The enriched broth was serially diluted prior to being pour plated on MRS agar. The plates were incubated at 37°C for 16 hours till colonies appeared. The plates were then overlaid with MRS soft agar inoculated with indicator organisms isolated from raw milk. All the plates were incubated at 37°C for 24 hours. Colonies showing zone of inhibition were considered as potential bacteriocin producers.

The bacterial strains that were selected as potential bacteriocin producers were subjected to morphological, cultural and biochemical characterization according to Bergey's Manual of Determinative Bacteriology (1997).

The strains that were selected as potential bacteriocin producers were grown in MRS broth at 37°C for 48 hours. Cells were separated by centrifugation at 5000 rpm for 10 minutes. The pH of the cell free supernatant was adjusted to 5.5 with sterile 0.2 N NaOH. Bacteriocin activity in the supernatant was then tested by agar well diffusion assay (Geis et al 1983). The antagonistic effects of the culture supernatants of bacteriocin producing LAB were tested on various Gram Positive and Gram Negative organisms listed in Table - 2. MRS agar was used for lactic strains, nutrient agar for gram positive and gram negative organisms and Saboraud Dextrose Agar was used for Candida albicans. All cultures were grown aerobically at 37°C for 48 hours. Inhibition zones around the wells were measured. The anti-microbial 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 (Nakamura et al 1983).

Figure 1

Table 2: Inhibition patterns of bacteriocin producer strains against different indicator organisms

To test the heat resistance, 100 ml of culture supernatant was heated for 10 minutes at 60°C, 70°C, 80°C, 100°C and 121°C and residual bacteriocin activity was detected against indicator organism (Ogubanwo et al 2003). Sensitivity of bacteriocin to different pH values was tested by adjusting the pH of culture supernatant in the range of pH 4 to 9 and then kept at room temperature for 4 hours residual activity was determined against the indicator organism (Karao Glu et al 2003).

Action of proteolytic enzyme was tested on culture supernatant by treatment with pepsin and trypsin each at a final concentration of 1mg per ml. It was then incubated at 37°C for 2 hours and residual activity of bacteriocin was assayed (Nakamura et al 1983).

Active cultures of producer organisms (1 % v/v) were inoculated in 100 ml aliquots of sterile composed media. Inoculated flasks were incubated at 15, 24, 48, and 72 hours and at the end of each incubation period, bacteriocin activity was observed by inoculating culture supernatant against indicator organism.

The effect of different concentrations of medium ingredients on bacteriocin production was evaluated using composed MRS medium. The carbon sources studied were glucose (1 – 4%) and lactose (1 – 4%) while nitrogen sources were tryptone (1 – 3%), peptone (1 – 3%) and yeast extract (0.5 – 2%).

Figure 2

Figure 1: Effect of concentration of Carbon and Nitrogen source on bacteriocin production

Two isolates designated as P1 and P2 have shown clear zone of inhibition against the indicator organism and they were selected as potential bacteriocin producers. The isolates were identified as Lactobacillus lactis and Lactobacillus plantarum (Table -1). The culture supernatant obtained from both producer strains were tested for antibacterial activity against different indicator organisms (Table - 2). Both the producer organisms have wide antibacterial activity against both Gram positive and Gram negative bacteria. Largest zone of inhibition was observed against Micrococcus lutae MTCC 106. Therefore, Micrococcus lutae MTCC 106 was used as an indicator organism in further characterization. Inhibitory activity of bacteriocins was seen against L acidophilus, Lactobacillus plantarum and Escherichia coli. However, none of the bacteriocins showed inhibitory activity against Candida albicans.

Figure 3

Table 1: Biochemical characterization of bacteriocin producer

The effect of incubation period on bacteriocin production was also studied and it was observed that at the end of 48 hours, the activity was found to be maximum (Table - 3). Further studies were carried out at different temperatures and pH to test sensitivity of bacteriocin. The bacteriocins of both isolates were stable at 100°C for 10 minutes. Bacteriocin of Lactobacillus lactis retained its activity even at 121°C for 10 minutes (Table - 3). This indicated that bacteriocins produced by this LAB belonged to heat stable low molecular weight group of bacteriocins. Bacteriocin of Lactobacillus lactis was stable in acidic to neutral range i.e. from pH 4 to 7 but it became inactive in the alkaline range (Table - 3) whereas bacteriocins of Lactobacillus plantarum remained active only in the acidic range from pH 4 to 6. Both the bacteriocins were completely inactivated by trypsin and pepsin treatment.

Figure 4

Table 3: Effect of heat treatment, pH and proteolytic enzymes on bacteriocin

The influence of culture medium components on the production of bacteriocin was investigated using Micrococcus luteus as an indicator organism. The results of our study revealed an increase in bacteriocin production in MRS medium containing 3% lactose concentration and 2% peptone concentration. Lactose was found to be better carbon source than glucose. Thus, variation in the concentration of constituents might have an influence on the amount of bacteriocins produced. Ogunbanwo et al (2003) obtained maximum bacteriocin production by supplementing 1% glucose and 1% peptone to normal MRS media. However, it is observed from the present study that lactose and peptone gave better bacteriocin production.

The financial assistance received to Mrs. M.P.Chitanand from University Grant Commission, Western Zone under Faculty Improvement Programme (FIP) to carry this work is highly acknowledged.