Casein hydrolysate

The following yeast strains were used in this study: BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0); BY4742 Δhap4 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; hap4:: kanMX4); BY4742 Δhxk2 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; Hxk2:: kan MX4); Candida utilis (CBS621) and Saccharomyces cerevisiae (yeast foam). Strains Δhxk2-Hap4p↑ and Δhap4-Hap4p↑ are BY4742 strains previously described (14 (link)).
Cells were grown aerobically at 28°C in the following medium: 0.175% yeast nitrogen base (Difco), 0.2% casein hydrolysate (Merck), 0.5% (NH₄)₂SO₄, 0.1% KH₂PO₄, 2% lactate (w/v) (Prolabo), pH 5.5, 20 mg.L⁻¹ L-tryptophan (Sigma), 40 mg.L⁻¹ adenine hydrochloride (Sigma) and 20 mg.L⁻¹ uracil (Sigma). When cells carried a plasmid, uracil was omitted in the growth medium [pTET-HAP4 (20 (link), 21 (link))]. Where indicated, glucose (60 mM) was added to the medium. Growth was measured at 600 nm in a Safas spectrophotometer (Monaco) in a 1 mL cuvette. Dry weight determinations were performed on samples of cells harvested throughout the growth period, washed twice in distilled water and weighed after extensive drying.

The cell suspension line of V. vinifera cv. Gamay Fréaux was a gift of Dr Francosis Cormier’s group¹⁴ it has been maintained at the Fondazione Edmund Mach in San Michele all’Adige, Italy. The cell culture was cultivated on B5 medium (Gamborg B5 Medium B5VIT, Duchefa B.V., The Netherlands) supplemented with 30 g L⁻¹ sucrose, 250 g L⁻¹ casein hydrolysate (Merck, Darmstadt), 0.1 mg L⁻¹ α-naphthaleneacetic acid (NAA) and 0.2 mg L⁻¹ kinetin (K) and 0.8% agar. Callus cultures were transferred every 28 days onto fresh solid sterile medium. The most red pigmented cell aggregates were selected for growth in liquid suspension cultures, obtained by transferring cell aggregates into 50 mL of liquid B5 medium in 250 mL Erlenmeyer flasks, continuously agitated on a rotary shaker at 110 r.p.m. at 25±2 °C under continuous light. The initial pH of the medium was adjusted to 5.5 with 0.1 m KOH before autoclaving. Cells were subcultured every 7 days with an inoculum dilution of 1:3.

The preparation of BM169 refers to studies of S. mansoni [12 (link), 13 (link)], using a liquid BME (Basal Medium Eagle) (Gibco) as a substitute for the powder BME in this study. We produced AB169 by adding 200 μM ascorbic acid (Sigma-Aldrich) and 0.2% V/V bovine washed red bloods cells (10% suspension; Hongquan Bio, Guangzhou, China) into BM169. AB169 (1640) was made by substituting the BME with RPMI-1640 (Gibco) in AB169. M-AB169 (1640) was obtained by replacing half the amount of the lactalbumin hydrolysate (Sigma-Aldrich) in AB169 (1640) with casein hydrolysate (Merck Millipore). M-BM169 (1640) was formulated by replacing BME with RPMI-1640 and incorporating casein hydrolysate in place of half of the lactalbumin hydrolysate present in the original BM169. Detailed compositions of the media are provided in Additional file 7: Table S1.

All experiments were done with V. harveyi strain ATCC 14126^T. It was routinely grown in artificial seawater (ASW) medium prepared by dissolving Instant Ocean^® sea salt (Instant Ocean Spectrum Brands, Blacksburg, VA, USA) in distilled water to obtain a final salinity of 35 g/L and supplemented with 0.1 M HEPES (Thermo Fischer Scientific Inc., Madrid, Spain) and 0.4% casein hydrolysate (Sigma-Aldrich, Madrid, Spain) to ensure the balanced growth of bacterial cultures. The pH of ASW medium was always adjusted to 7.0, 7.5, 8.0, 8.3 or 8.5 with NaOH or HCl, respectively, and then it was sterilized by filtration using TPP “rapid” 500 filtration unit (TPP Techno Plastic Products AG, Trasadingen, Switzerland). Analysis of V. harveyi growth and survival assays were performed in triplicate in 250 mL Erlenmeyer flasks beforehand cleaned with H₂SO₄ (97%, v/v), rinsed with deionized water and heated at 250 °C for 24 h to avoid any presence of residual organic substances.

The ability of the bacterial isolates to form biofilm in vitro on an abiotic surface was determined using a sterile 96-well flat bottom polystyrene plate as previously described (4 (link)). A Staphylococcus epidermidis strain was used as positive control and TSB as negative control. The effect of adding sugars as sucrose (5%) and lactose (5%) and milk compounds as skim milk powder (5%) (Oxoid^TM) and casein hydrolysate (3 mg/ml) was determined. All the additives and casein hydrolysate were purchased at Sigma, St. Louis, MO, USA.

A Trichoderma atroviride inoculum was plated on the center of a PDYC (24 g l⁻¹ potato dextrose broth (Difco, UK), 1.2 g l⁻¹ casein hydrolysate (Sigma-Aldrich), 2 g l⁻¹ yeast extract (Difco) agar plate and incubated for twenty-four hours in dark conditions. 3 ml of PDYC media were subsequently added to a new 10 cm culture plate. In the center of the plate a Whatman 50 filter paper cut to a diameter of 9 cm was placed over an 8 cm Whatman 1 filter paper. On top of these, a 0.5 cm square from the periphery of the fungal growth on the agar plate was placed in the center and incubated in the dark at room temperature for 48 h.