Winter vegetables (carrot, and cucumber) were collected from the retail market locations in Azimpur bazar in Dhaka city of Bangladesh. About 250 g each of carrot, and cucumber was obtained. The samples were wrapped separately in sterile polyethylene bags, and transported off the Industrial Microbiology Laboratory, Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka-1205, Bangladesh for further analysis.

Sample kept at room temperature before analysis. There are many ways to carry out natural fermentation process: Ten grams each, of fresh vegetables samples were soaked in 90 ml of normal saline solution (8.5 g NaCl/L), homogenized for 20 min, appropriately diluted in normal saline. From each sample, serial dilutions were made by following the method of Harrigan (1998) [11].

The typical colonies on MRS agar were sub-cultured on the same medium again and again until a pure growth for each culture isolate was obtained. Four or five times subcultured in this procedure on MRS Agar media.

The pure cultures were maintained in Industrial Microbiology Laboratory of Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhanmondi, Dhaka, at refrigeration temperature with a continuous sub culturing techniques on MRS agar after every 10 days in order to avoid any contamination and other problems.

Antimicrobial activity of the bacterial isolates against all the pathogenic microbes was ascertained by well diffusion method [17] under aerobic conditions. Agar plates were inoculated with 100 mL of each target microbes after growing them in a broth and diluting favourably. Wells (3 mm) were excise into the plates and 100 mL of cell-free culture supernatant fluid of the different strain was placed into every well. The inhibitory activity against Escherichia coli, E. coli 12079, E. coli fecalis, Acetobacter, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Pseudomonas, Pseudomonas aeruginosa, Salmonella typhi, Salmonella typhimoreum, Staphylococcus spp., Staphylococcus aureus was grown nutrient agar. Plates were kept at a cool temperature for 2 h and then incubated at 37˚C for 24 hours. The antimicrobial activity was identified by measuring the diameter of the inhibition zone around the wells.

The cells were harvested and the bacteriocin isolated from the cell free supernatant fluid as described by Green et al. (1997) [18]. Purification of bacteriocin from Lactobacillus spp. was given in the following.

50 ml of de Man, Rogosa and Sharpe (MRS) broth and 2 ml of Lactobacillus culture incubated at 37˚C for 14 hours in anaerobic condition. Then 100 ml MRS broth and 1% of Lactobacillus culture incubated at 37˚C for 24 hours in anaerobic condition. Centrifuged at 6000 rpm for 15 minutes at 4˚C. Cell free supernatant was concentrated to 100 ml in a rotary vacuum evaporator. Ammonium sulfate was added at 4˚C with stirring to 70% saturation. Centrifuged at 1000 rpm for 20 min at 4˚C. Precipitate was collected and resuspended in 3 ml distilled water at a centrifuge tube.

For the determination of protein profile of the lactic acid bacteria, strains previously identified by their phenotypic characteristics were submitted to SDS-PAGE as described by Laemmli [19].

1) Prepare in the following reagent

30% acrylamide-bisacrylamide solution, 10% ammonium per sulfate (APS) [Aliquots of 200 µl should be stored at −20˚C for single time use. APS decomposes slowly, therefore fresh solution should be prepared weekly.], 0.1% BMB (Bromophenol blue solution) or tracking dye, Destaining solution, Staining solution (Coomassie blue gel stain), Sample loading buffer, Electrophoresis buffer (pH 8.3), Stacking/Upper gel buffer (pH 6.8), Separating/Lower gel buffer (pH 8.8).

2) Preparation of separating gel (10%)

At first, clean and fresh glass plates were assembled in the gel casting chamber. A 10% separating gel was constructed by gently mixing the distilled water, lower gel buffer along with 10% SDS, 30% acrylamide-bisacrylamide solution, 10% SDS, 10% freshly prepared Ammonium per sulphate (APS) and TEMED according to given below. As soon as APS and TEMED were added to the mixture the freshly mixed solution was carefully poured immediately into the glass plate chamber with a sterile pasteur pipette without wasting a few seconds.

3) Preparation of stacking gel (5%)

After the polymerization of the separating gel has been completed, a 5% stacking gel mixture was prepared according to given below. Just like the separating gel, stacking gel mixture was also prepared by primarily mixing the distilled water, upper gel buffer along with 10% SDS and 30% acrylamide-bisacrylamide solution, 10% APS and TEMED. The gel mixture was then rapidly poured above the previously constructed separating gel.

4) Sample preparation

The protein sample (10 µL) was mixed with sample loading buffer at a ratio of 1:1 and was then boiled in a water bath for 3 minutes. During boiling precaution was taken so that the sample does not bump up. 5 μL tracking dye (0.1% Bromophenol blue) was then added to the boiled mixture.

5) Sample loading

After the polymerization of stacking gel has been completed, the comb was removed from the glass chamber with a soft hand so that the well divider did not crack. The glass chamber was then fixed in the electrophoresis unit and was placed gently in the buffer reservoir. The wells were filled with the running buffer by pouring running buffer inside two glass chambers of the electrophoresis unit up to the top edge of the glasses. Running buffer was also poured into the main buffer reservoir up to 1/3^rd of the height of the reservoir. 20 μL of sample was then added to the wells from the right side of the gel. 10 μL of molecular weight standard (BIO-RAD) was used as a marker.

6) Running the gel

After the loading process was done carefully, the electrophoresis unit was connected with the power pack adjusting at 20 mA current keeping the voltage supply free. The power supply was kept on for 1 - 1.5 h. As soon as the tracking dye reached the bottom level of the gel, the power supply was turned off.

7) Staining and destaining of the gel

After the run had been completed, the gel was released from the glass plates and immersed in a fresh staining solution (0.1% Coomassie Brilliant blue R 250). The gel was then shaken on a shaker (Memmert, USA) for approximately 2 h. Then the gel was taken out of the staining solution and flooded with destaining solution (10% Acetic acid) and placed on a rotary shaker for more 2 h. When the gel background became transparent, it was taken out of the destaining solution and immersed in distilled water.

Bacteria isolated from fermented vegetable cucumber & carrot was identified as Lactobacillus spp. by observing their colony morphology, physiological and as well as some biochemical characteristics. All isolates showed white and creamy colour on the MRS Agar plate (Figure 1). Colony morphology was observed creamy, little sticks and smooth round colonies.