologyobservation,catalase experiment and measure the p H value of bacteria liquid,screened three strains of lactic acid bacteria.At the same culture time,when p H value is 2,the survival rate of R-6 was 70.60%,significantly higher than the other two groups(P<0.05),when the p H value is 3,R-5 and R-6 were significantly higher than that of R-3(P<0.05),and when p H value is 4,the survival rate of R-5 was 87.83%,that was significantly higher than the other two groups(P<0.05);when the bile salt concentration of 0.2%,R-5 was 44.47%,the survival rate was significantly higher than that in the group of 42.93% and 39.87%(P<0.05).the hydrophobic rate of R-5 was 28.70%,was significantly higher than the other two groups(P<0.05),In the antibacterial activity against Escherichia coli and Staphylococcus aureus,R-5 and R-6 were significantly greater than that of R-3(P<0.05),R-5 and R-3 were significantly greater than that of R-6(P<0.05).So the comprehensive test results,selected R-5 as 16 S rDNA molecular identification.Conclusions1,Using the metagenome sequencing analysis of two groups of the V4 variable region of the the cecum bacterial,the results show that the diversity of the big bone chicken in the cage-free group was higher than that in the control group,and the community structure was more abundant than the control group.On the level of the phylum,the Bacteroidetes and Firmicutes were the advantage bacteriophyta of the chicken.2,Doing probiotic research by screened out the three strains of Clostridium strains from the Firmicutes.According to the experimental results,the D-19 probiotic is higher than the other two groups strains,the strains were 16 S rDNA molecular identification,the result was Clostridium butyricum.3,Doing probiotic research by screened out the three strains of lactic acid bacteria from the Firmicutes.According to the experimental results,the R-5 probiotic is higher than the other two groups strains,the strains were 16s rDNA,molecular identification,the result was Lactobacillus salivarius. |
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