ISOLATION OF BACTERIOCIN-PRODUCING LACTIC ACID BACTERIA AND IMMOBILIZATION IN MICRO-POROUS CALCIUM ALGINATE GEL BEADS FOR EFFICIENT PRODUCTION OF ANTI-MICROBIAL SUBSTANCES
The aim of this work was to isolate bacteriocin-producing Lactic Acid Bcateria (LAB) capable of inhibiting the growth of both Gram-positive and Gram-negative bacteria and to optimize their culture conditions. A total of fourty-two (42) acid-producing bacteria were isolated from different sources: 20 from akamu, 9 from ukpaka, 5 from nunu, 4 from fish and 4 from intestine of cow. Out of these 42 isolates, 11 had activity against Escherichia coli, Bacillus cereus, and Lactobacillus plantarum and were selected for further experiments. To determine the optimum pH of the medium suitable to harness the antimicrobial activities of these isolates against the target organisms, their pH was adjusted to pH 5.5, 3.5 and 8.0 before sterilization and a control set up without pH adjustment but was checked to be 6.0. Depending on the isolate, or the pH of its medium, the optimum pH varied but pH 6.0 (control experiment without pH adjustment) showed more occurrence as the highest for effective production and activity of antimicrobial substances against target organisms. To select the best isolates producing bacteriocins, the organic acids and hydrogen peroxide produced were eliminated. This was done by adjusting the pH of the Cell Free Supernatant (CFS) to 6.0 and addition of catalase respectively. Three (3) isolates; A12, A15 and N3 were selected for optimization studies among other isolates on the basis of their ability to produce bacteriocin-like substances inhibiting the target organisms especially Escherichia coli. The antimicrobial and bacteriocin production and activity of these 3 isolates were optimized by immobilizing in normal calcium alginate gel beads and micro-porous gel beads and comparing them to free cells. In all cases, immobilization in micro-porous gel beads showed highest antimicrobial and bacteriocinogenic effect against the target organisms especially Escherichia coli (p<0.05). There was no major difference between the antimicrobial and bacteriocinogenic effect of free cells compared to when immobilized in normal alginate gel beads (p<0.05). A12 isolate, having showed the highest antimicrobial and bacteriocin production against all target organisms especially Escherichia coli was identified to be Lactobacillus plantarum strain S4 using the 16S rRNA primer sequence. Lactobacillus plantarum is known to produce a bacteriocin called plantaricin. It is interesting to note that although the amount of bacteriocins produced was relatively low (IZD<1.0cm), higher productivity was achieved by immobilizing the bacteriocin-producing strains in micro-porous alginate gel beads.