Culture media for bacteriocinogenic lactic acid bacteria and Listeria innocua
Four bacteriocinogenic LAB strains of L. curvatus (Arla-10), E. faecium (JFR-1), L. paracasei subsp. paracasei (JFR-5) and S. thermophilus (TSB-8) were originally isolated from cheeses and yogurts in our lab. In brief, bacteriocinogenic LAB were isolated using MRS and M17 media. The agar diffusion bioassay was used to screen for bacteriocin or bacteriocin-like substances (BLS) producing LAB using Lactobacillus sakei and Listeria innocua as indicator organisms as described by Yang et al. (2012). Arla-10, JFR-1 and JFR-5 strains were cultured in MRS broth (10 g peptone, 8.0 g lab-lemco’ powder, 4.0 g yeast extract, 20 g glucose, 1 ml Tween’ 80, 2.0 g di-potassium hydrogen phosphate, 5.0 g sodium acetate 3 H2O, 2.0 g tri-ammonium citrate, 0.2 g magnesium sulphate 7 H2O, and 0.05 g manganese sulphate 4 H2O per liter of water, pH 6.2 ± 0.2) (Oxoid, Basingstoke, UK). TSB-8 strain was cultured in M17 broth (5.0 g tryptone, 5.0 g soya peptone, 5.0 g meat digest, 19.0 g di-dodium-β-glycerophosphate and 0.25 g magnesium sulphate per liter of water, 50 ml 10% (w/v) lactose, pH 6.9 ± 0.2) (Oxoid, UK). Li. innocua used as an indicator organism for the bacteriocins assay was cultured in Brain Heart Infusion broth (BHI, 7.7 g calf brain, infusion from 200, 9.8 g beef heat, infusion from 250, 10 g proteose peptone, 2.0 g dextrose, 5.0 g sodium chloride, and 2.5 g disodium phosphate per liter of water, pH 7.4 ± 0.2) (Fisher Scientific, ON, Canada). The bacteriocinogenic LAB strains were stored at − 80 °C in their culture broth adding 30% sterile glycerol (Goldman 1990).
Preparation of inoculums
One ml of frozen LAB (Enterococcus sp. or Lactobacillus sp.) was cultured in 20 ml MRS broth, pH 6.2, while Streptococcus sp. was cultured in M17 broth, pH 6.9, respectively, at 37 °C for 24 h. One ml of LAB culture was then sub-cultured in MRS broth overnight and cells were harvested using a centrifuging at 14,000g for 5 min (Sorvall RC6 PLUS, Thermo-electron Corporation, Asheville, NC, USA). The cell pellet was washed with saline solution (0.85% NaCl) and re-suspended using 0.85% NaCl to a final optical density (O.D.) of 0.4 at 600 nm measured with a spectrophotometer (Ultrospec 3100 Pro, Biochrom Ltd. England). The cell suspensions were used as the inoculum for the growth curve experiments. Li. innocua was grown in BHI broth at 37 °C for 16 h, and then centrifuged at 14,000g for 5 min to collect bacterial cells. A final density of Li. innocua at 5 × 105 CFU ml−1 was prepared using 0.1% (w/v) peptone water for bacteriocin assay study.
Combinations of growth condition for LAB growth curve study
Two culture media of MRS and BHI broths with initial pHs adjusted to 4.5, 5.5, 6.2, 7.4 and 8.5, respectively, were prepared with 0.5 N HCl or NaOH. Three different incubation temperatures at 20, 37 and 44 °C were set up for incubation of the bacteriocinogenic LAB strains.
The growth curves for the four bacteriocinogenic LAB in MRS and BHI broth at different initial pH and temperatures were obtained with the Bioscreen C® (OY Growth Curves Ab Ltd, Finland). Bioscreen C was able to measure bacterial growth kinetically and generate growth curves based on turbidity changes of samples. In brief, 15 μl of LAB cell suspensions (OD600 nm = 0.4) was inoculated into 285 μl (5% level (v/v) inoculum) of each treatment combination (culture medium and pH) in Bioscreen C multi-well plates. At 37 or 44 °C, the multi-well plates were incubated for 48 h, while at 20 °C, the plates was incubated for 72 h. OD values were measured every 20 min under brown filter with a wavelength of 600 nm. All assays were performed for four times, and the data was averaged. For statistical analysis, logistic model was used and latency times, slopes, and maximum OD values were calculated.
Relationships between LAB growth kinetics and bacteriocins production
Eight combinations of growth condition including MRS, pH 6.2 at 37 and 44 °C, MRS, pH 7.4 at 37 and 44 °C, BHI, pH 5.5 at 37 and 44 °C, and BHI, pH 6.2 at 37 and 44 °C were prepared for further investigation the impact of growth condition on LAB growth and bacteriocins activity.
0.5 ml of LAB cell suspensions (OD600 = 0.4) was inoculated to 9.5 ml of culture broth (5% level (v/v) inoculum) of each combination described above, respectively. At 37 °C, samples were taken at 6, 8, 10, 13, 16 and 24 h, while at 44 °C samples were taken at 5, 7, 9, 11, 13 and 18 h for determining LAB counts and bacteriocins activity (AU).
Measurement of pH, OD values and viability of LAB (CFU ml−1)
The pH and OD values of LAB samples were measured following the methods described by Yang et al. (2012). For determination of viability of LAB, a series of dilution were prepared with peptone water (0.1% peptone). MRS was used to culture Enterococcus sp. and Lactobacillus sp. while M17 was used for culturing Streptococcus sp. At each dilution, 50 µl of sample was spiral plated to their respective culture medium (agar) plates using a Whitely Automatic Spiral Plater (WASP2) (Don Whitley Scientific Limited, Shipley, England). The petri dishes were then incubated at 37 °C for 48 h and LAB colonies were counted using an aCOLyte colony counter (Synbiosis, Cambridge, England).
LAB bacteriocin activity (AU) test
The agar diffusion bioassay (Herreros et al. 2005) with modification by Yang et al. (2012) was used to determine LAB bacteriocin activity. At each sampling time the supernatant of LAB cultures was centrifuged at 14,000g for 5 min and LAB cells were moved. The cell free supernatants (CFS) were filtered through 0.22 µm syringe filters (Chromatographic Specialties Inc., ON, Canada) and then adjusted to pH 6.0 by sterilized 1 mol l−1 NaOH or 1 mol l−1 HCl to rule out inhibition effect resulting from organic acid. The neutralized supernatant was mixed with 1 mg ml−1 of catalase (Sigma-Aldrich Corporation, USA) at 25 °C for 30 min to eliminate the inhibitory effect of hydrogen peroxide. pH adjusted and H2O2 eliminated supernatants were filtered again to obtain bacteriocin like substance (BLS). 35 μl of untreated cell free supernatants (control) or BLS was added to the 5 mm wells in BHI agar plates (semi-solid with 0.7% agar) which contained Li. innocua at 5 × 105 CFU ml−1. The plates were incubated at 37 °C for 24 h to determine if there was any inhibitory zone. In this study, the agar diffusion bioassay was again used to measure bacteriocin activity with Li innocua as an indicator. To quantify the bacteriocin activity, CFS or BLS was serially diluted twofold with sterile deionized water. The bacteriocins’ antibacterial activity (AU) was defined as 2n × 1000 µl/35 μl, where n is the reciprocal of the highest dilution of CFS or BLS with inhibition of Li. innocua. The detailed procedures to determine the bacteriocin activity were described by Yang et al. (2012).
Statistical analysis
For growth curve test, a split–split plot design was performed with the four bacteriocinogenic LAB strains on the main plot while the two culture media and five initial pH values on the sub plot, which was split into three different temperatures. Bioscreen C with 10 × 10 layout multiwell plates was used and growth curves data were analyzed from fitted data of a logistic model. The logistic template formula is expressed as Y = A + C/(1 + EXP (− B * (t − M)), where A = the fitted initial level; B = the relative growth rate; A + C = the final population density; t = time points; M = the inflection point.
For bacteriocin production assay, analysis of growth curves was done from fitted data of spline model. Spline template equation is expressed as Y = a + bS (x; 2), where a = the intercept; b = the slope; S = the spline function; x = the measured values. All experiments were repeated four times.