Nutrient broth

A total of 207 lactic acid bacteria and 195 yeast isolates were obtained from thirteen Greek spontaneously fermented wheat sourdoughs [25 (link)]. The lactic acid bacteria isolates were identified as follows: Lactiplantibacillus plantarum (70 isolates); Levilactobacillus brevis (71 isolates); Companilactobacillus paralimentarius (30 isolates); Lvb. zymae (1 isolate); Latilactobacillus curvatus (6 isolates); Ltb. sakei (12 isolates); Leuconostoc citreum (1 isolate); Ln. mesenteroides (1 isolate); Lactococcus lactis (3 isolates); and Fructilactobacillus sanfranciscensis (12 isolates). The yeast isolates were identified as follows: Saccharomyces cerevisiae (161 isolates); Kazachstania humilis (2 isolates); Pichia fermentans (8 isolates); Pi. membranifaciens (18 isolates); and Wickerhamomyces anomalus (6 isolates). All isolates were stored at −20 °C in a Nutrient broth supplemented with 50% glycerol (Applichem, Darmstadt, Germany). Before experimental use, lactic acid bacteria and yeast isolates were grown twice in de Mann, Rogosa, and Sharpe (MRS) broth, and in Brain Heart Infusion (BHI) broth, and their purity was examined through streaking in MRS agar and BHI agar, respectively. All substrates were from LAB M (Lancashire, UK).

Motility assays were carried out in nutrient semisolid agar plates composed of 8 g/L of nutrient broth (AppliChem, Panreac, Barcelona, Spain), 4 g/L of agar (Liofilchem, Abruzzi TE, Italy), and 0.05 g/L of 2,3,5-triphenyltetrazolium chloride (Sigma Aldrich). Five different types of agar were used: a control agar and four semisolid agars, each supplemented with one SCFA at the highest subinhibitory concentration observed in this study. A saline suspension with a turbidity equivalent to 0.5 McFarland was prepared for each strain from the isolated colonies of nutrient agar plates incubated for 24 h at 37 °C. An inoculating loop was immersed in the Salmonella saline suspension, and then the strains were inoculated in the semisolid agar plates by stabbing. Motility agar plates were incubated at 37 °C for 48 h. After incubation, the ratio between the stabbing point and the end of the growth circle was measured. The experiments were carried out in triplicate.

On day two of the exposure phase, the immune system was challenged by the injection of 1 µl of heat-inactivated Escherichia coli (OD 0.5)^{25 (link),26 (link)}. Hemolymph was collected and stored at −20 °C. Bacterial test plates (ø 9 cm) were prepared by adding 0.8 ml of live Micrococcus luteus bacteria suspension (OD 0.5) to 150 ml of sterile broth medium (48 °C, 1.5 g Agar No. 1, Oxoid; 3.75 g nutrient broth, Applichem). For each test plate, five holes (ø 3.33 mm) were punched into the agar with a 1 ml pipette tip, and 1 µl of hemolymph solution was added to each hole. Plates were incubated at 38 °C overnight, and the diameter of inhibition zones was measured with a digital calliper^{25 (link),26 (link)}.