ABSTRACT
The characterization of a microbial isolate to genus level using conventional methods only amounts to a partial characterization of the isolate and has some limitations , apart from being laborious and time consuming, some microorganisms may however be misidentified at genus or species level. Bacteriocins are a group of potent antimicrobial peptides produced by lactic acid bacteria( LAB) and are ribosomally synthesized as primary metabolites. Their broad spectrum of inhibition suggests that these LAB strains serve as natural biopreservatives in various food products and may help to combat human pathogens. This study was aimed at optimizing bacteriocin production from molecularly identified Lactic Acid Bacteria (LAB) obtained from fermented maize (Ogi) by exploring the effects of nutrients and pH on the activity levels of bacteriocin.
White and yellow maize (500g each) were obtained from from Oba Market, Benin City and International Institute of Tropical Agriculture (IITA) Ibadan. Eight Lactobacillus plantarum were isolated from fermented maize made into ogi using standard microbiological methods. They were further characterized using molecular methods by Polymerase Chain Reaction (PCR) amplification of 16S rDNA genes to confirm their identities. Bacteriocin screening was determined using agar spot test and well diffusion method. Effect of enzymes, different pH, optimal temperature and different growth media were determined to optimize bacteriocin production
The results showed that the enumeration of LAB (log10 cfu/g) from the fermented maize samples ranged from 2.00± 0.01 - 7.11 ± 0.25, with treated maize having the highest LAB count and white maize having the least count. The molecular characterization by Polymerase Chain Reaction amplification of 16SrDNA genes confirmed the identities of the Lactic Acid Bacteria as Lactobacillus plantarum (SG 229), Lactobacillus plantarum (SG224) Lactobacillus fermentum (SG217). The isolated LAB species produced the following bacteriocin levels; 6500 Au/ml (SG 229), 3700Au/ml (SG224) and 3200Au/ml (SG 217) at pH 6.5. SG229 which produced the highest quantity of bacteriocin was optimized with Tryptone and Yeast extract as well as Tryptone and Malt extract and yielded 8800Au/ml and 8700Au/ml respectively from 6500Au/ml.
There was a significant reduction in bacteriocin activity after treatment with proteinase k and trypsin from 18mm to <5mm. LAB species grown in the presence of 2% w/v sugars resulted in low levels of bacteriocin production for SG229 for fructose and lactose (800Au/ml). While bacteriocin activity was also repressed with higher (from 0.5 to 50.0g/L) concentration of potassium ions. In the absence of glycerol, bacteriocin production was 6500Au/ml from SG229 control. Bacteriocin activity of 6500Au/ml was recorded without addition of vitamins while the addition of vitamins suppressed production level/potential to 2200 (vitamin B1) and 800 (vitamin C). Bacteriocin SG 229 had a molecular weight of 11kDa according to Tricine- SDS page while SG224 had a molecular weight of 9.5kDa. Bacteriocin produced inhibited the growth of Eschericia coli (18mm), Listeria monocytogens (17mm), Staphylococcus aureus (15.50mm) and Enterococcus faecalis (15mm).
