Studies On Some Properties Of Bacteriocins Produced By Lactobacillus Species Isolated From Agro-Based Waste
H Lade, M Chitanand, G Gyananath, T Kadam
antibacterial activity, indicator organisms, lactic acid bacteria
H Lade, M Chitanand, G Gyananath, T Kadam. Studies On Some Properties Of Bacteriocins Produced By Lactobacillus Species Isolated From Agro-Based Waste. The Internet Journal of Microbiology. 2005 Volume 2 Number 1.
Lactic acid producing bacteria (LAB) were isolated from vegetable waste. These organisms were studied for bacteriocin production. Among the isolated cultures
Bacteriocins are highly specific antibacterial proteins produced by strains of bacteria active mainly against some other strains of same or related SPP (Gaur
Fruits and vegetable waste provide a good source for isolating LAB having antagonistic and probiotic properties. However, work on bacteriocinogenic LAB from agro-based waste has been limited. Therefore the present studies are carried out to examine LAB population isolated from damaged and spoiled vegetable wastes collected from local market yard. Bacteriocins produced by these organisms are characterized with respect to inhibition spectrum, effect of physical and chemical parameters and effect of cultural conditions on baccteriocin production.
Materials and Methods
Vegetable waste containing spoiled cabbage and cucumber was collected from local market yard. It was then crushed, homogenized with kitchen blender, and inoculated in the deMan Ragosa and Sharpe (MRS) broth for enrichment of resident LAB. The tubes were incubated at 37°C for 24 hours.
Screening for bacteriocin producers
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.
Identification of bacteriocin producers
The bacterial strains that were selected as potential bacteriocin producers were subjected to morphological, cultural and biochemical characterization according to
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
Study of physical and chemical properties of bacteriocin
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
Effect of proteolytic enzyme on bacteriocin activity
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
Effect of incubation period on bacteriocin production
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.
Effect of different concentrations of carbon and nitrogen source on bacteriocin production:
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%).
Results and Discussion
Detection of bacteriocin producers
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
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
The influence of culture medium components on the production of bacteriocin was investigated using
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.