Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin AMA-K to Listeria sp.

Otimização da produção de bacteriocina por Lactobacillus plantarum AMA-K isolado de Amasi, um produto lácteo fermentado de Zimbabwe, e estudo da adsorção da bacteriocina AMA-K à Listeria innocua, Listeria monocytogenes e Listeria ivanovii subsp. ivanovii

FOOD MICROBIOLOGY

Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin AMA-K to Listeria sp.

Otimização da produção de bacteriocina por Lactobacillus plantarum AMA-K isolado de Amasi, um produto lácteo fermentado de Zimbabwe, e estudo da adsorção da bacteriocina AMA-K à Listeria innocua, Listeria monocytogenes e Listeria ivanovii subsp. ivanovii

Svetoslav D. Todorov^*

Department of Microbiology, University of Stellenbosch, Stellenbosch, South Africa

ABSTRACT

Bacteriocin AMA-K produced by Lactobacillus plantarum AMA-K inhibits the growth of Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Listeria spp. Growth of strain AMA-K in BHI, M17, soy milk and molasses was similar to growth in MRS. The effect of organic nitrogen sources, carbohydrates, glycerol, K₂HPO₄ and KH₂PO₄, MgSO₄, MnSO₄, tri-ammonium citrate, Tween 80, vitamins and initial pH on bacteriocin AMA-K was determined. The mode of action of bacteriocin AMA-K was studied. The effect of bacteriocin AMA-K to actively growing Listeria innocua LMG13568, L. ivanovii subsp. ivanovii ATCC19119 and L. monocytogenes ScottA was determined. Adsorption of bacteriocin AMA-K to target cells at different temperatures, pH and in presence of Tween 20, Tween 80, ascorbic acid, potassium sorbate, sodium nitrate and sodium chloride were studied. Bacteriocin AMA-K shares high homology to pediocin PA-1.

Key-words: Bacteriocin AMA-K; Lactobacillus planatrum; Amasi.

RESUMO

A bacteriocina AMA-K produzida por Lactobacillus plantarum AMA-K inibe a multiplicação de Enterococcus spp, Escherichia coli, Klebsiella pneumoniae e Listeria spp. A multiplicação da cepa AMA-K em BHI, leite de soja e melaço foi semelhante à multiplicação em MRS. O efeito de fontes de nitrogênio orgânico, carboidratos, glicerol, K₂HPO₄ e KH₂PO₄, MgSO₄, MnSO₄, citrato de triamônio, Tween 80, vitaminas e pH inicial sobre a bacteriocina AMA-K foi determinada. O modo de ação da bacteriocina AMA-K foi estudado. O efeito da bacteriocina AMA-K sobre Listeria innocua LMG13568, Listeria ivanovii subsp.ivanovii ATCC19119 e Listeria monocytogenes Scott A foi determinado. A adsorção da bacteriocina AMA-K às células-alvo em diferentes temperaturas, pH e na presença de Tween 20, Tween 80, ácido ascórbico, sorbato de potássio, nitrato de sódio a cloreto de sódio foi avaliada. A bacteriocina AMA-K apresenta grande homologia a pediocina PA-1.

Palavras-chave: bacteriocina AMA-K, Lactobacillus plantarum, amasi.

INTRODUCTION

Amasi is a traditional fermented milk product consumed in different regions of Southern African, including Zimbabwe, South Africa and Lesotho. The product is an unsweetened curd with a consistency slightly thicker than yoghurt and with a pH between 3.6 and 4.2. Although normally consumed with thick corn-meal porridge, Amasi is also consumed between meals with ground sorghum, similar to muesli.

Traditionally Amasi is produced from unpasteurised bovine (cow's) milk and is allowed to ferment spontaneously in an earthenware (clay) pot or gourd ("calabash") for two to three days at ambient temperature. The microbial flora responsible for the fermentation is derived from the air, raw milk and walls of the containers. After coagulation, the whey is drained through a plugged hole at the bottom of the container.

Fermentation is dominated by lactic acid bacteria (Lactobacillus plantarum, Lactobacillus helveticus, Lactococcus lactis subsp. lactis, Leuconostoc lactis, Leuconostoc citreum, Leuconostoc mesenteroides subsp. dextranicum, Lactobacillus delbrueckii subsp. lactis, Lactobacillus casei subsp. casei and Lactobacillus casei subsp. pseudoplantarum) typically in the order of 10⁸ CFU/ml (4).

Lactic acid bacteria are known for their production of antimicrobial compounds, including bacteriocins or bacteriocin-like peptides (10). Bacteriocins of LAB are defined as ribosomally synthesized proteins or protein complexes usually antagonistic to genetically closely related organisms (10). In previous work the characterization of a bacteriocin produced by Lactobacillus plantarum AMA-K was described (43). Bacteriocins are generally low molecular weight proteins that gain entry into target cells by binding to cell surface receptors. Their bactericidal mechanism vary and may include pore formation, degradation of cellular DNA, disruption through specific cleavage of 16S rDNA, and inhibition of peptidoglycan syntesis (10,15).

In recent papers (23,29), specific environmental conditions, including those found in food, have been studied to determine their effect on the production of bacteriocins. Bacteriocin production changes dramatically upon altering of environmental conditions and optimum production may require a specific combination of environmental parameters (22). Little is known about the interactions these factors have on the production of a bacteriocin, especially in a complex food environment.

Apart from studies conducted on the effect of nitrogen and carbon sources on the production of plantaricin ST31 (37), plantaricin 423 (46), plantaricin UG1 (13), plantaricin KW30 (19), plantaricin-149 (18), plantaricin S (16), plantaricin ST13BR (44) and plantaricin A (8), little is known about the growth conditions required for optimal production of these bacteriocins. Studies conducted on bacteriocins from other lactic acid bacteria, e.g. pediocin AcH (5), pediocin PD-1 (30), enterocin 1146 (32), enterocin AS-48 (2), enterocin P (14), sakP (1), and bacteriocins produced by Leuconostoc mesenteroides L124 (26) have shown that production is often regulated by growth pH and temperature. In some cases, higher bacteriocin activity has been recorded at sub-optimal growth conditions (1,3,6,11,20,21,27,28,32,33,37).

The aim of this study was to determine the conditions needed for optimal production and study some aspects of mode of action of bacteriocin AMA-K produced by L. plantarum AMA-K isolated from Amasi.

MATERIAL AND METHODS

Bacterial strains and growth conditions

Strain AMA-K, isolated from Amasi produced in Gwanda, Kafusi, in the South-Western region of Zimbabwe, was classified as L. plantarum based on phenotypic and genotypic characteristics (43). The strain was cultured in MRS medium (Biolab, Biolab Diagnostics, Midrand, SA) at 30ºC and stored at -80ºC in spent MRS broth, supplemented with 15% (v/v) glycerol. MRS broth (Biolab) was used in all experiments, except growth optimization, in which case MRS broth (9) was modified as indicated.

Bacteriocin bioassay

Bacteriocin screening was performed by using the agar-spot-test method (34). Correction of the cell-free supernatant to pH 6.0 with 1M NaOH prevented the inhibitory effect of lactic acid. Antimicrobial activity was expressed as arbitrary units (AU/mL), calculated as a^bx100, where "a" represents the dilution factor and "b" the last dilution that produces an inhibition zone of at least 2mm in diameter. Activity is expressed per mL by multiplication with 100. One AU is defined as the reciprocal of the highest dilution showing a clear zone of growth inhibition (34). Listeria innocua LMG13568 was used as indicator strain.

Cell-free supernatant containing bacteriocin AMA-K was incubated at 25ºC for 68h and at regular intervals bacteriocin activity was determined as described before.

Bacteriocin production in different growth media and at different initial growth pH

An 18h-old culture of strain AMA-K was inoculated (2%, v/v) into MRS broth (Biolab), BHI broth, M17 broth (Merck), soy milk (10%, w/v, soy flour) and molasses (10%, w/v), respectively. Incubation was at 30ºC and 37ºC, respectively, without agitation, for 25h. Samples were taken every hour and examined for bacterial growth (OD at 600 nm), changes in culture pH, and production of bacteriocins (AU/mL). The agar-spot-test method was used, with L. innocua LMG13568 as target organism.

In a separate experiment, the effect of initial medium pH on the production of bacteriocin AMA-K was determined. Volumes of 300 mL MRS broth were adjusted to pH 4.5, 5.0, 5.5, 6.0 and 6.5, respectively, with 6M HCl or 6M NaOH and then autoclaved. Each flask was inoculated with 2% (v/v) of an 18h-old culture of L. plantarum AMA-K and incubated at 30ºC for 24h, without agitation. Changes in culture pH and production of bacteriocin AMA-K, expressed as AU/mL, were determined every hour as described elsewhere. All experiments were done in triplicate.

Effect of medium composition on bacteriocin production

L. plantarum AMA-K was grown in 10 mL MRS broth (Biolab) for 18h at 30ºC, the cells harvested by centrifugation (8000xg, 10min, 4ºC), and the pellet re-suspended in 10 mL sterile peptone water. Four ml of the cell suspension was used to inoculate 200 mL of the following media: (a) MRS broth (9), without organic nutrients, supplemented with tryptone (20.0g/L), meat extract (20.0g/L), yeast extract (20.0g/L), tryptone (12.5g/L) plus meat extract (7.5g/L), tryptone (12.5g/L) plus yeast extract (7.5g/L), meat extract (10.0g/L) plus yeast extract (10.0g/L), or a combination of tryptone (10.0g/L), meat extract (5.0g/L) and yeast extract (5.0g/L), respectively; (b) MRS broth, i.e. with 20.0g/L D-glucose; (c) MRS broth without D-glucose, supplemented with 20.0g/L fructose, sucrose, lactose, mannose, maltose and gluconate, respectively; (d) MRS broth with 5.0 to 50.0g/L glucose as sole carbon source; (e) MRS broth with 2.0 to 20.0g/L K₂HPO₄, 2.0 to 20.0g/L KH₂PO₄ or combination of 2.0g/L K₂HPO₄ and 2.0g/L KH₂PO₄; (f) MRS broth supplemented with 1.0 to 50.0g/L glycerol; (g) MRS broth without MgSO₄; (h) MRS broth without MnSO₄; (i) MRS broth without or supplemented with 5.0g/L and 10.0g/L tri-ammonium citrate; and (j) MRS broth without Tween 80 or supplemented with 0.5 to 2.0g/L.

In a separate experiment, the vitamins cyanocobalamin (Sigma, St. Louis, Mo.), L-ascorbic acid (BDH Chemicals Ltd), thiamine (Sigma) and DL-6,8-thioctic acid (Sigma) were filter-sterilised and added to MRS broth at 1.0 mg/mL (final concentration). All cultures were incubated at 30ºC for 24h. Activity levels of bacteriocin AMA-K were determined as described elsewhere. All experiments were done in triplicate.

Cell lysis

In a separate experiment, 20 mL cell-free supernatant containing bacteriocin AMA-K (12800AU/ml, pH 6.0) was filter-sterilized (0.20 µm, Minisart®, Sartorius) and added to 100 ml 3-h-old cultures (OD₆₀₀ = 0.1 0.2) of L. innocua LMG13568, Listeria monocytogenes ScottA and Listeria ivanovii subsp. ivanovii ATCC19119, respectively. Incubation was on BHI broth (Biolab) at 37ºC. Optical density readings were recorded at 600nm, hourly for 12h. The experiment was repeated with stationary-phase cells.

Adsorption of bacteriocin AMA-K to target cells

Adsorption of bacteriocin AMA-K to target cells was performed according to the method described by Yildirim et al. (47). The target strains (L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii subsp. ivanovii ATCC19119) were grown overnight in BHI broth at 37ºC and then centrifuged (8000xg, 15 min, 4ºC). Cells were washed twice with sterile 5mM phosphate buffer (pH 6.5) and re-suspended in the same buffer to OD at 600 nm equal to 1.0. The pH was adjusted to 6.5 with sterile 0.1M NaOH. Each cell suspension was mixed with an equal volume bacteriocin AMA-K (12800AU/ml, pH 6.5) and incubated at 37ºC for 1h. After removal of cells (8000xg, 15 min, 25ºC), the activity of unbound bacteriocin AMA-K in the supernatant was determined as described before. All experiments were done in duplicate.

The percentage adsorption of bacteriocin AMA-K to target cells was calculated according to the following formula:

AU/mL₁ refers to the bacteriocin activity after treatment; AU/mL₀ refers to the original (before treatment) activity.

Effect of pH and temperature on the adsorption of bacteriocin AMA-K

Bacteriocin AMA-K was added to L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii subsp. ivanovii ATCC19119, as described elsewhere, and incubated for 1h at 4, 15, 30, 37 and 45ºC, respectively (pH 7.0), and at 37ºC at pH 3.5, 5.5 and 7.0. Cells were harvested (8000xg, 15 min, 25ºC) and the pH of the cell-free supernatant adjusted to 6.0 with sterile 1M NaOH. Bacteriocin activity in the supernatant was determined as described before. The experiments were done in duplicate.

Effect of inorganic salts and organic compounds on adsorption of bacteriocin AMA-K to target cells

Cells of L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii subsp. ivanovii ATCC19119 were treated with 1% (m/v) Tween 20, Tween 80, NaCl, ascorbic acid, potassium sorbate and sodium nitrate. The pH of all samples were adjusted to 6.5 with 1 M NaOH or 1 M HCl. Bactericoin AMA-K was added to the treated cells, as described before, and incubated for 1h at 37ºC. The cells were harvested (8000xg, 15 min, 25ºC) and the activity of bacteriocin AMA-K in the cell-free supernatant determined as described before. The experiments were done in duplicate.

Identification of genes encoding bacteriocin production

DNA was isolated according to the method of Dellaglio et al. (12). Primers PEDRPO (5'-CAA GAT CGT TAA CCA GTT T-3') and PEDC1041 (5'-CCG TTG TTC CCA TAG TCT AA-3') were designed from the operon encoding pediocin PA-1 (Accession number M83924). PCR reactions were performed using a GeneAmp^® PCR Instrument System 9700 (Applied Biosystems, Foster City, USA). The following conditions were used: an initial denaturation step of 94ºC for 1 min, followed by 35 cycles of 1min at 94ºC, 30sec at 50ºC and 1min at 72ºC, and final extension at 72ºC for 5min. The amplified product was visualized in a 0.8% (w/v) agarose gel stained with ethidium bromide. A band corresponding to the correct size was purified from the gel using the QIAquick PCR purification kit (QIAGEN GmbH). Purified PCR product was subject to restriction enzyme digestion with XhoI and HindIII. The digested product was visualized in a 0.8% (w/v) agarose gel stained with ethidium bromide.

PCR purified products were ligated into pGEM-T^® Easy Vector (Promega, Madison, USA) and transformed into E. coli DH5a according to instructions of the manufacturer. Plasmids were isolated using a QIAGEN Plasmid Mini Kit and fragments sequenced on an automatic sequencer (ABI Genetic Analyzer 3130XI, Applied Biosystems) using bigdye terminator chemistry (Biosystem, Wanington, England). Sequences were analysed using DNAMAN for Windows^® (Lynnon Biosoft, Quebec, Canada).

RESULTS AND DISCUSSION

All data represent an average of two or three repeats. The values recorded in each experiment did not vary by more than 5% and single data points are presented in the figures without standard deviation bars.

The cell-free supernatant of L. plantarum AMA-K inhibited the growth of E. faecalis, E. mundtii, E. coli, K. pneumoniae, L. lactis subsp. lactis, L. casei, L. curvatus and L. sakei (54), and E. faecium, L. innocua, L. monocytogenes and L. ivanovii subsp. ivanovii. According to tricine-SDS-PAGE, bacteriocin AMA-K is approximately 2.9kDa (43).

Growth of L. plantarum AMA-K in BHI, M17, soy milk and molasses was very similar to growth in MRS (Fig. 1). The cell density of both strains increased from OD_600nm 0.03 to approximately 9.8 (dilution factor taken into calculation) during 36h (Fig. 1). Low levels of bacteriocin AMA-K activity (less than 200AU/mL) were detected after 8h of growth in MRS broth (Fig. 1). Optimal production of bacteriocin AMA-K (25600AU/mL) was recorded after 29h (Fig. 1), and only when incubated at 30ºC or 37ºC. Bacteriocin AMA-K is a primary metabolite. Similar results have been reported for bacteriocin ST151BR (35), plantaricin Y (7) and bacteriocins produced by P. acidilactici (31).

The activity of bacteriocin AMA-K did not decrease during 68h of incubation at 25ºC, suggesting that extracellular proteases have not been produced. Optimal bacteriocin production (12800AU/mL) was recorded in MRS broth with an initial pH of 6.0 and 5.5 (Table 1). In MRS broth (pH 6.5) supplemented with 1mM EDTA, bacteriocin AMA-K production was 12800AU/mL, suggesting that the bacteriocin was not effected by proteases. Low levels of bacteriocin activity were recorded when the strains were cultured in MRS broth with an initial pH of 5.0 and 4.5 (3200AU/mL and 6400AU/mL, respectively, Table 1). The culture pH after 24h growth was between 3.45 and 3.60. Similar results have been reported for other bacteriocins produced by L. plantarum (8, 16, 37).

Growth of L. plantarum AMA-K in BHI broth or M17 broth adjusted to pH 6.5 yielded only 1600AU/mL of bacteriocin AMA-K (Table 2). No bacteriocin production was recorded in 10% (w/v) soy milk or 2% (w/v) molasses (Table 2). Low levels of bacteriocin AMA-K (800AU/mL) were recorded when the strains were grown in 10% (w/v) molasses (Table 2). Specific nutrients are required for the production of the bacteriocin AMA-K. This phenomenon has been observed for other bacteriocins, e.g. bacteriocins ST194BZ, ST414BZ and ST664BZ, produced by different strains of L. plantarum (36, 41).

Tryptone (20g/L), or a combination of tryptone and yeast extract (1:0.6), added to basal MRS medium yielded a bacteriocin level of 12800AU/mL (Table 2). Growth in the presence of a combination of tryptone and meat extract (1:0.6) reduced bacteriocin production by 50%. Growth in the presence of yeast extract (20g/L) resulted in 6400AU/mL, or in the presence of meat extract (20g/L) or a combination of meat extract and yeast extract (1:1), resulted in bacteriocin production of 3200AU/mL (Table 2).

Tryptone is the key nitrogen source needed for optimal production of bacteriocin AMA-K. Similar results have been reported for the production of plantaricin 423 (46), bacteriocin ST712BZ (40) and for bacteriocins ST151BR and ST112BR (35,38). In the case of plantaricin 423, optimal bacteriocin production was recorded in MRS broth supplemented with bacteriological peptone, followed by casamino acids, tryptone and meat extract. Stimulation of bacteriocin production by yeast extract and meat extract has been reported for helveticin J (17). As far as we could determine, this is the first indication that tryptone is the key nitrogen source needed in the production of L. plantarum bacteriocins.

Growth of L. plantarum AMA-K in the presence of glucose (20.0, and 50.0g/L) yielded 12800AU/mL of bacteriocin AMA-K (Table 2). Increased bacteriocin production (25600AU/ml) was recorded in the presence of 30g/L glucose. Lower concentrations of glucose (5.0g/L and 10.0g/L) yielded 3200AU/mL and 6400AU/mL, respectively (Table 2). Growth in the presence of maltose (20.0g/L) and sucrose (20.0g/L) yielded the same activity as 20.0g/L glucose (Table 2). Growth in the presence of mannose (20.0g/L) and fructose (20.0g/L) reduced bacteriocin production by 75%, i.e. to 3200AU/mL. In the presence of 20.0g/L lactose and 20.0g/L gluconate reduction in bacteriocin AMA-K production was even higher, i.e. 1600AU/mL. These results are surprising, since this strain was isolated from a fermented milk product, but this indicates that bacteriocin production is dependent on combination of factors. Based on these results, the production of bacteriocin AMA-K is stimulated when cells are grown in medium supplemented with 30.0g/L glucose.

Bacteriocin AMA-K production was affected differently by the presence of different concentrations of K₂HPO₄ or KH₂PO₄. Optimal bacteriocin AMA-K production (12800AU/mL) was recorded in the presence of 2.0g/L K₂HPO₄. Higher concentrations K₂HPO₄ had a negative effect on bacteriocin AMA-K production (Table 2). However, replacing K₂HPO₄ with KH₂PO₄ resulted in reduction of bacteriocin AMA-K production (Table 2). A combination of K₂HPO₄ and KH₂PO₄ (2.0g/L and 2.0g/L) resulted in the reduction of bacteriocin AMA-K by 50% (Table 2). In the case of plantaricin UG1, 7.0g/L K₂HPO₄ resulted in increased activity (13). Different concentrations of K₂HPO₄ and KH₂PO₄ did not significantly affect production of bacteriocin ST712BZ, produced by L. pentosus ST712BZ (40). In the case of bacteriocin ST112BR, higher levels of activity was recorded when the medium contained 5.0g/L, 10.0g/L and 20.0g/L KH₂PO₄ (23). The optimal concentration of K₂HPO₄ required for plantaricin ST31 production was between 2.0g/L and 5.0g/L (37).

Production of bacteriocin AMA-K was the highest (12800AU/mL) in the absence or in presence of very low (1.0g/L) concentration of glycerol (Table 2). Glycerol concentrations of 5.0g/L and higher (up to 50.0g/L) led to progressively decreased levels of bacteriocin AMA-K production (Table 2). Similar results were reported for the production of bacteriocins ST151BR, ST112BR, ST712BZ and plantaricin ST31 (35,37, 38,40). An increase in glycerol leads to a lowering in water activity. The production of bacteriocin AMA-K may be influenced by osmotic stress or by binding of bacteriocin to the cell membranes or other molecules, initiated by presence of the glycerol.

Optimal bacteriocin AMA-K production was recorded in presence of 1.0g/L, 1.5g/L and 2.0g/L Tween 80. Lower concentrations of Tween 80 have a negative effect on bacteriocin AMA-K production (Table 2).

Production of bacteriocin AMA-K requires the presence of magnesium sulphate and manganese sulphate as a part of the production medium. Exclusion of these salts resulted in reduction of bacteriocin AMA-K production (Table 2). Similar results were recorded for the effect of tri-ammonium citrate. Normally this component is present in MRS medium at a level of 2.0g/L. At this concentration optimal bacteriocin AMA-K production was recorded. Exclusion of tri-ammnium citrate from the media formula resulted in reduction of bacteriocin activity to 3200AU/mL. However, increasing the concentration to 10.0g/L had a similar effect on bacteriocin AMA-K production (3200AU/ml, Table 2).

Reduction in bacteriocin AMA-K production to 6400AU/mL was recorded in the presence of Vit. B₁ or DL-6,8-thioctic acid (Table 2). However, a decrease to 3200AU/ml was recorded then MRS were supplemented with Vit. B₁₂ or Vit. C (Table 2). In the case of bateriocin ST194BZ, produced by L. plantarum ST194BZ, vitamins Vit. B₁₂ and Vit. B₁ had no effect on bacteriocin production, but addition of Vit. C or DL-6,8-thioctic acid had a negative effect, reducing the bacteriocin ST194BZ production with 50% (36).

The addition of 25 600 AU/ml bacteriocin AMA-K to a 3-h-old culture of L. innocua LMG13568, L. ivanovii subsp. ivanovii ATCC19119 and L. monocytogenes ScottA (OD_600nm = 0.1) resulted in growth inhibition for 9h (Fig. 2), suggesting that the mode of activity of bacteriocin AMA-K is bacteriocidal. Addition of the same concentration of bacteriocin AMA-K to stationary-phase cells of L. innocua LMG13568, L. ivanovii subsp. ivanovii ATCC19119 and L. monocytogenes ScottA, resulted in no significant growth inhibition (results not shown). This suggested that bacteriocin AMA-K is only active against actively growing cells.

Bacteriocin AMA-K was adsorbed at 75% to cells of L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii subsp. ivanovii ATCC19119. (Table 3). Different levels of adsorption was observed in our previous study for bacHV219, but in general highest levels were observed in adsorption to sensitive strains compared to strains resistant to the effect of bacteriocin (42). Similar results have been reported for pediocin N5p (24), viz. 100% adsorption to sensitive cells of O. oeni X2L, 80% to Lactobacillus hilgardii and O. oeni L10, and 70% to L. hilgardii 6D (24). Adsorption of pediocin N5p to resistant bacteria was below 20% (24). Buhnericin LB adsorbed 100% to sensitive cells of L. plantarum, Pediococcus dextranicus, O. oeni and E. faecalis, but also 100% to an insensitive strain of Pediococcus cerevisiae (47). In the case of plantaricin 423, adsorption ranged from 17% for Streptococcus caprinus ATCC 700066 to 67% for L. plantarum LMG 13556, L. curvatus DF38, L. innocua LMG 13568 and L. sakei DSM 20017 (39). Strains sensitive to plantaricin 423 adsorbed the peptide stronger (39).

Optimal adsorption of bacteriocin AMA-K (75%) to L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii subsp. ivanovii ATCC19119 were recorded at pH 7.0. lower levels of pH (3.5) resulted in reduction of the adsorption of bacteriocin AMA-K to this Listeria species to 50%. However, in an experiment at pH 5.5, reduction to 50% were recorded for adsorption to L. innocua LMG13568 and to L. monocytogenes ScottA, but no change in level of adsorption was recorded for L. ivanovii susbp. ivanovii ATCC19119 (Table 3). These results show the potential of the application of this bacteriocin at neutral or moderate acid pH. Differences in adsorption affected by pH rates may be due to specific interaction between bacteriocin AMA-K and the target strain. In the case of buchnericin LB, optimal adsorption to L. plantarum was recorded at pH 5.0 8.0 (47). Optimal adsorption of plantaricin 423 to E. faecium HKLHS was recorded between pH 8.0 and 10.0, and to L. sakei DSM20017 between pH 2.0 and 6.0 (39).

Temperature has effect on bacteriocin AMA-K adsorption to L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii susbp. ivanovii ATCC19119. At 30ºC and 37ºC levels of 75% adsorption were observed for all tested Listeira species (Table 3). At 45ºC increased adsorption of bacteriocin AMA-K was recorded to cells of L. ivanovii susbp. ivanovii ATCC19119 (87.5%) and to L. monocytogenes ScottA (100%) (Table 3). However, temperature of 4ºC and 15ºC resulted in reduction to 50% of adsorption of bacteriocin AMA-K to cells of L. innocua LMG13568 and L. ivanovii subsp. ivanovii ATCC19119 (Table 3). It was previously reported for bacHV219 that an increase in temperature from 25ºC to 60ºC had a negative effect on the adsorption of bacHV219 to E. faecium HKLHS. Forty percent adsorption was recorded between 25ºC and 60ºC, with optimum adsorption (80%) at 4ºC. Complete adsorption (100%) to E. faecalis E88 was recorded after treatment at 4ºC, 10ºC, 45ºC and 60ºC. A 20% decrease in adsorption was recorded at 37ºC (42).

Adsorption was affected by temperature. Similar results were observed for other bacteriocins. In the case of buchnericin LB, identical adsorption levels to cells of L. plantarum was recorded after treatment at 0, 10, 25, 50 and 80ºC (47). Changes in temperature had no effect on the adsorption of plantaricin 423 to E. faecium HKLHS (50).

Decreased adsorption of bacteriocin AMA-K to L. innocua LMG13568, L. monocytogenes ScottA and L. ivanovii susbp. ivanovii ATCC19119 was observed in the presence of Tween 20, Tween 80 and different concentrations of NaCl (Table 3). Ascorbic acid and potassium sorbate not effect adsorption of bacteriocin AMA-K to cells of L. innocua LMG1568, but reduced this process to cells of L. monocytogenes Scott and to L. ivanovii subsp. ivanovii ATCC19119 (Table 3). The presence of 1% sodium nitrate increased adsorption of bacteriocin AMA-K to cells of L. innocua LMG13568 and L. monocytogenes ScottA (Table 3). Increased adsorption of bacHV219 to E. faecium HKLHS was detected in the presence of Triton X-100, b-mercapto-ethanol, chloroform, NaCl, KH₂PO₄ and MgCl₂ (42). Adsorption of bacHV219 to E. faecalis E88 increased in the presence of Na-acetate, Na₂CO₃, Triton X-100, 80% ethanol, methanol, K₂HPO₄, KH₂PO₄, MgCl₂, KCl, Tris and NH₄-citrate (42).

An increase in the adsorption of plantaricin 423 to E. faecium HKLHS was observed in the presence of Triton X-100, Triton X-114 and chloroform (39). Adsorption of bacHV219 to E. faecalis E88 increased in the presence of Na-acetate, Na₂CO₃, Triton X-100, 80% ethanol, methanol, K₂HPO₄, KH₂PO₄, MgCl₂, KCl, Tris and NH₄-citrate. L. sakei DSM 20017 treated with NaCl, K₂HPO₄, KH₂PO₄, MgCl₂, KCl, KI, Tris, NH4-citrate, Na₂CO₃, SDS, b-mercapto-ethanol, 80% ethanol and methanol led to a reduction in the adsorption of plantaricin 423 (39). No change in adsorption was observed in the presence of Na-acetate or EDTA, whereas an increase in adsorption was observed in the presence of Triton X-100, Triton X-114 and chloroform (39). Adsorption of buchnericin LB to L. plantarum was reduced by NaCl, NH₄Cl, MgCl₂, KCl, KI and Tris. Treatment of cells with NH₄-citrate, Na-acetate, NaCO₃, EDTA SDS, triton-X, 2-mercapto-ethanol, 80% ethanol and 80% methanol had no effect on adsorption of buchnericin LB to L. plantarum (47). Adsorption of pediocin N5p to P. pentosaceus E5p increased in the presence of MgCl₂, MgSO₄, MnCl₂, MnSO₄, whereas NaCl, KCl, KI, NH₄Cl, CaCl₂, Na₃PO₄, Na₂SO₄, EDTA and ethanol had no affect on adsorption (31). Organic salts and Na-acetate reduced pediocin N5p adsorption to target cells. Adsorption of pediocin N5p increased with 25% in the presence of SDS (24).

L. plantarum AMA-K has a 1044bp fragment corresponding to that recorded for pediocin PA-1 (Fig. 3A). Digestion with XhoI and HindIII showed that the purified PCR product differed from that obtained for L. plantarum 423 (Fig. 3B). The PCR product from L. plantarum 423 was digested to 2 fragments by XhoI. However, the PCR product from L. plantarum AMA-K DNA was digested to 2 products only by HindIII. The difference in digested profiles were expected based on the genetic sequence of the genes of plantaricin 423 and pediocin PA-1 (45). The sequences of the PCR product using DNA from L. plantarum AMA-K was identical to that reported for pediocin PA-1 (25). Pediocin PA-1 biosynthesis involves a DNA fragment of approximately 3.5 kb, comprising the four genes pedA, pedB, pedC, and pedD (25). This results show that bacteriocin AMA-K share high homology to pediocin PA-1.

CONCLUSIONS

Bacteriocin AMA-K inhibits the growth of E. faecalis, E. mundtii, E. coli, K. pneumoniae, L. lactis subsp. lactis, L. casei, L. curvatus, L. sakei, E. faecium, L. innocua, L. monocytogenes and L. ivanovii subsp. ivanovii. Growth of strain AMA-K in BHI, M17, soy milk and molasses was similar to growth in MRS. Optimal bacteriocin production was recorded in MRS broth with an initial pH of 6.0 and 5.5. After 20h of incubation in all media compositions tested, MRS supplemented with tryptone (20.0g/L), tryptone and yeast extract (12.5g/L and 7.5g/L), glucose (20.0 and 50.0g/L), sucrose (20.0g/L), maltose (20g/L), glycerol (up to 1.0g/L), K₂HPO₄ (2.0 g/L), tri-ammonium citrate (5.0g/L) yielded 12800AU/mL. Glucose at 30g/L increased bacteriocin AMA-K production by 100%. Optimal adsorption of bacteriocin AMA-K (75%) to Listeria strains was recorded at pH 7.0. However, temperatures of 4ºC and 15ºC resulted in reduction of 50% of adsorption of bacteriocin AMA-K to cells of L. innocua LMG13568 and L. ivanovii subsp. ivanovii ATCC19119. Decreased adsorption of bacteriocin AMA-K to Listria strains was observed in the presence of Tween 20, Tween 80 and different concentrations of NaCl. Ascorbic acid and potassium sorbate did not effect adsorption of bacteriocin AMA-K to cells of L. innocua LMG1568, but reduced this process in cells of L. monocytogenes Scott and to L. ivanovii subsp. ivanovii ATCC19119. The presence of 1% sodium nitrate increased adsorption of bacteriocin AMA-K to cells of L. innocua LMG13568 and L. monocytogenes ScottA. Bacteriocin AMA-K shares high homology to pediocin PA-1.

ACKNOWLEDGMENTS

The research was supported by Claude Leon Foundation, Cape Town, South Africa. The author is grateful to Dr. Carol van Reenen for discussions about bio-molecular work and to Prof. Leon M.T. Dicks for using his laboratory facilities.

Submitted: August 14, 2007; Returned to authors for corrections: October 17, 2007; Approved: January 23, 2008.

• 1. Aasen, I.M.; Moreto, T.; Katla, T.; Axelsson, L.; Storro, I. (2000). Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687. Appl. Microbiol. Biotechnol., 53: 159-166.
• 2. Abriouel, H.; Valdivia, E.; Galvez, A.; Maqueda, M. (2001). Influence of physico-chemical factors on the oligomerization and biological activity of bacteriocin AS-48. Curr. Microbiol., 42: 89-95.
• 3. Audisto, M.C.; Oliver, G.; Apella, M.C. (2001). Effect of different complex carbon sources on growth and bacteriocin synthesis of Enterococcus faecium Int. J. Food Microbiol, 63: 235-241.
• 4. Beukes, E.M.; Bester, B.H.; Mostert, J.F. (2001). The Microbiology of South African traditional fermented milks. Int. J. Food Microbiol., 63: 189-197.
• 5. Biswas, S.R.; Ray, P.; Johnson, M.C.; Ray, B. (1991). Influence of growth conditions on the production of a bacteriocin, pediocin AcH by Pediococcus acidilactici H. Appl. Environ. Microbiol., 57: 1265-1267.
• 6. Bogovic-Matijasic, B.; Rogelj, I. (1998). Bacteriocin complex of Lactobacillus acidophilus LF221 production studies in MRS-media at different pHvalues and effect against Lactobacillus helveticus ATCC 15009. Process Biochem., 33: 345-352.
• 7. Chin, H.S.; Chin, J.S.; Kim, J.M.; Yang, R.; Yoon, S.-S. (2001). Detection and antibacterial activity of a bacteriocin produced by Lactobacillus plantarum. Food Sci. Biotech., 10: 335-341.
• 8. Daeschel, M.A.; McKeney, M.C.; McDonald, L.C. (1990). Bacteriocidal activity of Lactobacillus plantarum C-11. Food Microbiol., 7: 91-98.
• 9. De Man, J.C.; Rogosa, M.; Sharpe, M.E. (1960). A medium for the cultivation of lactobacilli. J. Appl. Bacteriol., 23: 130-135.
• 10. De Vuyst L.; Vandamme E. (1994). Bacteriocins of lactic acid bacteria (Blackie London, United Kingdom pp. 539
• 11. De Vuyst, L.; Callewaert, R.; Crabbe, K. (1996). Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiol., 142: 817-827.
• 12. Dellaglio, F.; Bottazzi, V.; Troatelli, L.D. (1973). Deoxyribonucleic acid homology and vase composition in some thermophylic lactobacilli. J. Gen. Microbiol., 74: 289-297.
• 13. Enan, G.; Essawy, A.A.; Uyttendaele, M.; Debevere, J. (1996). Antibacterial activity of Lactobacillus plantarum UG1 isolated from dry sausage: Characterization, production and bactericidal action of plantaricin UG1. Int. J. Food Microbiol., 30: 189-215.
• 14. Herranz, C.; Martinez, J.M.; Rodriguez, J.M.; Hernandez, P.E.; Cintas, L.M. (2001). Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH. Appl. Microbiol. Biotechnol., 56: 378-383.
• 15. Heu, S.; Oh, J.; Kang, Y.; Ryu, S.; Cho, S.K.; Cho, Y.; Cho, M. (2001). Gly gene cloninig and expression and purification of Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. Glycines 8ra. Appl. Environ. Microbiol, 67, (9) 4105-4110.
• 16. Jimenez-Diaz R.; Rios-Sanchez R.M.; Desmazeaud M.; Ruiz-Barrba J.L.; Piard J.C. (1993). Plantaricin S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl. Environ. Microbiol., 59: 1416-1424.
• 17. Joerger, M.C.; Klaenhammer, T.R. (1986). Characterization and purification of helveticin J and evidence for chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J. Bacteriol., 167: 439-446.
• 18. Kato, T.; Matsuda, T.; Ogawa, E.; Ogawa, H.; Kato, H.; Doi, U.; Nakamura, R. (1994). Plantaricin-149, a bacteriocin produced by Lactobacillus plantarum NRIC 149. J. Ferment. Bioeng., 77: 277-282.
• 19. Kelly, W.J.; Asmundson, R.V.; Huang, C.M. (1996). Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum. J. Appl. Bacteriol, 81: 657-662.
• 20. Kim, W.S.; Hall, R.J.; Dunn, N.W. (1997). The effect of nisin concentration and nutrient depletion on nisin production of Lactococcus lactis Appl. Microbiol. Biotechnol., 50: 429-433.
• 21. Krier, F.; Revol-Junelles, A.M.; Germain, P. (1998). Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation. Appl. Microbiol. Biotechnol., 50: 359-363.
• 22. Leal-Sánchez, M.V.; Jiménez-Díaz, R.; Maldonado-Barragán, A.; Garrido-Fernández, A.; Ruiz-Barba, J.L. (2002). Optimization of bacteriocin production by batch fermentation of Lactobacillus plantarum LPCO10. Appl. Environ. Microbiol., 68: 4465-4471.
• 23. Leroy, F.; De Vuyst, L. (2003). A combined model to predict the functionality of the bacteriocin-producing Lactobacillus sakei strain CTC494, Appl. Environ. Microbiol., 69: 1093-1099.
• 24. Manca de Nadra, M.C.; Sandino de Lamelas, D.; Strasser de Saad, A.M. (1998). Pediocin N5p from Pediococcus pentosaceus: Adsorption on bacterial strains. Int. J. Food Microbiol., 39: 79-85.
• 25. Marugg, J.D.; Gonzalez, C.F.; Kunka, B.S.; Ledeboer, A.M.; Pucci, M.J.; Toonen, M.Y.; Walker, S.A.; Zoetmudler, L.C.M.; Vandenbergh, P.A. (1992). Cloning, expression, and nucleotide sequence of genes involved in the production of pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0. Appl. Environ. Microbiol, 58: 2360-2367.
• 26. Mataragas, M.; Metaxopoulos, J.; Galiotou, M.; Drosinos, E.H. (2003). Influence of pH and temperature on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. Meat Sci., 64: 265-271.
• 27. Matsusaki, H.; Endo, N.; Sonomoto, K.; Ishizaki A. (1996). Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: Relationship between production of the lantibiotic and lactate and cell growth. Appl. Microbiol. Biotechnol., 45: 36-40.
• 28. Mortverd-Abildgaard, C.I.; Nissen-Meyer, J.; Jelle, B.; Grenov, B.; Skaugen, M.; Nes, I.F. (1995). Production and pH-dependent bacteriocidal activity of lactocin S, a lantibiotic from Lactobacillus sake L45. Appl. Environ. Microbiol., 61: 175-179.
• 29. Motta, A.S.; Brandelli, A. (2003). Influence of growth conditions on bacteriocin production by Brevibacterium linens Appl. Microbiol. Biotechnol., 62: 163-167.
• 30. Nel, H.A.; Bauer, R.; Vandamme, E.J.; Dicks, L.M.T. (2001). Growth optimisation of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1. J. Appl. Microbiol., 91: 1131-1138.
• 31. Nieto-Lozano, J.C.; Reguera-Useros, J.I.; Pelaez-Martinez, M.C.; De la Torre, A.H. (2002). Bacteriocinogenic activity from starter cultures used in Spanish meat industry. Meat Sci., 62: 237-243.
• 32. Parente, E.; Ricciardi, A. (1994). Influence of pH on the production of enterocin 1146 during batch fermentation. Lett. Appl. Microbiol, 19: 12-15.
• 33. Parente, E.; Ricciardi, A.; Addario, G. (1994). Influence of pH on growth and bacteriocin production by Lactococcus lactis subsp. lactis 140VWC during batch fermentation. Appl. Microbiol. Biotechnol, 41: 388-394.
• 34. Todorov, S.D.; Dicks, L.M.T. (2005). Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria. Enzyme Microb Technol., 36: 318-326.
• 35. Todorov, S.D.; Dicks, L.M.T. (2004). Effect of medium components on bacteriocin production by Lactobacillus pentosus ST151BR, a strain isolated from beer produced by the fermentation of maize, barley and soy flour. World J. Microbiol. Biotechnol., 20: 643-650, 2004.
• 36. Todorov, S.D.; Dicks, L.M.T. (2005). Effect of growth medium on bacteriocin production by Lactobacillus plantarum ST194BZ, a strain isolated from boza. Food Technol. Biotechnol., 43: 165-173.
• 37. Todorov, S.; Gotcheva, B.; Dousset, X.; Onno, B.; Ivanova, I. (2000). Influence of growth medium on bacteriocin production in Lactobacillus plantarum ST31. Biotechnol. Biotechnol. Eq., 14: 50-55.
• 38. Todorov, S.D.; Dicks, L.M.T. (2004). Influence of growth medium on bacteriocin production by Lactobacillus pentosus ST112BR, a strain isolated from traditional South African beer. Proceedings of National conference with foreign participation, 3-4 Juny, 2004, Stara Zagora, Bulgaria. Volume IV, Human medicine. Part 1. Clinical and molecular biology, microbiology. Physiology and pharmacology. Publishing group: Stara Zagora Union of Scientists. 132-136.
• 39. Todorov, S.D.; Dicks, L.M.T. (2006). Parameters affecting the adsorption of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423 isolated from sorghum beer. Biotechnol. J., 1: 405-409.
• 40. Todorov, S.D.; Dicks, L.M.T. (2007). Bacteriocin production by Lactobacillus pentosus ST712BZ isolated from boza. Braz. J. Microbiol, 38: 166-172.
• 41. Todorov, S.D.; Dicks, L.M.T. (2006). Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza. Biologia, 61: 269-274.
• 42. Todorov, S.D.; Botes, M.; Danova, S.T.; Dicks, L.M.T. (2007). Probiotic properties of Lactococcus lactis subsp. lactis HV219, isolated from human vaginal secretions. J. Appl. Microbiol., 103: 629-639.
• 43. Todorov, S.D.; Nyati, H.; Meincken, M.; Dicks, L.M.T. (2007). Partial characterization of bacteriocin AMA-K, produced by Lactobacillus plantarum AMA-K isolated from naturally fermented milk from Zimbabwe. Food Control, 18: 656-664.
• 44. Todorov, S.D.; Van Reenen, C.A.; Dicks, L.M.T. (2004). Optimization of bacteriocin production by Lactobacillus plantarum ST13BR, a strain isolated from barley beer. J. Gen. Appl. Microbiol., 50: 149-157.
• 45. Van Reenen, C.A.; Van Zyl, W.H.; Chikindas, M.L.; Dicks, L.M.T. (2002). Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae Int. J. Food Microbiol., 81: 29-40.
• 46. Verellen, T.L.J.; Bruggeman, G.; Van Reenen, C.A.; Dicks, L.M.T.; Vandamme, E.J. (1998). Fermentation optimisation of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423. J. Ferment. Bioeng., 86: 174-179.
• 47. Yildirim, Z.; Avºar, Y.K.; Yildirim, M. (2002). Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactobacillus buchneri Microb. Res., 157: 103-107.

Publication Dates

• Publication in this collection
  11 Apr 2008
• Date of issue
  Mar 2008

History

• Accepted
  23 Jan 2008
• Received
  14 Aug 2007