Mrs medium

Lactobacillus casei CC16 was isolated from the intestine of common carp, and grown in De Man, Rogosa and Sharpe (MRS) medium (Thermo Fisher Scientific, Oxoid, UK) at 30 °C without shaking. The Escherichia coli-Lactobacillus shuttle vector pPG-1 and pPG-2 have been described in our previous study [44 (link)]. The competent cells, Escherichia coli MC1061, were grown in Luria-Bertani (LB) medium for cloning of the plasmids at 37 °C with shaking. Chloramphenicol (Cm) was utilized at a final concentration 10 μg/mL when it was necessary. Aeromonas veronii TH0426 strain was isolated from the farmed yellow catfish Pelteobagrus fulvidraco as described by Kang et al. [45 (link)].

L. paracasei ATCC 334 (formerly referred to as Lactobacillus casei ATCC 334) and a nonpathogenic murine E. coli isolate (13 (link)) were grown at 37°C in aerobic atmosphere in MRS medium and TS medium (Thermo Fisher), respectively. Bacteria in stationary-growth phase were harvested by centrifugation, washed with PBS, and resuspended in cell culture medium and water for cellular and animal experiments, respectively. To identify specific effectors, bacteria were incubated in m-ICcl2 medium for 16 h, and bacterial pellets were collected, subjected to heat treatment (110°C, 30 min), and resuspended. Culture supernatants (CS) were filtered (pore size, 0.22 µm). Medium supernatants resulting from 16-h cell-bacterium cocultures were filtered (pore size, 0.22 µm) and used as conditioned medium (CM).

The bacterial species used in this study were selected to represent a diverse group of intestinal bacteria. Both Gram-positive and Gram-negative bacteria were selected, as well as microorganisms classified as beneficial and potentially pathogenic microflora. Lactobacillus rhamnosus B-445, Enterococcus faecalis JCM 1513, Escherichia coli, Bifidobacterium adolescentis DSM 20083, Bifidobacterium longum DSM 20088, and Enterobacter cloacae PCM 533 were used in this study. L. rhamnosus was cultured in MRS medium (OXOID LTD., Hampshire, UK), E. faecalis and E. cloacae in nutrient broth (BTL, Łódź, Poland), E. coli in LB broth (BTL, Łódź, Poland), and the Bifidobacterium strains were cultivated in modified Garche’s medium containing peptone, 20 g/L; lactose, 10 g/L; sodium acetate, 6 g/L; Na₂HPO₄ × 12 H₂O, 2.5 g/L; yeast extract, 2 g/L; KH₂PO₄, 2 g/L; L-cysteine hydrochloride, 0.4 g/L; MgSO₄ × 7H₂O, 0.12 g/L; pH 6.4. All the test strains were incubated for twenty-four hours at 37 °C.

A growth curve for Lactobacillus plantarum NRRL B-4496 was initially prepared within MRS broth (Oxoid) for approx. 24 h at 32 °C under anaerobic conditions. For fermentation experiments cell cultivation lasted until the late exponential phase of growth (approx. 10 h), followed by centrifugation (10 000×g, 20 min, 4 °C; centrifuge model 5810R; Eppendorf, Mississauga, ON, Canada), and then washing twice with peptone solution. The resulting pellet was used as the inoculum for fermentation. Lactobacillus plantarum was added to a 25% (m/V) PPF solution (400 mL) in an Erlenmeyer flask at a content of 7 log CFU per g PPF, which was then incubated under anaerobic conditions at 32 °C for 11 h. Enumeration of L. plantarum was carried out by plating onto MRS medium (Oxoid) at 37 °C for 48 h under anaerobic conditions. Anaerobic conditions were maintained by placing the experiments within a rectangular jar with Anaerogen anaerobic gas generating kit (Thermo Scientific, Waltham, MA, USA). Aliquots (60 mL) were taken at 0, 1, 5, 9 and 11 h of fermentation and then freeze-dried for 48 h using a freeze dryer (Labconco, Freezone 12, Kansas City, MO, USA). All dried samples were then ground using a coffee grinder (model 80365; Hamilton Beach Custom Grind™, Glen Allen, VA, USA). Fermentation experiments were run in triplicate, yielding three separate fermented PPF for each time point.

Lactobacillus fermentum 222 and L. plantarum 80 were originally isolated from a spontaneous cocoa bean heap fermentation process carried out in Ghana [7 (link)]. The strains were stored at −80 °C in de Man-Rogosa-Sharpe (MRS) medium (Oxoid, Basingstoke, United Kingdom), supplemented with 25 % (v/v) glycerol as a cryoprotectant. To obtain cell pellets, the pure strains were grown overnight in MRS medium at 37 °C, followed by cell harvesting through centrifugation (21,036 × g, 15 min, 4 °C) of 2-ml cultures.

10 g of Mozzarella di Bufala Campana (MBC) samples were diluted in 90 mL sodium citrate solution (2% w/v) and homogenized in a BagMixer400 (Interscience, France). 60 μL of homogenate was inoculated in 50 mL of MRS medium (Oxoid Ltd., England) and incubated at 37°C for 48 h under anaerobic conditions (Anaerocult A, Merck, Germany), to obtain a bacterial titer of about 1 × 10¹⁰ Cfu/mL, corresponding to OD₆₀₀ = 3. Bacterial counts were obtained by serial dilution in quarter-strength Ringer's solution, followed by plating on MRS agar. Plates were incubated at 37°C for 48 h under anaerobic conditions. Independent colonies displaying different morphologies were isolated from the plates, grown as described above, and stored at −80°C in 15% (v/v) glycerol. Lactobacillus rhamnosus GG (LGG, ATCC53103) was used as probiotic control where indicated.