Samples, isolation of Lactobacillus strains, foodborne pathogens, media, and growth conditions
All media and materials used in this study were obtained from (Merck Co. Darmstadt, Germany). For isolation of Lactobacillus strains, 50 samples of yogurt (0.5 kg each) were obtained from local vendors in Rasht city, Guilan province, Iran. 50 g of each sample was placed into sterile stomacher bags and then were diluted (1:10) with MRS broth, processed to enrich the LAB by stomaching, sealed, and incubated overnight at 30 °C.
Lactic acid bacteria were isolated by 5-fold serial dilutions. At first, 1 mL of sample in stomacher bags was added to 9 mL of sterile (0.90% NaCl) and then serially diluted. Then, 0.1 mL aliquots of each sample were plated onto MRS agar. The plates were incubated at 37 °C for 24-48 h in anaerobic jars using the AnaeroGen anaerobic system (Oxoid, Thermo Fisher Scientific, USA) in microaerobic conditions (5% CO2). MRS agar plates were used to identify the initial growth of the LAB in each sample. The resulting isolates were randomly selected and were streaked twice on fresh MRS agar for obtaining purified colonies. The isolated strains were maintained by culturing them in MRS broth medium at 30° C and then storing them at -80 °C in MRS broth containing 50% (v/v) glycerol for further studies (Bin Masalam et al., 2018; Bosch et al., 2012; Kargozari et al., 2015).
Micrococcus luteus PTCC 1408 was used as an indicator for primary evaluating the antimicrobial susceptibility tests. Lactobacillus casei ATCC 39392 was used for the evaluation of antimicrobial activity in submerged culture conditions. Also, Foodborne pathogens including Listeria monocytogenes PTCC1294 (native strain), Bacillus cereus PTCC1857 (native strain), Staphylococcus aureus ATCC 29213 and, Escherichia coli PTCC1276 (native strain) were used for evaluation of the antimicrobial activity of produced bacteriocin by the isolated strain. All bacteria were obtained from Persian Type Culture Collection (PTCC, Tehran, Iran).
Phenotypic, biochemical, and molecular characterization of the isolates
For identification and characterization of the LAB isolates, phenotypic characterization including Gram staining, colony size, and morphology, biochemical tests such as carbohydrate fermentation, catalase activity, growth at 15°C and 45°C, growth at 4% and 6.5% NaCl, and motility were carried out according to the Bergey’s Manual of Systematic Bacteriology (Vos et al., 2011).
For taxonomic characterization of L. curvatus isolate, the 16S rRNA analysis was performed. The genomic DNA was extracted from single colonies of the bacterial strains, using Qiagen DNeasy blood and tissue kit (QIAGEN N.V, Germany) (following the manufacturer’s instructions).The bacterial 16S rRNA loci were amplified using the forward
27f: 5'-GAGTTTGATCCTGGCTCAG -3' and reverse
1505r: 5’- GATACGGCTACCTTGTTACGA -3' primers. The PCR program was performed in a Techne FTC51S5D Thermocycler (Staffordshire, UK) for 35 cycles under the following temperature profile: 95°C for 3min (1 cycle); 93 °C for 45 s, 58 °C for 1min and 72 °C for 90 s (35 cycles); and 72 °C for 10min at the end of the final cycle. The PCR product was purified using a GF-1 PCR Cleanup kit (Vivantis technologies, Malaysia). The purified products were sequenced using the following primers:
27f: 5'-GAGTTTGATCCTGGCTCAG -3', 16r339: 5’- ACTGCTGCCTCCCGTAGGAG -3’
16f358: 5’- CTCCTACGGGAGGCAGCAG-3’, 704f: 5’- GTAGCGGTGAAATGCGTAGA- 3'
1505r: 5’- GATACGGCTACCTTGTTACGA -3'
The Sanger’s dideoxy chain-termination sequencing method was applied using an ABI 3730XL DNA analyzer (Thermo Fisher Scientific Inc. USA). The similarities between 16S rRNA sequences were compared in the National Center for Biotechnology Information
(NCBI), Ribosomal Data Base and EzTaxon databases. The phylogenetic tree based on 16S rRNA gene sequence was constructed against different related Lactobacillus strains according to partial sequence analysis. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 28 nucleotide sequences. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There were a total of 1478 positions in the final dataset. Phylogenic dendrograms were generated through the neighbor-joining method with bootstrap test (1,000replicates) using MEGA X (Molecular Evolutionary Genetics Analysis software version 10.1.) (Kumar et al., 2018; Tamura et al., 2004).
Evaluation of bacteriocin production by L. curvatus LAB-3H
The growth kinetics and antimicrobial activity of bacteriocin produced by the selected
L. curvatus strain LAB-3H was evaluated using an MRS broth medium. 250 mL of MRS broth at pH 6.8 was inoculated with (1% v/v) cell suspension of L. curvatus LAB-3H and incubated at 37 °C without agitation; in the presence of nitrogen for 48 h. Bacterial cell density, antimicrobial activity against the indicator strain L. casei ATCC 39392, and pH were measured every 4 hours during fermentation.
Determination of the antibacterial activity of produced bacteriocin by L. curvatus LAB-3H against selected foodborne pathogens
For the determination of the antibacterial activity of produced bacteriocin by L. curvatus LAB-3H against selected foodborne pathogens, the method of (Thirumurugan et al., 2013) was used. L. curvatus LAB-3H was grown in MRS broth (Merck Co. Darmstadt, Germany) at pH 6.8 and maintained aerobically at 35°C for 48 h. Then, bacterial cells were removed from the growth medium by centrifugation (10000X g for 30 min at 4°C) and passed through the Whatman membrane filter (0.2 µm diam. 47 mm). The cell-free supernatant was with adjusted pH 6.0 using 1N NaOH and used as crude bacteriocin. Bacteriocin activity was determined by the agar well diffusion method. At first, suspension of four foodborne pathogens including L. monocytogenes PTCC1294, B. cereus PTCC1857, S. aureus ATCC 29213 and,
E. coli PTCC1276 were prepared using turbidity standard McFarland 0.5. Then, 150 𝜇L of these suspensions were inoculated onto the medium by the streaking plate method, and four wells were punched in each inoculated agar medium plate using a sterile Cork borer (6mm diameter). Then, 100 𝜇L aliquots of prepared cell-free extract of L. curvatus LAB-3H were pipetted into their designated wells on the plates. All inoculated plates were incubated at 37°C for 18-24 h. The diameter of each inhibition zones was measured twice using a ruler; then, the average was taken to represent the antibacterial activity (Bin Masalam et al., 2018; Thirumurugan et al., 2013). The activity of cell-free supernatant was explained in arbitrary units per ml (AU/ml). A unit activity of the bacteriocin was defined as an arbitrary unit (Mathur et al., 2017); 1 AU is a unit area of inhibition zone per unit volume, in this case, mm2/ml(Usmiati Marwati, 2009). The bacteriocin activity was calculated using the following formula: Bacteriocin activity (mm2/ml) =Lz-Ls/V which the parameters are LZ=clear zone area (mm2), Ls=well area (mm2), and V=volume of the sample (ml) (Bin Masalam et al., 2018).
Determination of minimum inhibitory concentration
For the determination of minimum inhibitory concentration, the standard broth dilution method for the crude bacteriocin, and selected antibiotics was used in 70192 Mueller Hinton broth (Sigma-Aldrich) medium, according to CLSI M7-A8 and M100-S28-2018 guidelines. Serial two-fold dilution (50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 µg/ml) of the cell-free extract of bacteriocin, and antibiotics (64, 32, 16, 8, 4, 2 and 1µg/ml) with adjusted bacterial concentration (108 CFU/ml 0.5 McFarland’s standard) were used to determine MIC in MH broth. The control contained only inoculated broth and was incubated for 24 h at 37° C. The MIC endpoint is the lowest concentration of antimicrobial components where no visible growth is seen in the tubes. The visual turbidity of the tubes was distinguished, both before and after incubation to approve the MIC value.
Purification of bacteriocin produced by L. curvatus LAB-3H
Bacteriocin produced by L. curvatus LAB-3H was purified using a two-step procedure, including ammonium sulfate precipitation and cation-exchange chromatography suggested by
(Srinivasan et al., 2013; Zhang et al., 2018) with some modifications.
L. curvatus strain LAB-3H was inoculated in 1-liter MRS broth and incubated at 37 °C for 20 h. Cells were discarded by centrifugation at 12,000 rpm for 5 min. Then, cell-free supernatant was subsequently precipitated using 45, 55, 65, 75, and 85% ammonium sulfate, after that the precipitates were collected by centrifugation at 10000 rpm for 20 min and dissolved in double-distilled water. The dissolved proteins were filtered through a 0.22-µm filter and dialyzed using a 1000 Da cut-off membrane (Solarbio, China). The AKTA purifier (GE Healthcare, Uppsala, Sweden), equipped with an SP Sepharose Fast Flow column, was used for further purification of the bacteriocin protein. The system was equilibrated with 50 mmol/L sodium phosphate buffer (pH 6.0), and the column was eluted with 50 mmol/L sodium phosphate buffer, containing 1 mol/L NaCl, at a flow rate of 2 mL/min. The elution procedure was monitored by a UV detector at 280 nm. Fractions with the maximum antimicrobial activity were collected and identified (Srinivasan et al., 2013).
SDS–PAGE analysis of purified bacteriocin was performed with 16% resolving gel and 10 % stacking gel. The gel was cut into two parts: (i) gel was stained with Coomassie brilliant blue R-250 to determine the molecular size of bacteriocin and (ii) another gel was fixed in 20% (v/v) isopropanol and 10% (v/v) acetic acid for 2 h, and rinsed in sodium phosphate buffer (pH 6.0) for 1 h. After that, the gel was overlaid with a soft agar medium inoculated with the indicator strain for 48 h.
In this study, all data were expressed as mean ± standard deviation (SD) of triplicate independent experiments. All statistical analyses were performed with SPSS/PC software, version 20.0.