Proteose peptone

For the pyocyanin assay, 3 mL supernatant from a 24 h culture in pyocyanin production broth (PPB; 2% proteose peptone [Oxoid, UK], 1% K₂SO₄, 0.3% MgCl₂⋅6H₂O) was extracted by chloroform, and further re-extracted with 0.2 M HCl, a red layer was obtained. The optical density was determined at 520 nm (Kong et al., 2005 (link)). Elastase activity in the supernatant was processed using the elastin-Congo red (ECR) assay as previously described (Yang et al., 2012 (link)). Briefly, filtered supernatant from a 6 h culture in peptone tryptic soy broth (PTSB; 5% peptone, 0.1% tryptic soy broth) was added to a new tube containing 10 mg of ECR (Sigma, United States), 900 μL 10 mM Tris HCl (pH 7.5), and 1 mM CaCl₂. The tubes were incubated for 4 h at 37°C with agitation. At last, unreacted substrate was removed by centrifugation. The optical density was determined at 495 nm. Five replicates were in each group and all experiments were performed three times independently.

The standard and optimal conditions for the culturomics approach were used, based on previous work performed in our laboratory, notably for research on the human gut microbiota [14 ]. This technique started with pre-incubation: a special liquid media comprising 15 g/l brain heart infusion (Becton, Dickinson and Company, Sparks, MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l Bacto yeast extract (Becton, Dickinson and Company, Sparks, MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l proteose peptone (Oxoid Ltd, Basingstoke, Hampshire, England), 1000 ml of sterile water (Fresenius Kabi France, 5 Place de Marivel, 92310 Sèvres, France) and 5 % (v/v) sheep blood in aerobic and anaerobic conditions at 28 °C for 1 month. We inoculated them on 5 % (v/v) sheep blood agar (bioMérieux, Marcy l’Etoile, France) after performing ten serial dilutions from 1/10 to 1/10^-10, allowing the growth of fastidious bacteria in order to isolate a maximum of bacterial species. This operation was carried out every 5 days from day 1 to day 25 (i.e. D1, D5, D10, D15, D20 and D25). Bacterial colonies were then isolated on 5 % (v/v) sheep blood agar after 24 h, and submitted to mass spectrometry (MALDI-TOF MS) for identification. Bacteria not identified by MALDI-TOF MS were then submitted to molecular biology for taxonomic determination by 16S sequencing.

A 50-mL sample of ruminal fluid was taken from each foetus and maintained under anaerobic conditions (Oxoid AnaeroJar with an AnaeroGen™). A 1-mL aliquot of the ruminal fluid was transferred to anaerobic solid medium and cultured at 37 °C for 48–72 h. The anaerobic solid medium had the following composition (per litre of distilled water): 15 g agar (Oxoid), 10 g peptone (Oxoid), 10 g yeast extract, 8.8 g Oxoid Lab-Lemco beef extract powder, 10 g proteose peptone (Oxoid), 12 g dextrose, 10 g KH₂PO₄, 12 g NaCl, 20 g soluble starch, 1.2 g l-cysteine hydrochloride and 0.3 g sodium thioglycollate with a pH (at 25 °C) of 7.3 ± 0.1. Colonies were isolated and subcultured 5 times onto new agar media plates, except for the control plates (n = 3) which showed no microbial growth. Colonies were subcultured on fresh media and DNA extracted. The extracts for 5, 6 and 7 months were combined prior to next-generation sequencing of the 16S rRNA genes to characterise the taxonomic structure.

Saliva was collected from six healthy adult donors, who had natural dentition but no periodontal disease, had no active caries, and were not taking antibiotics in the past 3 months. Donors were instructed not to brush their teeth for 24 h or intake food or drink for 2 h before saliva donation. Non-stimulating saliva was collected and kept on ice. The saliva from each of the six donors were mixed and diluted in sterile glycerol to a concentration of 70%, then stored at −80°C.
The saliva-glycerol stock was added with 1: 50 final dilutions to SHI media as inoculum (Tian et al., 2010 (link); Li et al., 2017 (link)). The SHI media contain proteose peptone 10 g/l, trypticase peptone (Oxoid) 5.0 g/l, yeast extract (Oxoid) 5.0 g/l, KCl 2.5 g/l, sucrose 5 g/l, hemin 5 mg/l, VitK 1 mg/l, urea 0.06 g/l, arginine 0.174 g/l, mucin (type II, Sigma) 2.5 g/l, sheep blood 5%, and N-acetylmuramic acid (Sigma) 10 mg/l. Sterilized glass disks were placed into a 24-well plate, and 1.5 ml of inoculum was added to each well, incubated under anaerobic conditions (37°C, 5%CO₂) for 8 h. Then, disks were transferred to new 24-well plates with fresh SHI media and incubated for another 16 h, for a total of 24 h.

Overnight-grown L. monocytogenes strains 10403S, EGDe, 850658 and M7 were harvested by centrifugation at 5000 × g for 10 min at 4°C, and then washed once in PBS (10 mM, pH 7.4). The bacterial pellets were re-suspended in BHI broth (pre-adjusted to pH 3.5 by using HA, AA, CA and LA, respectively) and incubated for 60 min at 37°C. Similar experiments were employed for 30 min survival in the synthetic human gastric fluid [8.3 g proteose peptone (Oxoid), 3.5 g D-glucose, 2.05 g NaCl, 0.6 g KH₂PO₄, 0.11 g CaCl₂, 0.37 g KCl, 0.05 g bile salts (Sigma), 0.1 g lysozyme and 13.3 mg pepsin (Sigma), all L⁻¹; adjusted to pH 2.5 with HCl] as described previously (Cotter et al., 2001a (link); Cheng et al., 2013b (link)). The survival bacterial cells were plated onto BHI agar after appropriate dilutions. The plates were incubated at 37°C for 24 h and survival rates are reported as the mean of three independent experiments, each performed in duplicate.

The fermentation medium was composed of the following ingredients for 1 kg of final solution: 40 g of trehalose (Treha™; Tokyo Japan); 10 g of proteose peptone (Oxoid; Waltham, MA, USA); 5 g of yeast extract (Humeau; La-Chapelle-sur-Erdre, France); 5 g of Tween 80 (VWR; Leuven, Belgium); 0.41 g of MgSO₄ (Merck; Darmstadt, Germany); 0.056 g MnSO₄ (Merck; Darmstadt, Germany); and water to reach a total of 1 kg of solution. All medium components were sterilized together at 121 °C for 20 min. Fermentations were carried out on C. maltaromaticum CNCM I-3298 pre-cultures. Pre-cultures were prepared by inoculating 10 mL of sterilized fermentation medium with 100 µL of C. maltaromaticum CNCM I-3298 stock culture and were incubated for 13 to 16 h at 30 °C. An amount of 1 mL of the resulting culture was transferred into 50 mL of fresh medium and then incubated again for 11 h under the same conditions. The resulting culture was then used to inoculate the bioreactor. Inoculation was performed at an initial concentration of approximately 10⁷ CFU mL⁻¹.