| By 16S rDNA genomic molecule technique,6 strains of Lactobacillus with the bacteriostatic ability,isolated from the muscovy ducks intestine were identified.In vitro,these strains were assessed about probiotic function.The influence on adherence to Caco-2 and the adhesion molecule was also studied by taking the advantage of the Lactobacillus.The study contained two aspects:1.Isolation and identification of Lactobacillus from muscovy ducks intestine30 healthy muscovy ducks(averagely weighted about 3.5 kg,300 d)were anatomized,with their intestinal flora isolated.The experiments were conducted as follows:1)isolatation and purification of Lactobacillus by MRS;2)observation of the isolated Lactobacillus;3)similarity comparison of 16S rDNA gene sequence of isolated strains in NCBI.The results showed that:1)The isolated strains included bacillus and coccus;2)6 strains of isolated Lactobacillus was highly homologous with Lactobacillus rhamnosus,Lactococcus lactis subsp.Lactis,L.salivarius,Streptococcus lactis,Enterococcus columbae and L.murinus,respectively.Gram staining showed that 6 isolated strains were all G+,and they were named CM1,CM2,CM3,CM4,CM5 and CM6.6 strains' ability to produce lactic acid was fine.The supernatant's pH value was 3.85,3.51,3.67,3.39,3.37 and 3.35,respectively.All 6 strains had certain tolerance to the acid,bile salts and protease,and showed a high viability with digestive enzyme's existence.The test about antibiotics sensitivity showed that 6 strains of Lactobacillus were generally sensitive to penicillin and tetracycline antibiotic,and drug-resistant to ofloxacin,macrodantin and cotrimoxzole.2.Study on the probiotic characteristics of isolated LactobacillusThe experiments were conducted as follows:1)detecting the bacteriostatic effect by Oxford cup method;2)detecting the adhesion ability of Lactobacillus to Caco-2 by SEM;3)testing the mRNA level to the adhesion molecule which related to Caco-2 by RT-PCR.The results showed that:1)6 isolated strains showed a certain bacteriostatic effect on Escherichia coli,Salmonella typhimurium and Staphylococcus aureus.The bacteriostatic effect on E.coli was not significant(P>0.05).To S.typhimurium,CM3 showed a strong bacteriostatic effect,and antibacterial circle diameter was about 19.41 mm.To S.aureus,CM6's bacteriostatic effect was much stronger than the others,the diameter was about 17.26 mm.2)6 strains of Lactobacillus had certain adhesion abilities to Caco-2.After detecting the bacterial characteristics including hydrophobicity and auto-aggregation,CM3 and CM4 showed the highest binding rate with 99.79%and 99.72%.CM3's auto-aggregation ability was 90.16%,which was stronger than the others;3)The results about gene expression showed that the mRNA level to F-actin all significantly increased in the 3 treatment groups:CM3(P<0.001),S.typhimurium and CM3+S.typhimurium(P<0.05).The mRNA level to CaM and E-cad significantly deceased(P<0.05)in the 2 treatment groups as S.typhimurium and CM3+ S.typhimurium,compared to the control group.The mRNA level to CaM,E-cad and F-actin significantly decreased in the S.typhimurium and CM3+S.typhimurium group(P<0.01),compared to the CM3 group.The results indicated that CM3 had the strongest bacteriostatic effect.Bacillus including CM1,CM3 and CM6,compared to the coccus including CM2,CM4 and CM5,had a significant difference in the adherence to Caco-2(P<0.05).The pH values of the 6 isolated strains were all acidic,while the hydrophobicity of CM3 and CM4 were the strongest,and the auto-aggregation of the CM3 was stronger than the others.By the tests on the growth performance,acid production,tolerability,bacteriostatic effect and adherence to Caco-2,an ideal strain CM3 was screened out,which could be used in the clinical trials and feeding of animals. |
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