Isolation and screening of LAB
LAB were isolated from 7 commercial cheeses: [Tre Stelle® bocconcini cheese (TSB); Tre Stelle® fromage romano cheese (TSR); Saputo® feta cheese (SF); Agropur® signature rondoux pure goat cheese (ASR); Agropur® signature OKA cheese (OKA); Arla® fontina cheese (Arla) and Jarlsberg® firm ripened cheese (JFR)]; plus 3 commercially available yogurts: (Danone, Activia®; Astro® BioBest yogurt; and Bioghurt® Liberte yogurt), and one in- house produced yogurt.
A 25 g sample of cheese was weighed into filtered stomacher bags (Fisher Scientific, Nepean, ON, Canada) and mixed with 225 ml of sterile 0.1% (w/v) peptone water (Fisher Scientific). Samples were blended at 280 rpm for 3 min (400C stomacher circulator, Seward, England). For yogurt samples, 1 ml of sample was added to 99 ml of sterile 0.1% peptone water. All samples were serially diluted and 50uL of each dilution was spiral plated onto de Man, Rogosa and Sharp (MRS) agar, (Oxoid, Basingstoke, UK) and M17 agar (Oxoid). MRS plates were incubated at 37°C under both aerobic and anaerobic conditions for 48 h and M17 plates at 44°C under anaerobic condition for 48 h. All gram positive, catalase negative (3% v/v H2O2) isolates were purified and observed under a light microscope. All isolates were coded and stored in MRS or M17 broth containing equal amounts of 30% sterile glycerol at - 80°C.
Antimicrobial activity and enzymatic testing of bacteriocin producing LAB
The agar diffusion bioassay described by Herreros et al. ([2005]) was used to screen for bacteriocin producing LAB among the 138 isolates. L. innocua (ATCC 33090TM) and Lact. sakei (ATCC 15521TM) were used as indicator bacteria. L. innocua was incubated overnight in Brain Heart Infusion broth (BHI, Fisher Scientific, ON, Canada) at 37°C and Lact. sakei was cultured anaerobically in MRS broth at 37°C.
One ml of each indicator organism (5 × 105 cfu ml-1) was inoculated into 15 ml of semisolid BHI or MRS agar (BHI or MRS broth plus 0.7% bacteriological agar) maintained at 50°C and then poured into a petri dish. After solidification, three wells (5 mm diameter) were cut and 35 μl of cell-free supernatant (CFS) from each LAB isolate and appropriately adjusted was added to each well. CFS were prepared as follows: one ml of frozen LAB isolate was cultured overnight in 20 ml MRS or M17 broth then 1 ml culture was sub-cultured overnight in 20 ml MRS broth. Cells were removed by centrifuging at 14,000 g for 5 min (Sorvall RC6 PLUS, Thermo-electron Corporation, Asheville, NC, USA). The supernatant was filtered through a sterile 0.22 μm syringe filter (Chromatographic Specialties Inc., ON, Canada) and 35 μl of the unadjusted aliquot of CFS was added to the first well. The remaining CFS was adjusted to pH 6.0 with 1 mol l-1 NaOH in order to rule out possible inhibition effects due to organic acids. 35 μl of the pH adjusted CFS was filtered and added to the second well. The neutralized CFS was then treated with 1 mg ml-1 of catalase (Sigma-Aldrich Corporation, USA) at 25°C for 30 min to eliminate the possible inhibitory action of H2O2 and filtered, then was placed in the third well. If inhibitions zones were found in the third well, the isolates were considered to be able to produce BLS.
The BHI or MRS plates were incubated at 37°C aerobically for 24 h or at 37°C anaerobically for 24 h, respectively. Inhibition zones were measured using an electronic caliper with digital display (MastercraftMD, Miami, FL, USA). Screenings for bacteriocin producing LAB were repeated twice for each isolate.
To confirm production of a proteinaceous compound, CFS displaying antimicrobial potential after acid neutralization and H2O2 elimination were treated with 1 mg ml-1 of proteolytic enzymes, including proteinase K (33 U mg-1), α-chymotripsin (66 U mg-1), and trypsin (105 U mg-1) (Sigma- Aldrich Corporation, USA) at 37°C for 2 h (Bonadè et al. [2001], Herreros et al. [2005]). Antimicrobial activity of treated CFS was determined by the agar diffusion bioassay as described above.
Identification of BLS producing LAB using 16S rRNA gene sequencing
Near full-length 16S rRNA gene sequencing was used to identify unknown bacteriocin producing LAB strains based on the method of Abnous et al. ([2009]). Briefly, genomic DNA was extracted from isolates using the UltraCleanTM Microbial DNA Isolation kit (MO Bio laboratories, Inc., Carlsbas, CA, USA). Universal primers F44 (5’RGTTYGATYMTGGCTCAG-3’) and R1543 (5’-GNNTACCTTKTTACG ACTT-3’) (Abnous et al., [2009]) were used for the amplification of the 16S rRNA gene by PCR. PCR reactions were carried out using a Biometra thermal cycler (Montreal Biotech Inc., Kirkland, QC, Canada) with the following cycle parameters: an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and elongation at 72°C for 1 min. A final elongation step was performed at 72°C for 5 min. PCR amplicons were separated by agarose gel electrophoresis (0.8% w/v) and visualized by staining with ethidium bromide.
The PCR products were cloned into the psc-A-amp/kan vector using the StrataClone PCR Cloning kit (Stratagene, La Jolla, CA) and transformed into E. coli according to the manufacturer’s instructions. Transformants were grown overnight in Luria-Bertani broth supplemented with 100 μg ml-1 ampicillin. Plasmids were extracted and purified from selected E. coli clones with the UltraClean TM mini plasmid prep kit (MO Bio Laboratories) according to the manufacturer’s recommendations and then sequenced using the Big Dye Terminator v3.1 cycle sequencing kit. A homology search of the sequences was conducted using the BLAST program at the NCBI database.
Thermal stability of bacteriocins produced by LAB isolates
Based on identified LAB species, isolation source and the size of inhibition zones, eight LAB isolates were chosen for thermal stability tests. The pH adjusted and H2O2 eliminated CFS described above were treated at 80 and 100°C for both 60 and 90 min, and at 121°C for 15 min. pH adjusted and H2O2 eliminated CFS without any heat treatments served as a controls. Residual antimicrobial activity of heat-treated CFS was determined by the agar diffusion bioassay compared to the control using L. innocua as the indicator bacteria.
Antibacterial effects of LAB isolates in vitro
The antibacterial effects of eight selected LAB isolates on six common food borne pathogens or spoilage organisms at 5 and 20°C were investigated using the agar diffusion bioassay described above. Targeted indicator organisms and their respective media used were as follows: L. innocua (BHI, DifcoTM, Spark, MD), E. coli K-12 (ATCC 10798TM) (Tryptic Soy Broth, DifcoTM), B. cereus (ATCC 14579TM) (Nutrient Broth (NB), DifcoTM), Ps. fluorescens (A7B) (NB), Erw. carotovora (ATCC 15713TM) (NB) and Leuc. mesenteroides subsp. mesenteroides (ATCC 8293TM) (MRS, DifcoTM). All strains were cultured aerobically for 24 h at their optimal growth temperatures: 26°C for Erw. carotovora, Ps. fluorescens and Leuc. mesenteroides; 30°C for B. cereus; and 37°C for E. coli and L. innocua. LAB isolates were sub-cultured and the CFS was prepared as previously described. Following inoculation, plates were incubated at 5 and 20°C for 7 d and 24 h, respectively. Inhibition zones were measured as before.
Antifungal effects of LAB isolates in vitro
The microdilution method described by Lavermicocca et al. ([2003]) with some modifications was used to test the eight LAB isolates against P. expansum (Pex 03–10.1), B. cinerea (B94-b) and M. fructicola (Mof 03–25) at 5 and 20°C. Fungi were obtained from the culture collection at our Research Centre, AAFC. The stock cultures were stored as spore or mycelial suspensions in 15% glycerol (v/v) at −80°C. Conidia of P. expansum were collected from 4 d-old cultures grown on potato dextrose agar (PDA) at 25°C. Conidia of B. cinerea were collected from 12 d-old cultures grown under a 12 h light/dark cycle on Pseudomonas Agar F at 22°C. Conidia of M. fructicola were collected from 12 d-old cultures grown on modified V-8TM medium at 25°C. Using sterile distilled water, the density of spore suspensions were diluted to 2 × 105 spores ml-1 as determined using a haemocytometer (Hausser Scientific, PA, USA).
Microdilution tests were performed in sterile 96-well micro dilution plates (Costar 3370, Corning Incorporated, Corning, NY, USA). 200 μl of test solution consisting of 185 μl LAB CFS inoculated with 15 μl of conidial suspension was dispensed into the wells. Microdilution plates were incubated at either 20 or 5°C and the optical density (OD) at 580 nm was recorded at specific time intervals using a microtiter plate reader/ spectrophotometer (Spectra MAX 190, Molecular Devices, CA, USA). For the 20°C plates, OD values were measured at 0, 24, 40, 48 or 72 h, while at 5°C, they were measured at 0, 48, 72, 96 or 120 h. For each fungus, both positive controls containing 185 μl MRS broth and 15 μl conidial suspension and negative controls containing 185 μl LAB CFS and 15 μl dead conidial suspension were prepared and monitored. Experiments were repeated three times.
In vivo testing of selected LAB on microflora naturally occurring on fresh-cut onions
Ent. faecium, Strep. thermophilu s and Lact. casei were chosen for in vivo tests. Fresh-cut yellow onions processed at a commercial facility were supplied by Nova Agri Inc. (Canning, NS). Ent. faecium and Lact. casei were incubated anaerobically in MRS broth at 37°C for 16 h. Strep. thermophilu s was incubated anaerobically in M17 broth at 43°C for 16 h. LAB were sub-cultured twice and then centrifuged at 14,000 g for 5 min. Pellets of LAB were washed using sterile distilled water, centrifuged, re-suspended and inoculated onto the batches of diced onions using a calibrated TLC sprayer to give a final concentration of 5 × 105 cfu g-1. Diced onions inoculated with sterile distilled water served as a control. Samples were left to dry for 10 min, then 100 g was transferred to food grade bags (Golden Eagle-VH-62 190) and sealed using an electric sealer (FoodSaver V2490, Tilia International Inc. US). The densities of naturally occurring (indigenous) LAB, Pseudomonas sp., yeasts and moulds, Listeria sp. and Enterobacteriaceae were enumerated following 0, 3, 6, 9 and 12 d of storage at 4°C. LAB were cultured anaerobically on MRS Agar at 37°C for 48 h; Pseudomonas sp. on Pseudomonas selective medium at 30°C for 48 h; yeasts and moulds on PDA supplemented with chloramphenicol (DifcoTM) at 25°C for 48 h; and Enterobacteriaceae on Violet Red Bile Agar (VRBG)(DifcoTM) at 37°C for 48 h. Colonies were counted using an aCOLyte automated colony counter (Synbiosis, Cambridge, England) and expressed as cfu g-1 of diced onions.
In vivo testing of selected LAB on L. innocua inoculated on fresh-cut onions
L. innocua was sub-cultured into BHI broth and incubated at 37°C for 24 h. The selected LAB strains and inoculation procedures were the same as described above. The final density of L. innocua inoculated on fresh-cut onions was 5 × 103 cfu g-1. After drying for 10 min, the LAB species were inoculated respectively onto the fresh-cut onions at a density of 5 × 105 cfu g-1. A batch of fresh-cut onions inoculated with L. innocua alone was used as the control. Samples (100 g/ bag) were stored at 4°C and Listeria sp. and LAB were enumerated following 0, 3, 6, 9 and 12 d. Listeria sp. was cultured on Listeria selective medium at 35°C for 48 h and LAB on MRS agar at 37°C for 48 h anaerobically.
Statistical analysis
The initial screening for bacteriocin producing LAB from 138 LAB isolates was repeated twice. For antimicrobial tests, a three repetition split-split plot design was used with the eight LAB isolates on the main plot versus the six bacteria (or three fungi) on the sub plot, which was then split into two temperatures (5 and 20°C). For the in vivo testing, non-inoculation or inoculation of L. innocua was designed on the main plot with the three LAB strains used to investigate the treatment effect on naturally occurring microflora and introduced L. innocua on the onions. The sub plot was represented by sample removal at days 0, 3, 6, 9 and 12. Data were analyzed using the ANOVA directive and standard errors of mean (SEM) option of GenStat® (12th Edition, VSN International Ltd, Hemel Hempstead UK, 2009). The results of a Principal Components Analysis (PCA) were discussed in terms of component scores.