Soya peptone

Twenty colonies per each medium were selected for further confirmatory tests. MRS medium with vancomycin (pH 5.6) and cultivation under aerobic conditions at 30°C was used for selective enumeration of L. plantarum after cultivation in broth, then the purity of L.plantarum cultures on this medium was monitored by colony morphology, Gram staining, and microscopy (Olympus CX23; Olympus, Tokyo, Japan) (Veselá et al., 2019 (link)).
Pure isolates of B.bifidum were cultivated in Wilkins–Chalgren broth supplemented with soya peptone (5 g/L, Oxoid). Tests were conducted for morphology, Gram staining, and fructose‐6‐phosphate phosphoketolase (specific enzyme for Bifidobacteriaceae family) activity (F6PPK test) in order to confirm the selectivity of the BSM agar for bifidobacteria (Bunesova et al., 2015 (link)).

The standards and reagents used in the HPCE analyses were from Sigma–Aldrich (St. Louis, MO, USA), as well as the glycerol and the salts used for the growth medium, whereas soya peptone was from Oxoid (United Kingdom). The methanol used as a solvent for the biomass extraction was from Carlo Erba (Italy).

Bifidobacterial strains (n = 115) of the species B. adolescentis, B. animalis, B. bifidum, B. breve, B. dentium, B. longum, and B. catenutalum, and B. pseudocatenulatum were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany) or the strain collection of the Department of Microbiology, Nutrition, and Dietetics (CZU, Czechia) (Table 1). Strain identity was confirmed with MALDI-TOF MS (Bruker Daltonik GmbH, Germany) according to Modrackova et al. (2019) [23 (link)]. MALDI-TOF MS failed to distinguish B. catenulatum and B. pseudocatenulatum, and to identify subspecies of B. longum. Subspecies of B. animalis were classified in previous studies [11 (link),24 (link),25 (link)]. Strains were routinely cultured in Wilkins–Chalgren broth (Oxoid, UK) supplemented with soya peptone (5 g L⁻¹, Oxoid), L-cysteine (0.5 g L⁻¹), and Tween 80 (1 mL L⁻¹, both Sigma-Aldrich, USA) (WSP broth) in an oxygen-free carbon dioxide environment at 37 °C for 24 h. Stock cultures were stored at –80 °C in 30% glycerol and were reactivated in WSP broth for 24 h to obtain working cultures. Purity was routinely confirmed by phase-contrast microscopy.

Bifidobacterium strains were routinely cultured at 37 °C in Wilkins-Chalgren Anaerobe Broth (Oxoid, Basel, Switzerland) supplemented with soya peptone (5 g L⁻¹, Oxoid), Tween 80 (1 mL L⁻¹, Sigma-Aldrich, Buchs, Switzerland), and fresh sterile filtered l-cysteine hydrochloride (0.5 g L⁻¹, Sigma-Aldrich). Carbohydrate utilization profile of bifidobacteria was investigated in API 50CHL Medium (10 g L⁻¹ bovine/porcine origin polypeptone, 5 g L⁻¹ yeast extract, 1 mL⁻¹ Tween 80, 2 g L⁻¹ dipotassium phosphate, 5 g L⁻¹ sodium acetate, 2 g L⁻¹ di-ammonium citrate, 0.2 g L⁻¹ magnesium sulphate heptahydrate, 0.05 g L⁻¹ manganese sulphate monohydrate, 0.17 g L⁻¹ bromocresol purple; BioMérieux, Genève, Switzerland). The pH of the API medium was adjusted to 7.5 to obtain a final pH of 7 after autoclaving at 121 °C for 15 min. Carbohydrates (concentration as indicated) were filter sterilized and added after autoclaving. Fresh sterile filtered l-cysteine hydrochloride was always added before cultivation (0.5 g L⁻¹). Glucose, lactose, and L-fucose were obtained from Sigma-Aldrich, 2′-fucosyllactose (2′-FL, Fucα1-2Galβ1-4Glc), 3′-fucosyllactose (3′-FL, Fucα1-3Galβ1-4Glc), 3′-sialyl-lactose (3′-SL, NeuAcα2-3Galβ1-4Glc), 6′-sialyl-lactose (6′-SL, NeuAcα2-6Galβ1-4Glc), Lacto-N-neotetraose LNnT (Galβ1-4GlcNacβ1-3Galβ1-4Glc) were donated by Glycom A/S (Lyngby, Denmark).

Nonomuraea jiangxiensis strain A7611 was cultured in 5 mL SV2 media, (For 1 L, add 15 g glucose (1st BASE, Singapore, Singapore), 15 g glycerol (VWR, Radnor, PA, USA), 15 g soya peptone (Oxoid, Basingstoke, Hampshire, UK), and 1 g calcium carbonate (Sigma-Aldrich, St. Louis, MO, USA), pH was adjusted to 7.0) for 3 days at 28 °C with shaking performed at 200 rpm. Saturated seed cultures were diluted in fresh fermentation media: CA09LB (For 1 L, add 10 g meat extract (Sigma-Aldrich, St. Louis, MO, USA), 4 g yeast extract (BD Biosciences, Franklin Lakes, NJ, USA), 20 g glucose (1st BASE, Singapore, Singapore), and glycerol 3 g (VWR, Radnor, PA, USA), pH was adjusted to 7.0) in a 1:20 volume ratio and fermented with 200 rpm shook at 28 °C in the dark. The cultures were pelleted after 9 days followed by lyophilization of the separated biomass and supernatant. The dried samples were extracted by MeOH then filtered through filter paper (Whatman Grade 4, Maidstone, Kent, UK). MeOH was removed under reduced pressure to give a crude extract of a combined weight of 15.70 g. The crude extract consists of broth extract 84.4% (13.25 g) and biomass extract 15.6% (2.45 g).

Because of the potential for using myxobacteria as biological control agents, seven phytopathogenic prey organisms were chosen, representing a taxonomically diverse set of organisms that infect a variety of economically important plant hosts (Table 2). Gram-negative organisms were grown in LB broth (10 g/L tryptone (Fisher), 10 g/L NaCl, 5 g/L yeast extract) for 18–24 h at 37 °C, 180 rpm. Gram-positive and fungal organisms were grown in tryptone Soy Yeast Extract (17 g/L tryptone, 3 g/L soya peptone (Oxoid), 6 g/L yeast extract, 5 g/L NaCl, 2.5 g/L K₂HPO₄, 2.5 g/L glucose) or YEPS (10 g/L yeast extract, 20 g/L tryptone, 10 g/L saccharose (Fisher)), respectively, for 40 h at 30 °C, 180 rpm. Strains were maintained at 4 °C for up to two weeks on agar plates before re-plating and stored long-term as liquid glycerol stocks at −80 °C.

Bifidobacterium animalis subsp. lactis BB-12 (BB12) was isolated from a commercial preparation Biopron Respiron (Valosun, Trinec, Czechia) on modified Wilkins–Chalgren agar (Oxoid, Basingstoke, UK) supplemented with soya peptone (5 g/L; Oxoid), mupirocin (100 mg/L), and acetic acid (1 mL/L) in anaerobic jars with AnaeroGen sachets (Oxoid) and incubated at 37 °C for 48 h, as we described elsewhere [46 (link)]. Salmonella enterica serovar Typhimurium strain LT2 (S. Typhimurium or LT2) [77 (link)] was from a collection of microorganisms from the Institute of Microbiology of the Czech Academy of Sciences (Novy Hradek, Czechia). It was cultivated on meat-peptone agar slopes (blood agar base; Oxoid) at 37 °C overnight. Then 8 log CFU/mL BB12 and LT2 suspensions in PBS were prepared for application to animals.