Isolation of Lactobacillus plantarum LMG P-26358 and bacteriocin assays
Lb. plantarum LMG P-26358 was initially isolated from French artisanal cheese as follows: approximately 1g of cheese was homogenized in 9 mls of maximum recovery diluent (MRD), serially diluted and plated on MRS (de Man, Rogosa, Sharpe) agar (Difco Laboratories, Detroit, MI, USA) and grown at 30°C for 2-3 days. Colonies that developed were overlaid with ~10 ml of GM17 agar [M17 medium (Difco Laboratories) supplemented with 0.5% (w/v) glucose] inoculated at 0.25% with an overnight culture of L. innocua (DPC6579). The plates were incubated for another 18 h at 37°C and inspected for zones of inhibition of the overlaid culture. Colonies showing a clear zone of inhibition were sub-cultured into fresh MRS broth having first been removed from the agar overlay using a sterile scalpel blade. Pure cultures were obtained by streaking onto MRS agar. Bacteriocin assays and estimation of bacteriocin activity in activity units (au)/ml were performed by the agar well diffusion assay (WDA) as described by Ryan et al. .
The sensitivity of a strain to plantaricin 423 was scored according to the diameter of the zone of inhibition surrounding the well. Each assay was performed in triplicate. Other media used in the study included BHI (Brain-Heart Infusion) broth (Oxoid Ltd., Basingstoke, Hampshire, England), RCM (Re-inforced Clostridial Medium) (Merck, Darmstadt, Germany), LBS (Lactobacillus Selective) agar (Difco Laboratories), PCA (Total Plate Count) (Difco Laboratories), SLB (Sodium Lactate Broth) as described by Drinan and Cogan . All strains were stocked in 50% glycerol at -20°C.
Identification of Lactobacillus plantarum LMG P-26358
The antimicrobial strain was identified by analyzing the 16S rDNA sequence. Genomic DNA was isolated from 1.5 ml of overnight MRS broth culture using the method of Hoffman and Winston  with slight modification as described by Mills et al. . The 16S rDNA primers CO1 (AGTTTGATCCTGGCTCAG) and CO2 (TACCTTGTTACGACTT) were used to amplify a product of ~1500 bp using an annealing temperature of 60°C. PCR amplification was performed in a Hybaid PCR express unit (Hybaid Ltd., Middlesex, UK) according to the manufacturer’s specifications using Biotaq DNA polymerase (BioTaq, Bioline Ltd., London, UK). The PCR product was purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and sequenced with an automated DNA sequencer (Beckman Coulter Genomics, Hope End, Takeley, UK). The 16S rDNA gene sequence was analysed using BLAST  to identify the closest bacterial neighbour.
Effect of proteinase K, pH and temperature on plantaricin 423 and stability of production
Proteinase K sensitivity was evaluated as follows: cell-free supernatant was harvested from 1 ml of overnight culture and exposed to a final concentration of 50 mg/ml proteinase K (Sigma-Aldrich, Poole Dorset, UK) and incubated for 2 hours at 37°C. The agar WDA was then performed against L. innocua DPC6579 with the proteinase K-treated sample and untreated cell-free supernatant as control. In order to determine the effect of pH on bacteriocin activity, the pH of cell-free supernatants was adjusted to pH values ranging from 1-10 using 1 M NaOH or 1 M HCl and incubated for 2 hours at 25°C before performing the agar WDA. In a separate experiment the effect of temperature on bacteriocin activity was assessed by incubating the cell-free supernatants at 40, 50, 60, 70, 80, 90 and 100°C for 30 min after which activity was assessed against L. innocua. Stability of bacteriocin production was assessed by sub-culturing Lb. plantarum LMG P-26358 (2%) in MRS broth twice a day for a 10 day period and performing the agar WDA against L. innocua each day. All experiments were performed in triplicate.
HPLC purification and mass spectrometry
The bacteriocin was purified and the molecular mass determined as follows: 50 μl of stock culture was grown overnight at 37°C in 5 ml of MRS broth. Forty ml of MRS was inoculated at 1% from the overnight culture and incubated for 6-7 hours at 37°C and this was then used to provide a 1% inoculum for 2 L of MRS broth. Following overnight incubation at 37°C, the culture was centrifuged at 14,160 x g for 15 minutes and the supernatant discarded. Cells were mixed with 250 ml of 70% isopropanol, 0.1% trifluroacetic acid (TFA) and stirred for 3 hours at room temperature. Cells were re-centrifuged and the cell-free supernatant assayed for anti-listerial activity. The isopropanol was removed from the cell-free supernatant using a Buchi rotary evaporator (Buchi, Switzerland) and the resulting prep was passed through a 5 g, 20 ml Strata C18-E SPE column (Phenomenex, Cheshire, UK), the column was washed with 20 ml of 30% ethanol and bacteriocin was eluted with 20 ml of 70% isopropanol, 0.1% TFA. Isopropanol was removed from 20 ml of the bacteriocin-containing sample and this was then applied to a C12 Proteo reverse phase HPLC column running a 25-40% acetonitrile, 0.1% TFA gradient over 35 minutes. Two HPLC runs were typically done per 2 L prep. Mass spectrometry was performed on the anti-listerial fractions using an Axima TOF2 MALDI TOF mass spectrometer (Shimadzu Biotech, Manchester, UK). A 0.5-µl aliquot of matrix solution (Sinapinic acid, 10 mg/ml in 50% acetonitrile-0.1 % (v/v) TFA) was deposited onto the target and left for 5 seconds before being removed. The remaining solution was allowed air-dry and the sample solution was deposited onto the pre-coated sample spot. Matrix solution (0.5-µl) was added to the deposited sample and allowed air-dry. The sample was subsequently analysed in positive-ion linear mode. The purified peptide was resuspended in 0.1 M phosphate buffer (pH 6.8) and stored at -20°C. The activity in au/ml was determined by WDA as described previously .
Identification of genes encoding plantaricin 423
N-terminal sequencing of the peptide was performed by Aberdeen Proteomics (Aberdeen University, Aberdeen, UK). Based on amino acid sequence similarity to plantaricin 423, primers designed to the structural gene, plaA, (423A5: AAATACTATGGTAATGGGG & 423A3: CATGGAAAGTGCTAATTA) as described by van Reenan et al.  and primers designed to the whole operon in this study (423F: ATGATGAAAAAAATTGAAAAA & 423R: CTTGATTATGAATTAACCGT) were used for PCR amplifications. DNA was amplified in a Hybaid PCR express unit. The Expand High Fidelity PCR system (Roche Diagnostics Ltd., East Sussex, UK) was used to amplify the products according to the Roche Diagnostics applications manual. The PCR product representing the whole operon was purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The purified product was cloned into the TOPO XL PCR Cloning kit (Invitrogen, Paisley, UK). Clones were sequenced with an automated DNA sequencer (Beckman Coulter Genomics, UK) using the M13-Forward and Reverse priming sites on the pCR-XL-TOPO vector. Restriction enzymes were purchased from New England Biolabs (Hertfordshire, UK) and used according to manufacturer’s instructions. The sequence was annotated using ORF Finder (NCBI) and analysed using BLAST .
Mode of action of plantaricin 423
The mode of action of plantaricin 423 against L. innocua was performed as described by Deraz et al. . Briefly, a 1% inoculum of the culture was grown overnight. The following day the cells were harvested and resuspended in 0.1 M potassium phosphate buffer (pH 7.0). The sample was then divided into test and control and 2560 au/ml of a purified preparation of plantaricin 423 was added to the test. Both samples were incubated at 37°C for 8 hours. Optical density (600 nm) and cell numbers (colony forming units (cfu/)ml) were determined at 2 hour intervals. Each experiment was performed in triplicate and percentage killing was calculated according to the method of Deraz et al.  as follows:
% Killing = [((initial viable cells)-(final viable cells))/(initial viable cells)] x 100.
Minimum inhibitory concentration (MIC) and specific activity of plantaricin 423
The MIC was determined according to the method of Gravesen et al.  and Ramnath et al. . Briefly, 5 μl of a two-fold serial dilution of cell-free supernatant (2560 au/ml plantaricin 423) or purified peptide were spotted onto GM17 plates seeded with 0.25% L. innocua. The MIC was calculated as the minimal concentration that produced a visible zone after 18 hours at 37°C. The experiment was performed in triplicate. The specific activity was experimentally measured by generating a standard curve of bacteriocin concentration in mg/ml versus au/ml. The experiment was performed in triplicate with two different purified preparations of plantaricin 423. In both experiments the R2 value was above the 95% confidence level.
Laboratory-scale cheese manufacture with Lactobacillus plantarum LMG P-26358
Cultures which included L. lactis DPC4268 (cheese starter), L. lactis CSK65 (nisin producer) and Lb. plantarum LMG P-26358 were inoculated into 1 L of whole milk heated to 32°C as follows: Vat 1 = 0.75 % L. lactis DPC4268 (cheese starter); Vat 2 = 0.75 % L. lactis DPC4268, 0.75 % L. lactis CSK65 (nisin producer); Vat 3 = 0.75 % L. lactis DPC4268, 0.75% Lb. plantarum LMG P-26358; Vat 4 = 0.75% L. lactis DPC4268, 0.5% Lb. plantarum LMG P-26358, 0.5% L. lactis CSK65. A streptomycin-resistant derivative of L. innocua (DPC6578) was added to each sample vat at a level of 103 cfu/ml. Thirty min after inoculation Chymax rennet (Hansens, Little Island, Cork, Ireland) was added according to manufacturer’s instructions and the curd was cut at the appropriate time. The temperature was then elevated from 32°C to 38.5°C over a 30 min period. At pH 6.2, the whey was drained and the temperature was reduced to 32°C. When the curd reached pH 5.2, the curd was further drained and pressed into moulds overnight. The cheeses were then incubated at 20°C for 16 hours after which they were vacuum-packed and ripened at 12°C for 4 weeks. L. innocua DPC6578 was enumerated in each cheese on a weekly basis by homogenising 1 g of cheese in 2% sterile trisodium citrate and plating serial dilutions on GM17 agar containing streptomycin (500 μg/ml). Lb. plantarum LMG P-26358 was enumerated by plating on LBS agar. Each cheese trial was performed in triplicate.
Industrial-scale Gouda cheese manufacture with Lactobacillus plantarum LMG P-26358
The starter cultures applied in the manufacture of industrial-scale Gouda cheese were produced by CSK Food Enrichment, The Netherlands. In each vat, L. lactis CSK 976 was used as the main starter culture. The vats were set up as follows: Vat 1 = L. lactis C976, L. lactis biovar diacetylactis C975 (nisin producer); Vat 2 = L. lactis C976, Lb. plantarum LMG P-26358; Vat 3 = L. lactis C976, L. lactis biovar diacetylactis C975, Lb. plantarum LMG P-26358; Vat 4 = L. lactis C976, L. lactis biovar diacetylactis C975, Lb. plantarum LMG P-26358. Vat 4 differed from Vat 3 in that it contained double the amount of Lb. plantarum LMG P-26358. Gouda cheese was manufactured at the pilot plant of Nizo Food Research Ede, The Netherlands. In each experiment 1500 L of milk was used per vat to produce 15 x 10 kg cheese wheels with 51% fat, 41 % moisture and 3% salt content. The milk was bactofugated at 68°C for 13 sec and pasteurized at 73°C for 13 sec, then the cheese milk was cooled down to 32°C. At this point nitrate (35% sol) and calcium choride (33% sol) were added in the amounts of 55 and 70 g, respectively, per 1500 L. The different starter/adjunct were added as frozen pellets that resulted in bacterial counts of 106-108 in fresh, unripened cheese. The amount of calf rennet, Ceska®-Lase 150 IMCU (CSK Food Enrichment, Ede, The Netherlands) added was 20 g per 1500 L cheese milk. After a coagulation time of 35 min, the curd was cut for 20 min and then 40 % of the whey was removed. The remaining lactose was washed out of the curd by adding hot water. The curd was further stirred for 33 min at a constant temperature of 35°C. After whey drainage, the cheese was pressed for 90 min and brined for 72 hours. The pH of the cheeses after one day was 5.2. The cheeses were coated with Ceska®-Coat during 18 weeks of ripening at 13°C. Cheese samples were evaluated for bioactive properties at different stages of ripening (day 1, day 14, weeks 6, 12 and 18).
Microbiological analysis of the cheese
Samples extracted using a borer were collected in sterile bags. After discarding 1 cm of outer surface, the sample (app. 10 g) was homogenized with a 2% sterile trisodium citrate solution at 45°C (1:10 dilution). The homogenized samples were diluted in physiological water, and the plate count technique was used to determine the bacterial counts. Lb. plantarum counts were determined using commercially available MRS agar medium for selective enumeration and total lactococci counts were determined in commercially available M17 medium containing 1% lactose as the carbon source. Colonies were counted after anaerobic incubation for 3 days at 37°C or 30°C for lactobacilli or lactococci, respectively. All determinations were performed in duplicate.
The industrial cheeses were assayed for antimicrobial activity by homogenising 1 g of cheese in 9 mls of MRD to which 104 cfu/ml of L. innocua DPC6578 was added from an overnight culture. A control was also set up which did not contain any cheese. The cheese/Listeria slurry was then incubated at room temperature over 24 hours and Listeria counts were determined at 5 and/or 24 hours by plating serial dilutions on GM17 containing streptomycin (500 μg/ml). A plug of each cheese was also overlaid with L. innocua to determine if plantaricin 423 was active.
Detection of nisin and plantaricin 423 in industrial cheeses by MALDI-TOF mass spectrometry
Cheese samples were subjected to mass spectrometry to detect the presence of bacteriocins. Briefly, 20 ml of 70% isopropanol, 0.1% TFA was added to 1 g of cheese sample and mixed at room temperature for 3-4 hours. Cheese mixtures were centrifuged and 20 ml of 70% isopropanol, 0.1% TFA was added to the supernatant. The isopropanol was removed using rotary evaporation and suspensions were re-centrifuged to spin down particulate matter. The pH of the supernatant was adjusted to 4-5 by adding approximately 30 μl of 7.5 N NaOH and then passed through a 5 g 20 ml Phenomenex Strata C18-E SPE column, columns were washed with 20% ethanol and bacteriocins eluted with 70% isopropanol, 0.1% TFA. The isopropanol was removed from the 70% isopropanol, 0.1% TFA samples before applying to a semi prep C12 Proteo Jupiter RP-HPLC column running a 25-48% acetonitrile, 0.1% TFA gradient over 35 minutes. Eluent was monitored via UV at 214 nm. Fractions were collected at 1 min intervals and nisin- and plantaricin-containing fractions were analysed by MALDI-TOF mass spectrometry as previously described to confirm their presence.
A spray-dried powder of Lb. plantarum LMG P-26358 was generated by growing the strain to 8 L in 20% RSM with 0.5 % yeast extract and 0.2 g/l MnSO4.4H2O. The fermentate was concentrated to 40% total solids in a single-effect falling-film evaporator (Anhydro F1-Lab) before spray-drying. Concentrates were then dehydrated in a pilot-scale Anhydro spray drier (Model Lab 3) at an inlet temperature of 187°C and an air outlet temperature of ~ 85°C. The powder was assayed for viable cells by plating on PCA and LBS, and counts were compared across both sets of plates to determine the cfu/g of Lb. plantarum LMG P-26358. Anti-listerial activity was assessed by adding the powder at 1% (w/v), 5% (w/v), 10% (w/v) and 15% (w/v) to 104 cfu/ml of L. innocua DPC6578 in GM17 broth. The culture/powder mix was incubated for 6 hours at 37°C and samples were removed every 2 hours to enumerate Listeria by selecting on GM17 with streptomycin (500 μg/ml). Each experiment was performed in triplicate.