Antimicrobial Properties of Purified Bacteriocins Produced from Lactobacillus casei and Lactobacillus fermentum against Selected Pathogenic Microorganisms

Abstract

Aims: (1) To isolate bacteriocin samples produced from Lactobacillus using natural fermented foods which include: palm-wine, milk, locust beans, fufu (white solid food made from cassava), ogi (known as pap) and dairy fermented product (Yogurt); (2) extraction and purification of these bacteriocin samples using centrifugation and ammonium sulphate respectively and removal of impurities using dialysis. (3) to confirm the production of bacteriocin by performing antimicrobial assay against some selected pathogenic microorganisms. (4) to examine the effects of pH, heat and storage stability as well as biopreservative efficiency of the bacteriocin samples in pap, kunu (made from millet) and fresh orange juice; (5) to investigate the effect of viable antibiotics on the growth of isolates. Study Design: Data were analyzed using the statistical software package SPSS version 16.0 and standard errors of mean (SEM) for all the graphs plotted were represented with error bars while their characterization was designed using reciprocal of the highest dilution (2n) that resulted in the inhibition of the indicator lawn. Thus, the arbitrary units (AU) of the bacteriocin activity per milliliter (AU/ml) were defined as 2n× 1000/ 10µl. Place and Duration of Study: Microbiology Laboratory, Sacred heart Hospital, Abeokuta and Department of Microbiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria, between March 2010 and November 2012. Methodology: Bacteriocins, otherwise known as the antimicrobial compounds were produced from the Lactobacillus strains and then isolated from Nigerian fermented foods which include: palm wine, milk, yoghurt, locust beans, ogi and fufu. These foods were isolated using de Mann Rogosa and Sharpe medium. The isolated microorganisms (L. fermentum and L.casei) were identified phenotypically after isolation. Bacteriocins were extracted and purified from the Lactobacillus strains by centrifugation, followed by ammonium sulphate precipitation and dialysis. The antimicrobial activities of the crude bacteriocins were tested against nine selected pathogenic clinical isolates collected from the University College Hospital, Ibadan. The tested isolates were Shigella dysenteriae, Shigella flexneri, Enteropathogenic Escherichia coli type 1, Enteropathogenic Escherichia coli type 2, Enterohaemorrhagic E. coli type 3, Salmonella typhi, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. Results: The bacteriocins of Lactobacillus fermentum and Lactobacillus casei showed a broad range of activities and had higher significant effect (P< 0.05) on the selected pathogenic microorganisms. The effects of pH on the bacteriocins were active in range of 2 to 6. Bacteriocins produced by Lactobacillus casei were stable at 800C for 15 minutes while bacteriocins produced by Lactobacillus fermentum were stable at 1000C for 15 minutes. It was observed that these bacteriocins can be stored between -200C and 40C and they had significant difference on the selected pathogenic microorganisms (P<0.05). The preservative activities of the bacteriocins tested on different foods showed that the bacteriocin of Lactobacillus fermentum had maximum reduction on bacterial population. Lactobacillus fermentum and Lactobacillus casei isolates were resistant to erythromycin of 70% and 100% for cotrimoxazole, ciprofloxacin, augmentin and amoxicillin. Conclusion: This study showed that bacteriocins from fermented foods could be used as an effective control for pathogenic microorganisms as they were able to exhibit antimicrobial activity against the test organisms when investigated for bacteriocin production and when characterized.