ISOLATION AND PARTIAL PURIFICATION OF BACTERIOCIN PRODUCED BY LACTOBACILLUS CRISPATUS KLB46

초록

Lactobacillus crispatus KLB46 isolated from human vagina produces a pH and heat-stable bacteriocin that has a narrow inhibitory spectrum. After a culture of L. crispatus KLB46 was grown in MRS broth for 15 h at a constant pH 5.5, cell was removed by centrifugation. The bacteriocin present in the supernatant fraction was concentrated by adding 80% ammonium sulfate. The precipitate pelleted by centrifugation was dissolved in 20 mM sodium phosphate buffer (buffer A), dialyzed against same buffer. The concentrated sample was applied to a CM cation-exchange column equilibrated buffer A. After subsequential washing of buffer A and 0.1 M NaCl, and eluted with linearly increasing NaCl gradient (0.1 to 1M) in buffer A. Active fractions obtained were pooled dialyzed and then lyophilized. For the activity measurement of bacteriocin, washed SDS-polyacrylamide gel was overlaied with MRS agar containing indicator strain for direct detection of antimicrobial peptide and estimated molecular masses ranging 4 Kd and 6.5 Kd.