M17 broth

Lactococcus lactis subsp. cremoris strains JM1 and JM2 were cultured in M17 broth (Oxoid) supplemented with 0.5% (w/v) lactose at 30°C without agitation overnight. PFGE plugs were then prepared and restricted with SI nuclease (Thermo Fisher Scientific, Ireland) as previously described (Bottacini et al., 2015 (link)).
A 1% (wt/vol) PFGE agarose gel was prepared in 0.5X TBE [89 mM Tris-borate, 2 mM EDTA (pH 8.3)] buffer and the PFGE plugs were melted in and sealed with molten agarose in 0.5X TBE buffer. A CHEF-DR III pulsed-field system (Bio-Rad Laboratories, Hercules, CA, United States) was used to resolve the DNA fragments at 6 V/cm for 18 h in 0.5X TBE running buffer maintained at 14°C with linear increment (interpolation) of pulse time from 3 to 50 s. DNA ladder (Chef DNA lambda) was included in each gel (number 170-3635; Bio-Rad Laboratories). The gels were stained in ethidium bromide (10 mg/ml) (25 μl/500 ml dH₂O) for 120 min under light-limited conditions and destained in distilled water for 60 min. Gels were visualized by UV transillumination.

A retail-purchased semi-hard cheese made from pasteurized ewe’s milk was used for bacteriophage screening. Different E. faecalis strains (18a, 19a, 23a, 28a, 63c, BA62, HFS 25, HFS59, CECT 4039, and CECT481^T), were used as challenge hosts; the capacity of these strains to produce tyramine and putrescine has been reported (Ladero et al., 2009b (link), 2012b (link)). To determine the host range of bacteriophage 156, additional E. faecalis strains were assayed (Table 1). To ensure that the phage do not infect technological relevant species, L. lactis (12 strains) and Lactobacillus casei (3 strains) strains were also tested (data not shown). All bacterial strains were grown in M17 broth (Oxoid, Spain) supplemented with 0.5% glucose (GM17) without aeration, except those of L. casei that were grown in MRS (Oxoid). In host strain assays, the culture medium was supplemented with 10 mM Mg₂SO₄ and 10 mM Ca(NO₃)₂ (MC-GM17 or MC-MRS). Phage titres were determined in double-layer agar plates, mixing 100 μl of serial dilutions in SM buffer (20 mM Tris–HCl pH 7.5, 1 mM Mg₂SO₄, 100 mM NaCl) with 100 μl of an overnight culture of the appropriate host strain. Plates were incubated at 37°C for 18 h and the resulting plaques counted. Unless otherwise stated, all reagents were purchased from Sigma-Aldrich (Spain).

Chickpeas (Cicer arietinum variety Kabuli), Yellow peas (Pisum sativum), Faba beans (Vicia faba), Red beans (Phaseolus vulgaris variety Kidney), green lentils (Lens culinaris) were generously donated by Pulse Canada (Manitoba, Canada). α-Glucosidase from Saccharomyces cerevisiae (≥100 units/mg protein), dipeptidyl peptidase IV human (recombinant expressed in Sf9 cells), α-amylase (from porcine pancreas, type VI-B, ≥5 units/mg solid), pancreatin (from porcine pancreas, 8xUSP specification), pepsin (from porcine gastric mucosa, ≥250 units/mg solid), Calcoflour white stain (calcofluor white M2R 1 g/L, evans blue, 0.5 g/L), Toluidine blue O, gallic acid, vanillin and catechin were purchased from Millipore Sigma (Burlington, MA, USA). Lactobacillus plantarum ATCC® 8014™ was purchased from Cedarlane (Burlington, ON, Canada). DeMan, Rogosa and Sharpe (MRS) broth, M17 broth and Bacteriological agar were purchased from Oxoid (Nepean, ON, Canada). DC^TM Protein Assay Kit II and Ladder Precision Plus Protein dual color standards were purchased from BioRad (Mississauga, ON, Canada). GelCode blue safe protein stain was purchased from Fisher Scientific (Toronto, ON, Canada).

The accession numbers of the phages examined in this study can be found in Supplementary Table S1. A total of 45 strains of the bacterial species L. lactis were used in this study (Supplementary Table S2). Bacterial strains were cultured overnight at 30 °C in M17 broth (Oxoid, Hampshire, UK) supplemented with either 0.5% w/v lactose (LM17) in the case of strains A-T and 1-20, or 0.5% w/v glucose (GM17) for all other strains.

Bacterial strains used in this study are listed in Tables 3 and 4. All bacterial strains in Table 4 were grown in brain heart infusion (BHI) (Oxoid) at 37°C, except for strains of Lactococcus lactis, which were propagated in M17 broth (Oxoid) supplemented with 0.5% (wt/vol) glucose (GM17) at 30°C, and Escherichia coli NEB 5-alpha (New England BioLabs), which was grown in LB (Oxoid) containing 100 μg/ml ampicillin at 37°C with shaking. All strains used for the determination of the antimicrobial spectrum (Table 3) were grown in BHI at 30°C.

Bacterial strains and phages used in this study are listed in Table 1. Lactococcal strains were grown at 30 °C without agitation in M17 broth (Oxoid Ltd., UK) supplemented with 0.5% glucose. Phage lysates for the various biological characterisation assays were induced from (at least) three separate overnights of NZ9000-Cro_t712 lysogenized with TP901-1erm, or a particular mutant derivative using 0.5 μg/ml mitomycin C when the growing cultures reached an optical density at 600 nm (OD600) of approximately 0.2. Polyethylene glycol 8000 (PEG 8000) precipitation of phage particles (or component parts) of the TP901-1erm phage or a particular mutant derivative, and their subsequent purification, was performed as described previously31 (link).

S. pneumoniae TIGR4 (serotype 4) [28] and Streptococcus anginosus 009–1 (clinical isolate from sputum sample 009–1, this study) were grown as standing cultures in M17 broth (Oxoid) with 0.5% glucose (GM17) at 37°C. S. aureus HG001 [29] was grown in TSB at 37°C under shaking conditions (250 rpm).