Rcm agar

Propionibacterium acnes ATCC 6919 and Staphylococcus epidermidis ATCC 28319 were kindly provided by Dr. Mayri A. Diaz, Manchester University, UK. S. epidermidis was cultured aerobically on brain heart infusion (BHI) agar (LAB M limited, Lancashire, UK) and incubated at 37 °C for 18 h. We sub-cultured isolated colonies of S. epidermidis in BHI broth and incubated at 37 °C for 18 h aerobically. For P. acnes, we cultured the bacteria anaerobically on reinforced clostridial medium (RCM) agar (Oxoid Limited, Basingstoke, UK) for 48 h at 37 °C and ~5% CO₂ using anaerobic jar and anaerobic atmosphere generation bags (Sigma-Aldrich, St. Louis, MO, USA). We sub-cultured isolated colonies of P. acnes in RCM broth for 48 h at 37 °C under anaerobic conditions.

Bacterial strains used are listed in Table 1. E. coli was grown aerobically at 30 °C or 37 °C in Luria-Bertani (LB) broth or agar supplemented with 25 µg/ml chloramphenicol (LB_Cm25) or 100 µg/ml kanamycin (LB_Km100), respectively. C. pasteurianum was routinely grown at 37 °C in an anaerobic workstation (Don Whitley, Yorkshire, UK) in 2x YTG broth (16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, 5 g/l glucose, pH 6.2) or on RCM agar (Oxoid Ltd) supplemented with 15 µg/ml thiamphenicol (2x YTG_Tm15, RCM_Tm15), 20 µg/ml erythromycin (2x YTG_Em20, RCM_Em20) or 40 µg/ml uracil (2x YTG_Ura40, RCM_Ura40), if required. Selections using 5-fluoroorotic acid (FOA, 600 µg/ml; Sigma-Aldrich, Dorset, UK) and uracil (5 µg/ml) were carried out in modified clostridial basal medium containing 0.5% (w/v) CaCO₃ and 5% (w/v) glucose (CBM-S) (Steiner et al., 2012 (link)) or on standard clostridial basal medium (CBM) agar (O'Brien and Morris, 1971 ). All solidified media contained 1.5% [w/v] agar. Solvent/ acid profiling was undertaken in CGM (Sandoval et al., 2015 (link)), Biebl medium (Biebl, 2001 ) or 2x YT media supplemented with 60 g/l glycerol or glucose. Biebl medium was additionally supplemented with 1 g/l yeast extract for glycerol fermentations Table 2.

Raw milk was collected and stored at 20 °C before use. The volume was divided into aliquots of 20 mL, which were inoculated with 1000, 100, and 10 spores. A negative control was also added consisting of milk without spores. Each sample was treated with 20 mL of Buffer F (EDTA 100 mM (Sigma-Aldrich, St. Louis, MO, USA), sodium citrate 250 mM (Carlo Erba, Cornaredo, Italy), Tris HCl 200 mM (Sigma-Aldrich, St. Louis, MO, USA), Triton X-100 0.01%; final pH 7.5 (Sigma-Aldrich, St. Louis, MO, USA)) and incubated in a water bath at 65 °C for 1 h. After that, samples were vortexed for 10 s and centrifuged at 13,000 rpm for 30 min at 40 °C. The supernatant was discarded, paying attention to avoid the mixing of pellets with the residual fat. The experiment was performed in triplicates. Moreover, an aliquot of each sample was analyzed to check the right number of recovered spores, through the plating of decimal dilutions on RCM agar (Oxoid, Altrincham, UK).