Anaerobic jar

Samples of kimchi soup were used to inoculated de Man-Rogosa-Sharpe (MRS) agar plate medium (cat. No.: HB0384, Hope Bio-Technology Co., Ltd) and incubated under aerobic conditions at 37°C for 72 h (31 (link)). A lactic acid bacterium was isolated and designated as L. plantarum VHProbi^® E15 based on polyphasic analysis (32 (link)). C. acnes ATCC 11827 and ATCC 6919 were purchased from American Type Culture Collection, (Manassas, VA, USA). C. acnes ATCC 11827 and ATCC 6919 were maintained on a Reinforced Clostridial Medium (RCM, cat. no.: HB0316, Hope Bio-Technology Co., Ltd) and incubated in an anaerobic jar (Sigma-Aldrich^®) with an oxygen absorber CO₂ generator (AnaeroPack C-1, MGC^®, Japan) at 37°C for 48 h.

Bioprospecting for antimicrobial activity was performed using a modified cross-streak method (51 (link)). Briefly, C. lactis RW3-42 was inoculated from an overnight culture as a single streak with an inoculation loop in the center of an BHI medim agar plate and incubated aerobically for at least 3 days at 30°C. Indicator bacteria (Table 2) were cultivated overnight in 5 mL BHI medium and streaked in a line from the border of the plate toward C. lactis. The plates were then incubated for 24 to 48 h at 30 to 37°C, depending on the indicator bacteria. In the case of C. acnes, incubation was carried out in an anaerobic jar (Merck KGaA, Darmstadt, Germany) containing an AnaeroGen anaerobic incubation system (Thermo Fisher Scientific).

DeMan–Rogosa–Sharpe (MRS) agar was used to grow lactic acid bacteria (Lactobacillus casei ssp. paracasei). A series of dilutions using 0.1% (w/v) peptone water (Merck) were prepared by aseptically removing the diluent of culture until the dilution factor determined in the preliminary test was achieved. Using the spread plate method, 0.1 mL of the sample was transferred onto the agar, and plates were incubated at 37 °C for 48 hrs. Anaerobic conditions were established using an anaerobic jar (Merck) with Anaerocult^® A kit (Merck). Plates containing from 30 to 300 colonies were chosen for enumeration, which was expressed in colony-forming units per milliliter of product (CFU·mL⁻¹). Analyses were performed in triplicate.

Samples of 10 mL were taken from the Cornelian cherry juice at various time intervals (days 1, 7, 14, 21 and 28) during fermentation and cold storage. The samples were blended with 90 mL of sterile ¼ strength Ringer’s solution (Merck, Taufkirchen, Germany) and mixed in a stomacher blender and then subjected to serial decimal dilutions in a ¼ strength Ringer’s solution. L. plantarum ATCC 14917 cells were recorded on acidified MRS agar (Merck, Taufkirchen, Germany) at 37 °C for 72 h, anaerobically (Anaerobic jar, Anerocult C, Merck, Taufkirchen, Germany). All cell counts were expressed as the log of mean colony forming units (CFU) per mL of Cornelian cherry juice. The results are presented as means of three repetitions plus standard deviations.

Normalized cultures of E. faecalis and S. aureus were mixed (with equal CFU) in the ratio of 1:1 for dual-species biofilms and 8 µl of single- or dual-species cultures were inoculated in 200 µl of Tryptone Soy Broth in a 96-well flat transparent plates (Thermo Fisher Scientific, United States) and incubated under static conditions at 37 °C for five days unless otherwise stated. For anoxic experiments, plates were instead incubated in an anaerobic jar (Merck, Singapore) with a gas pack (Becton Dickinson, United States) and incubated at 37 °C for five days. Details on Crystal Violet staining and CFU determination is available in Supplementary Information. For biofilm oxygen consumption rate (OCR) assays, 5-day biofilms were grown directly in Seahorse XFe96 FluxPak 96-well plates, with 80 µl of inoculated media added per well. Planktonic cells were removed with three washes of 80 µl PBS using a 96-channel pipettor to prevent cross-contamination and the remaining biofilms were resuspended in 80 µl of PBS. After three baseline measurements were taken, 30 µl of fresh TSB was injected into the wells and OCR measured for 1 h using standard parameters.

In July 2018, fresh individual faecal samples from 5 subjects, 3 (2 adults and 1 young) housed under semi-natural conditions in Parco Natura Viva-Garda Zoological Park (Bussolengo, Verona, Italy) and 2 (adults) in Bioparco (Roma, Italy) were collected from the ground using a sterile spoon, put into a sterile plastic tube, and stored under anaerobic conditions in an anaerobic jar (Merck) at 4 °C. The anaerobic atmosphere was obtained using the GasPak EZ Anaerobic Pouch system (BD).
Samples of faeces were collected by the animal-care staff (keepers) during their routine cleaning of the enclosure and were taken promptly to the laboratory (within 2 h). Animals were free from intestinal infections and did not receive antibiotics or probiotics for two months before samples were collected.
The two diets consisted mostly of grass, fruits and vegetables, and protein-based feed. (For details, please see Supplementary Table S1).