Purification And Characterization Of Bacteriocin Produced By Streptococcus Thermophilus CGMCC1.1864

The bacteriocin producted by Lactic Acid Bacteria (LAB) was the most well kno-w n bio-preservative. In this study, five LAB strains (Lactobacillus plantarum, Strept-oc ccus thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. Bulga-r icus, and Lactobacillus delbrueckii subsp. Delbrueckii) were included to investiga-te their antimicrobial activity of the supernatants of fermentation culture with the a-gar diffusion method.Inhibitive effects of hydrogen peroxide and organic acid were ruled out in the subsequent experiments. Antimicrobial activity of the culture supernata-nt of Streptoc occus thermophilus CGMCC1.1864was detected. The treatment of proteases and a-amylase could completely abolish the antimicrobial activity of the supernatant, while no activity loss was observed when treated with catalase. These results demonstrat-ed that the antimicrobial substance belonged to bacteriocin family and was named a-s thermophilin ST9tentatively.Since thermophilin ST9could specially adsorb to the producing strain, pH-adso-rption method was chosen to purify ST9, and the regulation of pH5.5for adsorpti-on and pH1.8for desorption is the optimum condition.Subsequently, the SephadexG-25column chromatography was adopted to elimin-ate other proteins. The ST9was washed with distilled water at0.3mL/min. The opti-mum detection wavelength and the best sampling volume are respectively220nm a-nd1mL. The crude extraction was treated by the SephadexG-25column chromato-graphy secondly, with the elution flow rate0.5mL/min. Under these conditions, t-he electrophoretic purity of ST9was obtained, and it’s specific activity was5133.7AU/mg, as23times as the cell-free fermentation broth. Purified thermophilin ST9was subjected to Tricine-SDS-PAGE analysis and its molecular mass was determine d to be5.0kD.Heat stability test was performed with the antimicrobial substance and no activ-ity lost was observed after2h incubation at100℃or20min at121℃. However, its activity was reduced significantly if the boiling samples were transferred to-20℃freezer for storage immediately. It was also tolerable to the change of pH level s, no significant reduction of its antimicrobial activity was observed at pH2.0throu-gh pH9.0. The antimicrobial spectrum assays showed that the supernatant could in-hibit the growth of both Gram-negative bacteria and Gram-positive bacteria.The surface structure of ST9on the mica were studied by atomic force micros-cope(AFM). The experiment indicated that this bacteriocin is irregularly spherical,1- entous and self-assembly. The concentration has large influence for AFM images, a-nd the aggregating state is various in different concentration.