African Journal of Biotechnology Vol. 2 (7), pp. 179–184, July 2003   

ISSN 1684-5315  © 2003 Academic Journals

Full Length Research Paper

Influence of cultural conditions on the production of bacteriocin by Lactobacillus brevis OG1                

S.T.  Ogunbanwo*, A.I. Sanni, and A. A. Onilude

Department of Botany and Microbiology, University of Ibadan, Nigeria

*Corresponding author: Fax: 234-02-8103043; 8103118; e-mail: topzybanwo@yahoo.com

Accepted 5 June 2003

Bacteriocin produced by Lactobacillus brevis OG1 has large spectrum of inhibition against pathogenic, food spoilage microorganisms and various Lactic acid bacteria employed as test strains. The bacteriocin inhibited E coli NCTC 10418 and Enterococcus faecalis, but did not inhibit Candida albicans ATCC 10231 and Klebsiella sp. UCH 15.  The antibacterial activity appeared to be pronounced between early logarithmic and early stationary phase. Supplementation and/or replacement of nutrients demonstrated that larger quantities of bacteriocin could be produced by addition of yeast extracts (3.0%), NaCl (1.0-2.0%), glucose (1.0 %) and Tween 80 (0.5%), while addition of tri-ammonium citrate, sodium acetate, magnesium sulphate, manganese sulphate and potassium phosphate had no effect on production. Maximal activity in composed medium was achieved at initial pH of 5.5, and incubation period of 48h at 30-37⁰C.    

Key words: Bacteriocin, growth media, Lactobacillus brevis OG1, indicator organisms, antagonistic activity.

Traditional preparation of ogi

The cereal grains (Zea mays) are cleaned and steeped in water for 2 days in earthenware pot (or any suitable container). The water is decanted and the grains wet-milled before sieving with muslin cloth or fine wire-mesh. The pomace is discarded and the starch suspension is allowed to sediment during which fermentation is carried out for 2-3 days by the natural flora of the grains (Odunfa and Adeyele, 1985).

Bacterial Strains and Cultures

The strain of L. brevis OG1 used in this study was isolated from ogi and characterized using the API 50 CH strips (API System Biomerieux  SA, France). It was maintained as frozen stocks at –20⁰C in Hogness freezing medium and propagated twice in MRS medium (Oxoid; Onipath Ltd, Basingstoke, Hampshire, England) before use.  The bacterial strains used as indicator organisms are listed in Table 1.  All the LAB indicators organisms were cultivated in MRS medium while Enterococcus, Micrococcus, Bacillus, Listeria and Staphylococcus, were cultivated in brain heart infusion medium (Oxoid, UK).  Clostridium was cultivated on blood agar while Salmonella and Shigella were cultivated in Desoxycholate-Citrate agar (DCA) medium. Gram-negative bacteria were cultivated in tryptic soy broth medium (Oxoid), while Candida was cultivated in yeast extract agar.

Bacteriocin Assay

L. brevis OG1 was cultivated anaerobically (Oxoid Gas Generating Kit) in MRS broth (glucose, 0.25%; peptone 0.5%) for 72 hours at 30°C.  The pH and growth pattern of the test isolates was determined.  Extraction of bacteriocin was carried out using the method of Schillinger and Lucke (1989).  Inhibitory activity from hydrogen peroxide was eliminated by the addition of 5mg/ml catalase (C-100 bovine liver, sigma) (Daba et al., 1991).  The culture supernatant was purified to ensure that only bacteriocin (with the exception of other antimicrobial compounds such as lactic acid, hydrogen peroxide, diacetyl, ions etc) was assayed according to the method of Jimenez-Diaz et al. (1993). Bacteriocin was detected in the fraction at every stages of the purification.  Antagonistic activity against indicator organisms was determined using a well diffusion assay (Schillinger and Lucke, 1989).  The antimicrobial activity of the bacteriocin is defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and is expressed in activity units per ml (AU/ml).

Influence of medium component on the production of bacteriocin

The effect of medium ingredients on bacteriocin production was evaluated using composed media.  The supplements studied were tryptone (0.0 – 3.0%), yeast extract (0.0 – 3.0%), beef extract (0.0 – 3.0%), triammonium citrate (0.0 – 0.2%) sodium acetate (0.0 – 0.5%), MgSO₄·7H₂O (0.0 – 0.5%), MnSO₄·4H_­2O (0.0 – 0.1%), K₂HPO₄ (0.0 – 0.2%), NaCl (0.0 – 0.3%), glucose (0.0 – 3.0%) and tween 80 (0.0 – 1.0%).

Influence of growth conditions on the production of bacteriocin

The effect of incubation temperature and time on production of bacteriocin was carried out.  Three portions of composed media were inoculated (1% v/v) with an overnight culture of bacteriocin producing organism; incubated at 25, 30, 37 45 and 55⁰C for 48h and the absorbance values (580nm), pH and bacteriocin activities of cultures were determined.

To determine the effect of initial pH on production of bacteriocin, 100ml of composed media were adjusted to initial pH values of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, using 5 mMol^-1 hydrochloric acid or 5mMol1^-1 NaOH.  Each medium was inoculated (1% v/v) with an overnight culture of bacteriocin producing organism and incubated at 30⁰C for 48 h. Absorbance values (580 nm), pH and bacteriocin activities were determined (Yildirim and Johnson, 1998).

The effect of incubation period was studied in the same manner as described for pH. Active cultures of producer organism (1% v/v) were inoculated into 100ml aliquots of sterile composed media in Erlenmeyer conical flasks.  Inoculated flasks were incubated at 37⁰C for periods of 12, 24, 36, 48, 60 and 72 h. Individual flasks were kept for each incubation period.  At the end of each incubation period, bacteriocin activity, pH and absorbance values (580 nm) were determined (Balasubramanyam and Varadaraj, 1998).

The present study was primarily aimed at determining cultural conditions for obtaining better and stable bacteriocin production. L. brevis OG1 was able to produce bacteriocin, which had a wide inhibitory spectrum towards both Gram-negative and Gram-positive food spoilage and pathogenic bacteria. It inhibited 21 out of 32 indicator strains with the largest zone of inhibition (12mm) being against Enterococcus faecalis EFI.  The bacteriocin from L. brevis OG1 (producer organism) had no inhibitory effect on the organism itself (Table 1). In a mixed fermentation environment, production of bacteriocins may prove advantageous for a producer organism to dominate the microbial population (Graciela, 1995).

   Table1. Inhibition of various indicator organisms by bacteriocin produced L. brevis OG1.

The influence of culture medium components on the production of bacteriocins was investigated using E. faecalis EFI as indicator organism. Results show that bacteriocin was produced when nutrients were available for metabolic activity.  Larger amounts of the bacteriocin were synthesized only when the medium was supplemented with glucose (1.0%), Tween 80 (0.5%), yeast extract (2-3.0%) and NaCl (1-2.0%), while addition of tri-ammonium citrate, sodium acetate, magnesium sulphate, manganese sulphate and potassium phosphate had no effect on bacteriocin production (Table 2). Thus variation     in     the     concentration     of     constituents/supplementation of cultivation media might have an influence on the amount of bacteriocin produced by microorganisms.  Similar observations have been made previously. Daba et al. (1993) obtained similar results in the production of mensenterocin 5. Biswas et al. (1991) compared the production of pediocin ACH by Pediococcus  acidilactici H cultivated in TGE broth, MRS broth and several modifications of it.  TGE broth containing varied concentration (0-2%) of trypticase, glucose and yeast extract, produced maximum pediocin at the 1% level.  When cultivated in normal MRS broth, 15% less pediocin was obtained compared to the yield in TGE broth.  Sanni et al. (1999) also reported that highest bacteriocin activity was obtained when glucose and peptone were varied to 0.25% and 0.5% in the constituted MRS broth, while bacteriocin activity was not detected at 2% glucose and peptone level. Modification of nutrients of cultivation media should be considered for maximal production of bacteriocin that has potential use as a food biopreservative (Biswas et al., 1991). The question of production cost is an important issue to be taken into account when large-scale production of the bacteriocin for use as a food preservative is considered. Our results also show that bacteriocin could be produced in a relatively inexpensive medium.

Table 2. Effect of nutrient components on bacteriocin production by L. brevis OG1.