Bacterial strains and media
The LAB strain, which was isolated from curd, identified as L. fermentum LMEM22 by conventional phenotypic characterization (Halder and Mandal, 2018), and maintained in MRS broth as well as in MRS agar stab in freezing, was utilized in the instant study. The indicator bacteria used include the randomly selected gram-negative (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica serovar Typhi and Proteus vulgaris) and gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus) clinical bacterial isolates from the laboratory stock cultures, and Listeria monocytogenes MTCC657 standard strain, which were maintained in cystine tryptone agar stabs, in refrigeration.
Molecular identity of lactic acid bacterium
The identity authentication of the bacteriocinogenic LAB strain was done by 16S rRNA gene sequence and phylogenetic analyses. The 16S rDNA (≈1.5 kb fragment) was PCR amplified (from the genomic DNA extracted from the LAB) and thereafter sequenced using 16S rRNA specific primers (forward primer: 5′-AGHGTBTGHTCMTGNCTCAS–3′ and reverse primer: 5′-TRCGGYTMCCTTGTWHCGACTH–3′) from Chromous Biotech Pvt Ltd, India.
The sequenced data (partial 16S rRNA gene sequence: 672 bp) was aligned using ClastalW (Thompson et al.,, 1994), and in the NCBI (National Center for Biotechnology Information) GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) the closest known relatives of the sequence obtained (from the test LAB strain) were determined through nucleotides homology search, with BLAST (Basic Local Alignment Search Tool), for nucleotide sequences (BLASTN).
The phylogenetic tree, based upon 16S rRNA gene sequences was constructed within the SeaView version–4 software (Gouy et al., 2010),following maximum likelihood method with PhyML GTR model (bootstrap with 1000 replicates). The partial 16S rRNA gene sequence of the test LAB strain has been deposited with the NCBI Genbank accession No. MH380182.
Probiotic characterization
The probiotic features of the test L. fermentum LMEM22 strain was substantiated through physiological stressors tolerance tests and safety profiling. The L. fermentum LMEM22 was subjected to probiotic characterization by performing bile salt and low-pH (acid) tolerance tests according to Liong and Shah (2005), and sodium chloride tolerance test following Chowdhury et al. (2012), with some alteration as cited before (Halder and Mandal, 2015; Halder et al.,, 2017).
In order to check the viability of the LAB at different incubation hours, the L. fermentum LMEM22 strain, with ≈5 × 105 CFU/ml (5.698 log10 CFU/ml) inocula, was allowed to grow in presence of varied concentrations of sodium chloride (2, 4 and 6.5 %) and bile salts (0.125, 0.25 and 0.5 %), and under acidic environmental condition (at pH: 2, 3 and 4) in MRS broth, up to 24 h, at 35 oC. Aliquots (each containing 100 µl), from the above mentioned cultures with different physiological stressors, were removed at 0, 2, 4, 6 and 24 h, mixed with MRS agar and pour plated, and following incubation at 35 oC for 24 h, CFUs were enumerated.
The safety properties of the LAB, L. fermentum LMEM22 strain, were validated by their hemolytic, gelatinase and DNase activity (Halder et al.,, 2017; Yadav et al.,, 2016).
Isolation and quantification of bacteriocin
Bacteriocin from the L. fermentum LMEM22 strain was extracted following Ismail et al. (2013), with modifications as explained below. Following subcultures of L. fermentum LMEM22 strain twice in MRS broth, 100 µl was inoculated into 25 ml of MRS broth, and after incubation at 37°C for 48 h, cell free supernatant (CFS) was prepared by centrifugation (at 10,000 rpm, for 10 minutes at 4°C) and syringe filtration. The CFS was treated with 60% ammonium sulfate at 4°C for 24 h to get precipitated the bacteriocin, which by centrifugation (at 12,000 rpm for 15 minutes at 4°C), was extracted. The isolated bacteriocin was processed by washing with sterilized double distilled water and centrifuging (at 10,000 rpm for 10 minutes at 4°C) for thrice, in order to remove the impurities in bacteriocin. At the final stage, pure bacteriocin pellet was mixed with phosphate buffer solution (1 ml, pH 7.2), with 0.6 % SDS, and stored at 4°C for further usage.
The bacteriocin yield was quantified spectrophotometrically, following Lowry et al. (1951), using bovine serum albumin as the standard.
SDS-PAGE analysis of bacteriocin
The molecular weight of bacteriocin isolated from L. fermentum LMEM22 strain was approximated by glycine-SDS-PAGE (glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis (Laemmli, 1970), using a vertical slab gel apparatus (Tarsons, India) with 7.5% stacking and 12.5 % separating gels, and high range protein molecular markers (Hi-Media, India). Following electrophoresis for 4 h at 130 V, the gel was subjected to Coomassie brilliant blue (Hi-Media, India) staining (for 30 min) and thereafter de-staining in 20% (v/v) methanol / 7.5% (v/v) glacial acetic acid until the bands were clearly visible. The molecular weight of L. fermentum LMEM22 bacteriocin was calculated from the relative mobility of the molecular weight markers in the gel.
Antibacterial activity of bacteriocin
The antibacterial activity of L. fermentum LMEM22 bacteriocin was determined by agar-well diffusion following Tagg and McGiven (Tagg and McGiven, 1971), using 66.67-µg bacteriocin per well (6 mm diameter), prepared on nutrient agar plate, which was swabbed with the overnight grown culture of indicator bacterial strains in nutrient broth, and the ZDIs were measured (in nearest whole in millimeter), in order to interpret the effectiveness of bacteriocin, following the criteria mentioned earlier6, as less active, moderately active, or highly active through ZDIs ≤ 10 mm, 11—14 mm, and ≥ 15 mm, respectively.
For the determination of MICs of L. fermentum LMEM22 bacteriocin, against the indicator bacteria, broth dilution method was used as mentioned elsewhere (Wiegand et al.,, 2008), utilizing random bacteriocin concentrations of 30—200 µg/ml, in nutrient broth. Inoculation of indicator bacteria, from overnight grown nutrient broth cultures, was done; all incubations were at 35 oC for 24 h. The lowest bacteriocin concentration, which was required to inhibit the visible growth of test pathogenic bacteria, was defined as the MIC.
Enzymatic sensitivity of bacteriocin
The enzymatic effect on the bacteriocin isolated from L. fermentum LMEM22, against protease K, trypsin, and α-amylase, was evaluated following the protocol of Ge et al (Ge et al., 2016). The test bacteriocin was mixed with each of the enzymes (10 mg/ml) and incubated at 37 oC for 24 h, and afterward the mixture was boiled at 80 °C for 5 min for enzyme inactivation. The L. fermentum LMEM22 bacteriocin without any enzymatic treatment was used as the control, in determining the residual bacteriocin activity (RBA), following agar-well diffusion against gram-positive (Staphylococcus aureus and Listeria monocytogenes) as well as gram-negative (Acinetobacter baumannii and Escherichia coli) human pathogenic bacteria. The RBA was computed based on the ratio of bacterial growth inhibition (in terms of ZDI) with treatment and with the control (Ge et al., 2016).