PRODUCTION OF ANTIMICROBIAL SUBSTANCES BY LACTIC ACID BACTERIA (LAB) IMMOBILIZED IN CALCIUM GEL BEADS.
Twenty six lactic acid bacteria (LAB) were isolated from fresh fish. Ten isolates were confirmed to produce antimicrobial substances by agar well diffusion. Four isolates with the highest antimicrobial activities were selected. Based on standard microbiological and biochemical tests, the isolates were tentatively identified as Lactobacillus plantarum, L fermentum, L brevis, and L delbruki. The four isolates were screened for antimicrobial activity against selected target organisms, Escherichia coli, Listeria. monocytogenes, Bacillus. subtilis and Bacillus cereus, using agar well diffusion method. L. plantarum had the highest inhibitory activity and was used for further studies. L plantarum was immobilized in calcium alginate for production of antimicrobial substances. The effects of media components and CaCl2 concentration on bead stability were investigated. Furthermore, the effects of oxygen tension, bead diameter, cell loading and bead coating on cell growth and antimicrobial activities were investigated. The results showed that addition of 5%w/v CaCl2 resulted in the most stable beads in MRS media. The antimicrobial activity of the immobilized cell under aerobic condition was significantly higher than the free suspended cells. The activity of the cells immobilized in 3mm bead diameter was significantly higher than the activities obtained with 5mm, 7mm and 9mm beads (p<0.05). Also inoculum size of 6.8 x108 cfu/ml resulted in higher antimicrobial activity than the values obtained with 1.5 x 10 9 cfu/ml, and 2.3 x 109 cfu/ml respectively (p< 0.05). Coating of the gel beads significantly reduced cell leakage but the antimicrobial activity was lower than that of uncoated beads (p< 0.05). The present findings suggest that immobilization of cells in calcium alginate is a promising method for efficient production of antimicrobial substances by LAB.