Dahi was collected from different locations of Lahore and seventy (70) bacteria were isolated and purified.

Antibacterial Potential Assay:

Indicator organisms were taken from First culture bank of Pakistan, Faculty of Agricultural Sciences, University of the Punjab. These seventy bacterial pure cultures were further screened for antibacterial potential against sixteen indicator bacteria named as (Listeria monocytogenes, Escherichia coli, Salmonella typhi, Salmonella paratyphi, Yersinia pseudotuberculosis, Yersinia enterocolitica, Klebsiella pneumoniae, Klebsiella pneumoniae ssp. rhinosclerosis, Clostridium difficille, Bacillus subtilis, Staphylococcus caprae, and Staphylococcus carnosus, Micrococcus roseus, Micrococcus luteus, Shigella dysenteriae, and Pseudomonas. All indicator organisms were grown in tubes containing LB broth medium by inoculating loop full sample and incubating at 37 °C for 24 hrs.

The purified bacterial isolates were grown in respective MRS medium by inoculating loop full of pure culture in tubes containing 7 mL MRS and M17 broth at 37 °C for 24 hrs. It was centrifuged, and the pellet was separated from the supernatant. The pellet was separated from the supernatant after centrifugation. The pellet was used to extract DNA.

Agar well diffusion method was adopted to analyze the antibacterial spectrum of isolated bacteria P26 against indicator organisms [9]. Wells were made in an agar plate with a sterile borer of 5mm. Then 100 microliter of bacterial supernatant was filled in it. It was given time for proper absorption before incubation. The zone of inhibition was measured in mm. Kanamycin (100 mg/mL) was used as a standard. The zone of inhibition produced by antibiotic kanamycin and bacterial culture was compared.

Isolation of Bacteriocin:

Cell-free culture supernatant fluid (Cfcsf) for bacteriocin assay:

The bacterial isolate P26 was further screened for bacteriocin activity. The bacterial strain was grown in broth media and their supernatant was separated by centrifugation.

pH neutralization:

The pH of Cfcsf was set by using acid standard solutions of (Hydrochloric acid) and base (Sodium hydroxide) for the exclusion of the antimicrobial effect due to acids (organic) released by bacteria in the medium. The antagonistic action was tested by adopting the agar well diffusion method [10].

Treatment with catalase:

To check the hydrogen peroxide effect on antagonistic activity, the catalase enzyme 10 mg/mL was poured into it. It was held (37°C, 2 hrs.). Afterward, it was heated at 80 °C for enzyme inactivation and inhibitory activity as checked against indicator bacteria [10].

Confirmation of bacteriocin:

To confirm the presence of bacteriocin, a 1 mL sample was treated with filter-sterilized protease enzyme solutions for 2 h at 37 °C, trypsin proteinase Kin 20 mM Tris-HCl, pH 7.0 [11].

Antibacterial activity of bacteriocin P26:

After confirmation of bacteriocin, its antibacterial assay was performed against sixteen indicator organisms. Kanamycin 10mg/mL was used as standard. 

Characterization of Isolate P26

Phenotypical characterization:

Isolate P26 was identified morphologically based on colony shape, color, texture, margins, opacity, and elevation. Bacterial isolate (P26) was gram stained and their morphological features, movement (motility) were observed under a light microscope. Then different biochemical and enzymatic (Catalase, Sugar fermentation) tests were performed for their identification following Bergey's manual of systematic bacteriology.

Molecular characterization

Genomic DNA isolation:

Genomic DNA was extracted by the following method of Cardinal and others [12] with some amendments. Activated MRS cultures (10mL) were centrifuged (12000 rpm, 10 min.), the supernatant was discarded, and the pellet was washed with 1ml EDTA-NaCl solution then re-suspended in 560 µL 1xTE buffer (pH 8). 3µL of 1mg/mL proteinase K, 3µL lysozyme (20mg/ml), 10 % SDS 20 µL were added and after gentle mixing 100 µL of 5M NaCl solution and 80 µL, CTAB/NaCl solution was added at kept at 37°C for 30 minutes. Then 700 µL chloroform/isoamyl was added before centrifugation. The upper phase was shifted into a new Eppendorf tube. It was extracted twice with 700 µL chloroform/isoamyl alcohol. DNA was precipitated by the addition of sodium acetate 20 µL and absolute ethanol 500 µL. It was mixed and kept overnight at -20 ⁰C. By centrifugation (12000rpm, 15 min), a pellet of DNA was formed. Then it was washed with 200 µL of 70% ethanol. DNA was again centrifuged after washing. Ethanol was removed and dried at room temperature. Then it was mixed with distilled water. 100µg/mL RNase (1µL/20µL DNA) was added before incubation at 37 °C for 30 minutes. Then washed with alcohol (chloroform/isoamyl 24/1, v/v). The upper phase was shifted into a new Eppendorf tube. Absolute ethanol 50 µL and sodium acetate 3 µL was added and kept overnight at -20 °C overnight and then centrifuged for pellet formation. Pellet was dried. And 25 µL of distilled water was added and mixed. Then the band was observed under UV transilluminator by conducting electrophoresis (agarose gel).

Polymerase chain reaction:

PCR-based amplification of isolated bacterial DNA was achieved through universal 16S ribosomal RNA primers obtained from Macrogen, (South Korea). 27F: 5’ AGA GTT TGA TCM TGG CTC AG-3” and 1492R: 5’ CGG TTA CCT TGT TAC GAC TT-3’. The annealing temperature was calculated according to primer sequences and Taq polymerase to be used.  A thermal cycler was used for amplification. The reaction mixture contained (30 µL) contained Taq DNA polymerase buffer (contain MgCl₂ and dNTPs) 15μL, Oligo forward (10 picomole/μL) 3μL, Oligo reverse (10 picomole/μl) 3μL, Phire Taq 0.6 µL, sterile deionized water 7.4μL. The DNA fragments were amplified as follows: initial denaturation at 98°C for 5 min followed by 35 cycles of denaturation at 98°C for 20 sec, annealing at 72°C for 20 sec, elongation at 72°C for 1 min, and final elongation at 72°C for 5 min. The products were stored at 4 °C until analysis. Aliquots (5 µL) of the amplified products were visualized by agarose gel electrophoresis (1%) using a DNA marker (Gene ruler 1Kb DNA) under UV Trans illuminator. Sequencing results obtained were matched and compared with primer walking and the gaps were filled. The obtained sequences were compared with the NCBI Gene Bank database by Basic local alignment search tool (BLAST) analysis. The phylogenetic tree was constructed using MEGA 6 software with a maximum likelihood method based on 16S rRNA gene fragments and was evaluated by the bootstrap method with 1000 replicates.

Partial purification of bacteriocin:

Bacteriocin (P26) was partially purified with ammonium sulphate by adopting the method of [13] 2010 with some modifications.

Ammonium sulphate precipitation:

Sterilized broth media was inoculated with bacterial culture and incubated under proper conditions. By centrifugation, pellet and supernatant were separated. The supernatant was heated at 80°C for 30 minutes. Then filter through a 0.22um syringe filter to remove any remaining cell. Its pH was adjusted at 6.5 with 2N NaOH. Then ammonium sulphate salt was added up to 40%, 60%, 80% saturation and it was continuously stirred for 15 hours. The solution was stirred in a magnetic stirrer using magnetic beads. This was kept at 4 ºC overnight. Then it was refrigerated centrifuged at 10,000 rpm for 30 min for protein precipitation. The supernatant was discarded, and precipitates collected were dissolved in 50mM tris buffer (pH 6.8).

Brad ford test:

Turn on the spectrophotometer and let it warm up for 15 minutes. It was calibrated at 595nm. In Eppendorf, 20L of each sample was accurately pipetted out, and 50 L of 1M NaOH was added. 1 mL of Coomassie brilliant blue R-250 (Bio-Rad) was gently mixed in. It was allowed to sit at room temperature for 15 minutes. The sample was placed in 1mL disposable polystyrene cuvettes, and absorbance at 595nm was measured. Using a known concentration of bovine serum albumin, a standard curve was created. The sample's concentration was determined by comparing it to the standard curve [14].

Bacteriocin Characterization

The antagonistic activity in arbitrary unit/ml (AU/mL) was calculated as a   measure of bacteriocin production.

        AU/ml = Diameter of zone of clearance (mm) x1000 / Volume taken in well (µL)

Activity is expressed as the percentage of residual activity (AU/ml) measured against indicator organisms by agar-well diffusion assay

Quantity of supernatant filled in wells =100µL

For bacteriocin characterization, the culture strain was cultivated in MRS broth (24-48 h at 37°C). It was centrifuged and Cfcsf was separated from the pellet. It was considered a crude bacteriocin [14]. Partially Purified (crude) bacteriocin was characterized at various temperatures (50 °C, 70 °C and 100 °C for 30 minutes, and at 121°C for 5 min), pH ranges (3·0, 4·0, 5·0, 6·0, 7·0, 8·0, 9·0 or 10·0) and assayed for bacteriocins activity [10]. The crude bacteriocin was also characterized by assessing sensitivity to proteolytic and non-proteolytic enzymes. 

Results