Study On Isolation And Identification Of Cellulose Decomposing Bacterium In Jilin White Geese Intestinal Tract

Twenty 2-month healthy Jilin White Geese were selected in the experiment and fed four Weeks. Crudefiber of the diets was 8％. 15 geese with 4kg body weight were slaughtered and sterilized in0.25％bromogeramine after slaughtering then intestinal tract including duodenum, jejunum, ileum, andcaecum were collected immediately by necropsy. Chyme and scraps were respectively collected andcultured under sterile conditions in isolation medium at 42℃for 7-10 days in the test tube. Culture mixturewere serially diluted and streaked onto CMC-Na culture medium. The exuberant growing strains wereselected and isolated by streaking repeatedly. All 42 strains were selected, three of them from ileum chyme,seven of them fromileum scraps and eight of them from caecum chyme, twenty of them from scraps. Therewere no strains which was isolated on categorically anerobic condition.Proliferation was conducted by inoculating stored strains to CMC-Na plates and cultured at 42℃for48h. Colonies were stained with 1 mg/L Conge-red for 5 min followed by removing stain liquor. Diametersof stain circles were measured and relative enzyme activity was calculated as Diameter of dissolvingcircle/days. Eighteen of which was selected, one of them from ileum chyme, three of them from ileumscraps and four of them from caecum chyme, ten of them from scraps. Isolates with higher ratio wereselected and cultured at 42℃for in shaking bottles. Three from caecum scraps and one from ileum scrapswith highest relative enzyme activity were selected for enzyme activity assay.The experiment of Crude fiber degradation was carried out by mimicking caecum environment in vitrounder condition of artificial caecum juice, then single strain and compound strains were inoculatedrespectively in the three kinds of forage grasses (leymus chinensis, alfalfa and straw).Further study was made about four strains' characteristic of bacteriology, physiology, biochemics andmolecular biology. Finally, the strain was identified by comparing 16S rDNA partial gene secquence ofstrains with BLAST analysis.The results showed that there was a set of cellulose enzyme-producing bacteria in ileum and caecumof Jilin White Geese and they could make use of crude fiber.For the four strains, the optimal enzyme-producing temperature was about 42℃and the optimalenzyme-producing time was 48h. The strain (M6) from caecum scraps demonstrated the highest enzymeactivity (EA 51.4U) in liquid fermented culture medium which consisted of carboxymethyl cellulose-Naand bean stem powder. The most moderate temperature and pH for enzyme activity was at 60℃, pH4.0,while the rest three showed highest EA at 60℃, pH6. The strain (H1) from ileum scraps demonstrated thehighest enzyme activity (EA41.4U) at 60℃, pH6.0. The highest crude fiber degradation rate was 7.87％ under alfalfa inoculated H1×H2×H6 strains. Crude fiber degradation rate is higher than the other two kindsof forage grasses in the alfalfa, the compound strains were a little higher than the single strain; Strain M3belonged to Shigella according to the colonial morphology, biochemistry characteristic and 16S rDNApartial gene order. Sequence homology between 16S rDNA partial sequence of M3 and Shigella achieved99％, and then it was initially regarded as Shigella. Further more, Sequence homology between strain M6and Bacillus subtilis was 92％, they cound not be regarded as the same genus, further study should be doneto identify it.