Bhi broth

Four bacterial species (one strain/species) were used as the target microorganisms in the antimicrobial assays. Their codes, isolation origins, and some other relevant information are presented in Table 1. Two of them were Gram-positive (S. aureus, L. monocytogenes), whereas the other two were Gram-negative (S. enterica, Y. enterocolitica). Before their use in the assays, they were stored deep-frozen at −80 °C in a Brain Heart Infusion (BHI) broth (Lab M, Heywood, Lancashire, UK) containing 15% (v/v) glycerol. When needed for the experiments, each strain was resuscitated through its streaking on the surface of Tryptone Soy Agar (TSA; Lab M) and its incubation at 37 °C for 24 h (preculture). Working cultures were prepared by inoculating a discrete colony from each preculture into 10 mL of fresh Tryptone Soy Broth (TSB; Lab M) and then incubating at 37 °C for 24 h. The purity of each working culture was always confirmed through its streaking on TSA and incubation under the same conditions.

The bacterial strains that were used in this study were S. enterica str. FMCC_B137, which was kindly provided by Prof. George-John Nychas (Agricultural University of Athens, Athens, Greece), and L. monocytogenes str. AAL 20107, which was kindly supplied by Dr. Nikolaos Andritsos (Athens Analysis Laboratories S.A., Microbiology Laboratory, Metamorfosi, Greece). The strain of S. enterica belongs to serovar Typhimurium; it is an epidemic strain of phage type DT193 and was originally isolated from a human salmonellosis outbreak [25 (link)]. The strain of L. monocytogenes belongs to serovar 1/2b and was originally isolated from a mixed green salad [26 (link)]. Before use, both strains were maintained frozen at −80 °C in BHI broth (Lab M, Heywood, Lancashire, UK) containing 15% v/v glycerol. When needed, each strain was streaked on Tryptone Soya Agar (TSA; Oxoid, Thermo Fisher Specialty Diagnostics Ltd., Hampshire, UK) and incubated at 37 °C for 24 h (preculture). Working cultures were prepared by inoculating a single colony from each preculture into 10 mL of fresh TSB (Condalab, Torrejón de Ardoz, Madrid, Spain) and then incubating at 37 °C for 18 h. The final concentration of each working culture was ca. 10⁹ CFU/mL and its purity was always confirmed by streaking on TSA and incubating it at 37 °C for 24 h.

Two virulent L. monocytogenes strains of cheese origin, ISS G79 (serotype 1/2b) and ISS G185 (serotype 1/2a) plus L. monocytogenes no.10 (serotype 4ab), an avirulent reference strain, were used in accordance with previous studies [44] ,[46] ,[49] (link). Frozen (−30 °C) stock cultures of each strain in tryptic soy broth with 0.6% yeast extract (Lab M, Heywood, UK) plus 20% glycerol were activated twice (30 °C, 24 h) in 10 mL of brain heart infusion (BHI) broth (Lab M) before use. For inoculation purposes, fresh (24-h) 10-mL BHI cultures of the strains were centrifuged (3,200 × g for 15 min), the cells were washed with 10 mL of quarter-strength Ringer solution (Lab M), resuspended in the same diluent, and suspensions were combined as before [11] ,[44] ,[51] . Afterward the three-strain cocktail of L. monocytogenes was diluted to yield an inoculum in milk of approximately 10³ CFU/mL [48] .

The bacterial strains used in this work belonged to the species S. enterica, L. monocytogenes, and Y. enterocolitica (one strain/species). Some critical information on these strains is provided in Table 4. Before their experimental use, these were stored long-term at −80 °C in BHI broth (Lab M, Heywood, Lancashire, UK) containing 15% v/v glycerol. When needed for the assays, each strain was streaked on the surface of Tryptone Soya Agar (TSA; Oxoid, Thermo Fisher Specialty Diagnostics Ltd., Hampshire, UK) and incubated at 37 °C for 24 h (preculture). Working cultures were prepared by inoculating an individual colony from each preculture into 10 mL of fresh TSB (Condalab, Torrejón de Ardoz, Madrid, Spain) and then incubating at 37 °C for 24 h (thereby achieving a final concentration of ca. 10⁹ CFU/mL). The purity of each working culture was always confirmed by streaking a small volume of it (10–20 μL) on TSA and incubating it at 37 °C for 24–48 h.