Study on Isolation and Identification of Lactic Acid Bacteria in Fermented Camel Milk of Xinjiang and Its Bacteriocin
|School||Xinjiang Agricultural University|
|Course||Of Food Science|
|Keywords||fermented camel milk lactic acid bacteria isolation and identification 16S rDNA analysis bacteriocin antibacterial activity|
In this study, lactic acid bacteria （LAB） were isolated from traditional fermented camel milk of Kazaks in Xinjiang and identified by the tests of morphology, physiology and 16S rDNA analysis.The lactic acid bacteria which were able to produce bacteriocin were screened out. The antibacterial activity of bacteriocin from the lactic acid bacteria was studied and the substances from LAB were extracted and purified. The results were as follows:1.23 strains of LAB were isolated from a Shubat sample which was gathered from places such as Manasi, Wulabo, Nashan and Shuixigou （simplified characters:MLS, WLB, NSL and SXG） in Xinjiang. According to the morphological characters, physiological and biochemical properties,17 strains of Lactobacillus （73.91%） were identified as L.helveticus, L.casei, L.rhamnosus, L.delbrueckii subsp. Lactis, L.plantarum, L.fermentum and L.paracasei; 3 strains of Lactococcus （13.04%） were identified as L.lactis subsp.lactis; 2 strains of Enterococcus （8.70%） were identified as E.durans; and 1 strain of Streptococcus （4.35%） was identified as S. thermophilus. The dominant strains found were L.helveticus （17.39%） and L.lactis subsp. Lactis （13.04%）.2.16S rDNA sequences to 10 representative strains NSL2, MLS5, SXG1, NSL4, WLB3, MLS2, MLS6, WLB5, SXG5 and NSL7 were analyzed. The results showed the high similarity between representative strains and the controls in the NCBI database by comparison. The similarities were all greater than 96%. There was a 99% similarity between the strain of MIS5 and L.casei. The results of analysis of 16S rDNA sequences were coincident with the identification results of physiological and biochemical properties.3. According to the phylogenetic analysis of 16S rDNA sequence, the strain of MIS5 was a monophyletic group with L.casei CGMCC1.2435 （HM162414）, BL23 （HM162415） and NM89-3 （HM218375）, the branch of genetic distance of them was the nearest and the confidence was 97%. Strains of L.casei MIS5, L.paracasei MLS6, L.helveticus NSL2, L.rhamnosus SXG1, L. plantarum WLB3, L.fermentum MLS2 and L.delbrueckii subsp. Lactis NSL4 constituted a branch; the strain of E.durans SXG5 was a system and strain of S.salivarius WLB5 and L.lactis subsp. Lactis NSL7 also made up another branch.4.9 Strains were screened out and showed inhibitory activity against S.aureus、B.subtilis and E.coli by double-deck agar diffusion and punch methods. One strain of MLS5 exhibited the strongest antibacterial activity. The antimicrobial substance produced by MLS5 was initially identified as protein bacteriocin by the experiments of organic acids and hydrogen peroxide exclusion and protease susceptibility. The tolerance tests, with the results of temperature and pH, indicated that the bacteriocin still had thermal stability at 121℃for 20 minutes and antibacterial activity at pH3-6. The bacteriocin was generated from the mid-logarithmic growth phase and reached the maximum activity at the stationary phase. The bacteriocin produced by MLS5 exhibited a broad antibacterial spectrum. It can not only inhibit the Gram-positive bacteria, but also inhibit E. coli and Mucor. The fermentation curve of strain MLS5 showed that the strain had a capacity of producing bacteriocin and acid at 37℃.5. L.casei MLS5 was able to produce bacteriocin when it was incubated at 37℃for 22 hours in MRS broth. The bacteriocin was extracted and purified through both degree of saturation 50% ammonium sulphate precipitation and 732-cation exchange resin chromatography. The yield of bacteriocin was 15.14%. The specific activity was 1854.27IU/mg. and 31.96 times of supernate. The clear belts showed up after SDS-PAGE electrophoresis of purified bacteriocin; its molecular weight was between 14.4 kDa-20 kDa.