African Journal of Biotechnology Vol. 2 (8), pp. 219-227, August 2003

ISSN 1684-5315  © 2003 Academic Journals  

Full Length Research Paper

Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1

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

Department of Botany and Microbiology University of Ibadan, Nigeria

*Corresponding author: E-mail: topzybanwo@yahoo.com

Accepted 9 July 2003

Lactobacillus plantarum F1 and L. brevis OG1 isolated from Nigerian fermented food products, produced bacteriocins that had broad spectrum of inhibition against both pathogenic, food spoilage organisms and various lactic acid bacteria.  The test organisms exhibited activities of 6400 and 3200 AU/ml respectively against Escherichia coli NCTC10418 and Enterococcus faecalis EF1, but did not inhibit Candida albicans ATCC10231 and Klebsiella sp. UCH15. Comparison of the antimicrobial spectra and characterization of the two bacteriocins were not identical. Bacteriocin produced by L. brevis OG1 was the most heat stable at 121^oC for 60 min, while that of L. plantarum F1 was stable at 121^oC for 10 min. The bacteriocins produced by the test isolates maintained full stability after storage for 60 days at – 20⁰C; partial stability after storage for 120 days at 4⁰C; while activity was not detected after storage for 80 to 120 days at 37⁰C. Bacteriocin produced by L. brevis OG1 was stable at pH range of 2.0 to 8.0 while, that of L. plantarum F1 was found to be stable at pH 2.0 to 6.0. Their active principle was proteinaceous in nature since the bacteriocins were inactivated by proteolytic enzymes, but not by other non–proteolytic enzymes. mitomycin C and uv light did not affect the activity of the bacteriocins, while chloroform extraction completely destroyed their activity. Exposure to surfactant resulted in an increase in the bacteriocin titre, except Nonidet P-40, which led to total loss of bacteriocin activity. The bacteriocins were able to pass through cellulose membranes with 100,000 KDa and 1,000,000 KDa but could not pass through one with a 10,000 KDa and 1,000 KDa molecular weight cut off. The paper concluded that the ability of bacteriocins produced by the test isolates in inhibiting a wide-range of bacteria, is of potential interest for food safety and may have future applications as food preservative.

Key words:  Bacteriocins, lactic acid bacteria, indicator organisms, fermented foods, antagonistic activity.

Sample collection

Cassava (Manihot esculenta Crantz) tubers and maize (Zea mays) grains used in this study were obtained from retail market in South-Western Nigeria.

Traditional Fermentation of Cassava

Cassava tubers (1 kg) was peeled, cut into pieces and washed several times in water followed by soaking in water (submerged fermentation) for period of 3 days at ambient temperature (26°C ± 1°C).  The softened pulpy mass of the fermented cassava samples was disintegrated and passed through a clean coarse sieve to remove lumps and fibers after which the mass was allowed to sediment (Oyewole and Odunfa, 1990).

Traditional preparation of ogi 

The cereal grain (Z. mays) were 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

Lactic acid bacterial strains were isolated from cassava retting and traditional prepared ogi (Table 1).  For all samples, 10 g were added to 90 ml of sterile diluent’s containing 0.1% peptone water and homogenized for 30 s. From appropriate 10-fold dilutions; isolation of bacteria was carried out on MRS agar and incubated anaerobically at 30⁰C for 48 h. The cultures were purified by repeated streaking. Strains were characterized using the AP1 50CH strips and AP1 50 CHL medium (AP1 Systems, Biomerieux Sa, France). The food spoilage and pathogenic bacteria used as indicator organisms were obtained from the culture collection of Medical Microbiology, Laboratory, University College Hospital, Ibadan, Nigeria.

Table 1. Lactic acid bacteria isolated from fermented ogi and cassava retting.

Production of crude bacteriocin samples

Lactobacillus species were propagated in 1000 ml MRS broth (pH 7.0, glucose, 0.25% w/v, peptone, 0.5% w/v) for 72 h at 30⁰C anaerobically (Oxoid Gas Generating Kit) in triplicate. For extraction of bacteriocin, a cell-free solution was obtained by centrifuging (10,000 rpm for 20 min. at 4⁰C with Beckman L5050B) the culture and was adjusted to pH 7.0 by means of 1M NaOH to exclude the antimicrobial effect of organic acid, followed by filtration of the supernatant through a 0.2 mm pore-size cellulose acetate filter. The supernatant was dialysed for 24 h at 4⁰C (Schillinger and Lucke, 1989).  Inhibitory activity from hydrogen peroxide was eliminated by the addition of 5 mg/ml catalase (C-100 bovine liver, Sigma) (Daba et al., 1991).

Purification of bacteriocin samples

Ammonium Sulphate Precipitation: The crude bacteriocin samples produced were treated with solid ammonium sulphate (Mallinckrodth Chemical, Inc., Paris, KY, USA) to 0, 30, 35, 40, 45, 50, 55 and 60% saturation. The mixtures were stirred for 2 h at 4ºC and later centrifuged at 20,000 rpm for 1 h (4ºC). The precipitates were re-suspended in 25 ml of 0.05 M potassium phosphate buffer (pH 7.0).  Dialysis was followed in a tubular cellulose membrane (Specrapor, 1000 dalton MWco, Fisher Scientific Pittsburgh, PA USA) against 2 litres of the same buffer for 18 h in spectrapor No. 4 dialysis tubing.  Assay of the bacteriocin activity was carried out and titer was determined in both the precipitate and supernatant to know which one actually contain the bacteriocin (Fraction 1) (Jimenez-Diaz et al., 1993).

Trichloroacetic acid (TC) precipitation: Five percent (5%) equivalent of TC was added to 25 ml of Fraction 1 to precipitate  the protein (bacteriocin).  The mixture was centrifuged at 13,000 rpm for 10 minutes after which the supernatant was decanted.  The resulting pellet was dissolved in 2 ml of potassium phosphate buffer (Fraction 2).

Ultrafiltration studies: The Fraction 2 (Trichloroacetic acid precipitation) bacteriocin sample was resuspended to ¹/₃₀ volume in potassium phosphate buffer (50mM, pH 7.0). Several aliquots (1ml) were ultrafiltered through various filtron membranes (Filtron Technology Corp; Northborough, Mass), including 1,000,000, 100,000, 10,000 and 1,000 KDa. molecular exclusion sizes.  Bacteriocin activity was determined in retained and eluted fractions (Jimenez-Diaz et al., 1993) and protein concentration of the fractions were determined by the Bradford method (Bradford, 1976).

Determination of bacteriocin activity: A well diffusion assay procedure was used (Schillinger and Lucke, 1989; Takahiro et al., 1991). Aliquots of 50 ml from each bacteriocin dilution (see determination of bacteriocin titire) were placed in wells in plates seeded with the bioassay strain. The plates were incubated overnight at 30ºC for lactic acid bacteria indicators (anaerobically) and at 37ºC for non-lactic acid bacteria indicators (aerobically), and the diameters of the inhibition zone were taken (Rammelsberg and Radler, 1990).

Determination of bacteriocin titre: The titres of bacteriocin produced were quantified by two fold serial dilutions of bacteriocin in saline solution and aliquots of 50 ml from each dilution were placed in wells in plates seeded with the bioassay strain.  These plates were incubated anaerobically at 30⁰C for lactic acid bacteria indicators and aerobically at 37⁰C for non-lactic acid bacteria indicators for 18-24 h and examined for the presence of 2 mm or larger clear zones of inhibition around the wells. The antimicrobial activity of the bacteriocin was defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and was expressed in activity units per ml (AU ml-1) (Graciela et al., 1995).

Characterization of bacteriocin

The purified bacteriocin samples (Fraction 2) were characterized with respect to thermal and pH stability, susceptibility to denaturation by enzymes, stability during storage, extraction with organic solvent, treatment with dissociating agents and mitomycin C and uv light induction.

Heat Resistance: Purified bacteriocin (400ml) was exposed to various heat treatments: 40, 60, 80, 100 and 121ºC. Aliquot volumes of each Fraction were then removed after 0, 30, 60 or 90 min (ten Brink et al., 1994) and assayed for bacteriocin.

pH Sensitivity: Purified bacteriocin (400 ml) were adjusted to pH 2, 4, 6, 8, 10, and 12 with hydrochloric acid (HCl) and sodium hydroxide (NaOH), incubated for 4 h at room temperature (ten Brink et al., 1994) and similarly assayed.

Enzyme Treatments: Purified bacteriocin was assessed for its sensitivity to various enzymes.  Enzymes (all obtained from Sigma) and their respective buffers were lipase (type 1, 8.6 U/mg) in 0.05 M Tris hydrochloride (pH 8.0), 0.01 M CaCl₂; a-chymotrypsin (type 11, 47 U/mg) in 0.05 M Tris hydrochloride (pH 8.0) 0.01 M CaCl₂; pronase E (type xxvi, 4.1 U/mg) in 0.01m sodium borate, 0.05 M HCl, 5 mM CaCl₂, 1 mM CaCl₂ (pH 7.2); pepsin (3,2001 U/ml), in 0.2 M citrate (pH 6.0); catalase (2,000 U/mg), in 10 mM potassium phosphate (pH 7.0); phospholipase C (type 1, 10 U/mg), in 0.05 M Tris hydrochloride (pH 7.0),  0.01M  CaCl₂;  trypsin  (type  x,  15000 U/mg), in 0.05 M Tris hydrochloride (pH 8.0); lysozyme (Serva, 20600 U/mg), in 1N NaOH (pH 6.5); a-amylase (type IX, 1000 U/mg), in 1N NaOH (pH 6.5); dextranase in 0.2 M citrate (pH 6.1) and proteinase K (11.5 U/mg) in 1N NaOH (pH 6.5). Samples of bacteriocin  (500 ml) were incubated with 500mg of each enzyme per ml for 60 min at 37ºC except for samples containing trypsin, a-chymotrypsin and catalase, which were incubated at 25ºC.  Prior to being assayed for bacteriocin activity, preparations containing papain were adjusted to pH 6.0 and those containing trypsin and chymotrypsin were treated with trypsin-chymotrypsin inhibitor (Sigma) according to the manufacturer’s instructions (Wanda and Bonita, 1991).

Stability of bacteriocin during storage: Purified bacteriocin was stored at –20, 4 and 37ºC.  At different time intervals, samples were taken from the stored material (ten Brink et al., 1994) to determine bacteriocin activity.

Extraction of bacteriocin with organic solvents: Various organic solvents including iso–amylalcohol, chloroform, n–propanol, hexane, Di – ethyl ether, petroleum ether were added to purified bacteriocin in 1:1 ratio.  After thorough mixing, phase separation was achieved by centrifugation (10 min at 5000 rpm).  When propanol was used for the extraction, 50 g/l NaCl was added to the mixture in order to obtain phase separation. The organic phase and the aqueous phase were collected and solvent removed by evaporation at 45ºC.  The residue from the organic phase was resuspended in an amount of saline (8.5 g/l NaCl) equal to the starting volume of the original supernatant fluid (ten Brink et al., 1994). Bacteriocin activity of both preparations was then determined.

Effect of mitomycin C on bacteriocin activity: Mitomycin C was added at a final concentration of 1.0 mg/ml to purified bacteriocin and incubation was carried out at 30ºC.  Samples were removed at 60, 120, 240 and 360 min. and analysed by the well diffusion method (Wanda and Bonita, 1991).

Effect of uv light on bacteriocin activity: A 10 ml aliquot of purified bacteriocin was placed in a sterile petri dish and exposed to short – wave uv light from a 15 – W General Electric germicidal bulb at a distance of 30 cm. Times of exposure ranged from 0 to 5 min. (Wanda and Bonita, 1991). After each time interval, bacteriocin activity was analysed by the well diffusion method.

Effect of surfactant on bacteriocin activity: This was carried out by incorporating non-ionic (triton X100, tween 20, tween 80, nonidet P40), anionic (sodium dodecyl sulphate, deoxycholic acid) and dipolar  ionic   (N–hexadecyl–N,N–dimethyl-3-ammonio–1–propane sulphonate, N–lauroylsarcosine) surfactants.The surfactants were obtained from Sigma Chemical Co. and were added to purified bacteriocin at a concentration of 0.1 ml or 0.01 g of surfactant ml^-1 of bacteriocin solutions. These preparations were incubated at 30ºC for 60 min. (Kelly et al., 1996) and assayed for bacteriocin activity against indicator organisms by using titre evaluation.

The selected Lactobacillus strains (L. plantarum F1 and L. brevis OG1) produced bacteriocin, which showed inhibitory activity against one or more of the Gram-positive and Gram-negative target strains (Table 2). The two species of Lactobacillus had different profiles of inhibition, and the profiles were strain specific. L. plantarum F1 and L. brevis OG1 were characterized by the broad-spectrum inhibition of microorganisms.  All the bacteriocin produced by the test isolates inhibited E. coli (NCTC 10418) and Enterococcus faecalis (EF1) but did not inhibit Candida albicans. (ATCC 10231) and Klebsiella spp (UCH15).  The largest spectrum of inhibition was showed by L. plantarum F1, which inhibited 24 out of 32 indicator strains.  However, it was generally observed that bacteriocin from the producer organism had no inhibitory effect on the organism producing it.

Table 2. Inhibition of various indicator organisms by bacteriocin produced by lactic acid bacteria. 

Click here to view Table 2

The effects of heat, storage time, pH, enzymes and surfactants on bacteriocin activity were determined using E. faecalis (EF1) as indicator organism. The inhibitory compound produced by the test isolates was considered to be heat stable.  Bacteriocin produced by L. brevis OG1 was considered to be the most heat stable, as the activity (3200 AU/ml) remained constant after heating at 121ºC for 60 minutes, but declined thereafter (Figure 1), while bacteriocin produced by L. plantarum F1 activity (6400 AU/ml) remained constant after heating at 121ºC for 10 followed by subsequent decline. At 90 minutes there was no detectable bacteriocin activity in L. plantarum F1 (Figure 1).

            Figure 1. Effect of temperature on bacteriocin activity produced by the test isolates.

Effect of time and temperature of storage on bacteriocin activity was also carried out.  It was observed that all the bacteriocin produced by the test isolates maintained full stability after storage for 60 days at –20ºC; partial stability after storage for 120 days at 4ºC, while no activity was detected after storage for 80 to 120 days at 37ºC (Figure 2). Effect of pH on activity of bacteriocin was carried out.  It was observed that bacteriocin produced by L. brevis OG1was stable at pH 2 to 8, while for L. plantarum F1, it was found to be stable at pH 2 to 6 (Table 3).  

Figure 2. Effect of time and temperature of Storage on bacteriocin activity produced by the test isolates.

Table 3. Effect of pH treatment on bacteriocin activity (AU/ml) Produced by the test isolates.

Bacteriocin produced by the test isolates was tested for their sensitivity (loss of activity) to various enzymes.  The antimicrobial activity was lost or unstable after treatment with all the proteolytic enzymes, whereas treatment with lipase, catalase, phospholipase C, lysozyme, a-amylase, dextranase, mitomycin and uv-light did not affect the activity of bacteriocin produced by the test isolates. Bacteriocin produced by L. plantarum F1 showed decreased in activity when treated with pronase E (Table 4).

Table 4. Effect of enzymes, mitomycins and uv light treatments on activity (AU/ml) of bacteriocin produced by the test isolates.

       nd = non-detectable

Various organic solvents were tested for the extraction of bacteriocin produced by the test isolates. It  was observed that extraction with polar solvents such as hexane, di-ethyl ether, and petroleum ether did not result in the removal bacteriocin produced at the aqueous phase to the organic phase, while chloroform extraction completely destroyed the bacteriocins activity.  However, when different alcohols such as n-propanol and Iso-amyl alcohol were used in the extraction procedure, bacteriocin was removed from the aqueous phase and recovered from the organic phase (Table 5).

Table 5. Influence of organic solvents in the extraction of bacteriocin produced by the test isolates.

     Zone of inhibition (mm)

Table 6 showed the effect of dissociating agents on bacteriocin activity.  Exposure to surfactants resulted in an increase in the bacteriocin titre (by at least one to two fold dilutions) rather than any decrease in activity with the exception of nonidet P-40, which led to total loss of bacteriocin activity.  N-lauroyl sarcosine had little or no effect on bacteriocin activity produced by the test isolates.

Table 6.  Effect of surfactants on activity of bacteriocin (AU/ml) produced by the test isolates.

Purification steps of the bacteriocin are summarized in Table 7. The bacteriocin of L. plantarum F1 and L. brevis OG1 were recovered following the 60% saturation of the culture broths with ammonium sulphate with an increase to specific activity of 9.4 and 5.2 AU/mg protein respectively (Fraction 1). The second step in the purification protocol was Trichloroacetic acid precipitation of Fraction 1. At this stage of purification, the recovery was 18.3% for both isolates. The specific activity increased to 85.3 and 44.4 AU/mg protein for L. plantarum F1 and L. brevis OG1 respectively (Fraction 2). Finally, these fractions were subjected to ultrafiltration using various filtron membranes. The eluted and retained fractions were collected and assayed for bacteriocin activity. At this stag