Oxoid anaerogen

To obtain spores, Gapes agar plates were inoculated with 0.2 mL of C. beijerinckii cultures and plates were stored in an anaerobic jar. An anaerobic generator (Oxoid AnaeroGen, Thermo Scientific) and anaerobic indicator (Oxoid Resazurin, Thermo Scientific) were placed inside the jar to maintain and check for anaerobic conditions. The plates were incubated at 37 °C for 3 weeks. The spores were harvested by adding 4–5 mL sterile physiological water (0.9 % w/v NaCl) and scraping the plates. The spore suspensions were collected, glycerol (20 %, v/v) was added, and spores were stored at −20 or −80 °C for longer-term storage.
Spore activation was done by heat-shocking the clostridial spore suspensions in a boiling water bath for 1 min. For the toxicity tests, successfully sporulating spore suspensions were inoculated (0.6–1.0 % v/v depending on the amount of spores) into 50-mL fresh mCGM medium. The cultures were grown overnight at 37 °C without shaking (OD600 = 2; cells = rod-shaped and very motile). The OD600 was measured using Ultrospec 2000 (Pharmacia Biotech). A droplet of the preculture (5 μL) was dropped onto freshly poured mCGM agar plates containing 30 to 50 g/L isopropanol.

Stomach, ileum, and colon contents were collected from commercial male pigs in a slaughterhouse located in the province of Gelderland, the Netherlands. Immediately after the pigs were slaughtered, contents from each pig's gut location were collected and immediately stored under anoxic conditions, placing the gut content in sterile serum bottles previously flushed with N₂. After the bottles were sealed with a butylrubber stopper and aluminum crimp seals, a sterile needle coupled to a filter (0.2 μm) was inserted into the rubber stopper, and bottles were placed into an anaerobic jar containing anoxic gas generating sachets (Thermo Scientific™ Oxoid AnaeroGen). The collected samples were rapidly transferred to the laboratory, within 2 h at most. Once in the laboratory, the material from each gut location was weighted and samples from 3 pigs were pooled for each location inside of an anaerobic tent filled with a gas mixture of 96% N₂/4% H₂. Stomach, ileum, and colon inocula were separately prepared from each of the generated pools with a pre-warming step (39°C), anaerobic (N₂/CO₂) and sterile saline solution (0.9% v/v NaCl) in a ratio of 1:1, 1:10 and 1:10 (w/w), respectively. Inocula were prepared right before the experiment began to be used as fresh as possible.

The samples for culture-dependent quantitative microbiology analyses of the number of total culturable microorganisms (TCM) and the lactic acid bacteria (LAB) were prepared following the ISO 6887 standard. Leaven samples were taken after 48, 96, 144, 240, and 336 h of the fermentation process. The quantification of microorganisms was performed by spreading the 10-fold dilutions of the sample in growth medium (Bouillon agar (BTL, Warsaw, Poland) and MRS medium (BTL, Warsaw, Poland) for TCM and LAB, respectively). The colonies were counted according to ISO 7218 standard after 48 h of incubation at 37 °C. Plates for LAB enumeration were incubated under anaerobic conditions (AnaeroPack supplied with Oxoid™ AnaeroGen™, ThermoFisher, Waltham, MA, USA). All results are expressed as CFU/mL of red beet leaven.