African Journal of Biotechnology
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African Journal of Biotechnology Vol. 2 (7), pp. 179184, 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 |
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| Abstract | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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.
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| Introduction | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Lactobacilli are important organisms recognized for their fermentative ability as well as their health and nutritional benefits (Gilliland, 1990). They produce various compounds such as organic acids, diacetyl, hydrogen peroxide, and bacteriocin or bactericidal proteins during lactic fermentations (Lindgren and Dobrogosz, 1990). The antimicrobial properties of lactobacilli are of special interest in developing strongly competitive starter cultures for food fermentation. Lactobacilli exert strong antagonistic activity against many microorganisms, including food spoilage organisms and pathogens. Production of the primary metabolite, lactic acid and the resulting pH decrease is the main preserving factor in food fermentation. In addition, some strains may contribute to the preservation of fermented foods by producing other inhibitory substances, such as bacteriocins (Brink et al., 1994). Bacteriocins are antimicrobial proteinaceous compounds that are inhibitory towards sensitive strains and are produced by both Gram-positive and Gram-negative bacteria (Tagg et al., 1976). Research on bacteriocins from lactic acid bacteria has expanded during the last decades, to include the use of bacteriocins or the producer organisms as natural food preservatives. However, the antibacterial properties of lactic acid bacteria (LAB) have not been fully explored for use as biopreservative (Reddy et al., 1984) due to limited production techniques. Maximal bacteriocin production could be obtained by supplementing a culture medium with growth limiting factors, such as sugars, vitamins and nitrogen sources, by regulating pH or by choosing the best-adapted culture medium (Vignolo et al., 1995). In order to exploit the potential utilitarian benefit of the antimicrobial properties of Lactobacillus brevis OG1, the present study was undertaken to determine the optimum cultural conditions for greater bacteriocin yield in constituted growth media. |
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| Materials and Methods | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sample collection White maize (Zea mays) grains used in this study were obtained from retail markets in South-Western Nigeria.
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%), MnSO4·4H2O (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). |
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| Results and Discussion | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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.
Bacteriocin production by the test isolate displayed secondary metabolite kinetics. For example, bacteriocin was produced during the pre-and early exponential growth phases and reached a maximum level at late stationary phase. Growth beyond the stationary phase resulted in a decrease in bacteriocin activity (Figure 1a). This decrease could be due to the activity of extracellular endogenous proteinases induced during this growth phase. Some reports indicate that bacteriocins are produced throughout the experimental growth phase and not solely during late logarithmic or early stationary phase (Joerger and Klaenhammer, 1986; Piard et al., 1990).
a b
c
Figure 1. Effect of Incubation temperature,
pH and incubation period on the activity of bacteriocin produced by L.
brevis OG1.
The effect of incubation temperature, incubation period and initial pH of medium on production of bacteriocin was also investigated. The use of constituted medium at 300C incubation temperature, initial pH 5.5 and for 48 to 60 hours fostered the best production of bacteriocin by Lactobacillus brevis OG1 (Figures 1a - c). ACKNOWLEDGEMENTS The authors thank Dr S. I.
Smith of Genetics Laboratory, Nigeria Institute of Medical Research, Yaba,
Lagos, Nigeria for technical assistance |
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| References | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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