Studies on Isolation, Identification of Microorganisms and Their Antibacterial Factors from Koumiss
|School||Inner Mongolia Agricultural University|
|Course||Safety of animal products production|
|Keywords||Koumiss Lactic acid bacteria Yeast DNA sequence analysis Identification Antibacterial substance Isolation and purification Factor of anti-mycobacterium tuberculosis|
For researching the medical value of the koumiss, microorganisms in koumiss, collected from Xilinguole region, were isolated, identified morphologically, physiologically and molecular biologically.The antibacterial abilities of the microorganisms, isolated from koumiss, were studied and the antibacterial substances in the media, incubated microorganisms, were extracted, purified and tested.47 strains of lactic acid bacteria and 19 strains of yeasts were isolated and identified morphologically and physiologically. In the lactic acid bacteria, 26 were lactobacillus, which were belonged to 9 species of Lactonostoc and 1 was still not identified yet, 21 were lactococcus, which were belonged to 3 genus, 6 species. 18 of 19 yeasts belonged to 9 genus, Endomyce,Lodderomyces,Pichia,Torulaspora,Kloeckera,Saccharomycodes,Saccharomyces,Brettanomyces,Pachytichospora and 1 was not identified yet. 2 strains of lactococcus, named BY-3-2 and SQ-3-2, were identified molecular biologically by comparing with 16S rDNA sequence. The results indicated that they were E. faecium and E. durans,respectively. 1 strain of yeast, named HJ-5, was also identified molecular biologically by comparing with 26S rDNA D1/D2 sequence. It was Saccharomyces cerevisiae. The results of identification were same when the methods were in the level of morphology and physiology or the level of molecular biology.The antibacterial ability of the 47 strains of lactic acid bacteria was screened. 2 of them had stronger ability to inhibit the growth of bacteria. They were named BY-3-2 and SQ-3-2. The antibacterial substances in the media were extracted, purified and the characteristics of the substances were tested. The optimal growth temperature of BY-3-2 was 37℃, the lowest pH was 4.41. BY-3-2 began to produce antibacterial substances after 8 h incubation, reached to highest level at 12 h of incubation. The antibacterial substances in the media incubated BY-3-2, could be precipitated with 50% ammonia sulphate. The precipitate was then partitioned with iron CM-52 and Sephadex G-100 chromatography. The second partition had the activity of antibacterial. After a electrophoresis, three bands were achieved, the molecular weight were between M33,800Da and M42,800Da. The substances had a higher antibacterial activity between pH 4.0-6.0 and were not sensitive to temperature, but sensitive to amylase and proteinase. The optimal growth temperature of SQ-3-2 was 30℃, the lowest pH was 3.78. Antibacterial substances reached to highest level at 13 h of incubation. The antibacterial substances in the media incubated SQ-3-2, could be precipitated with 70% ammonia sulphate. The precipitate was then partitioned with iron CM-52 and Sephadex G-100 chromatography. After a electrophoresis, two bands were achieved, the molecular weight were between M22,700Da and M23,200Da. The substances had a higher antibacterial activity in a lower pH and were sensitive to temperature, but not sensitive to amylase and proteinase.In pH 6.5, optimal nitrogen and carbon sources could increase the output and activity of antibacterial substances. Treated BY-3-2 and SQ-3-2 with heat, chemical and heat plus chemical, their antibacterial abilities were not affected significantly(P>0.05).The antibacterial ability of the 19 strains of yeasts was screened also. 1 of them had stronger ability to inhibit the growth of bacteria. It was named HJ-5. The antibacterial substances in the media were extracted, purified and the characteristics of the substances were tested. The optimal growth temperature of HJ-5 was between 26-28℃. It began to produce antibacterial substances after 6 h incubation, reached to highest level at 42 h of incubation. After super-filtrated of the incubation medium, the substances, the molecular weight above 30kDa, had the highest activity of antibacterial. In the conditions of pH2 and pH8, among the organic solvent,ethyl ether had the best extraction efficiency. The antibacterial ability of the substances extracted with ethyl ether increased 1.38 times. The antibacterial substances produced by HJ-5 were not belonged to the six kinds of antibiotics. They were sensitive to pH. When pH was under 7.0, the activity was high. As the pH increased, the activity of antibacterial decreased significantly. The tolerance to temperature of antibacterial activity was also related to pH. In the condition of pH 2, antibacterial substances could tolerance to 121℃, 20min.The factors of anti- mycobacterium tuberculosis in koumiss were lipids, the ability of factor was higher, when they were extracted with middle pH condition. Yeast in koumiss also had some ability of inhibition to the growth of mycobacterium tuberculosis.