Butyric acid

The wild-type Candida albicans strain SC5314 (Noble and Johnson 2005 (link)) was used throughout this study. Stock cultures were preserved in 35% glycerol and maintained at −80°. Cells were grown in De Man, Rogosa, and Sharpe (MRS) media (Sigma-Aldrich) at 37° in a shaking incubator at 150−200 rpm. MRS medium was chosen, because it was previously shown to support the growth of both C. albicans (Kohler et al. 2012 (link)) and lactobacilli (De Man et al. 1960 ). Individual acids, i.e., hydrochloric, lactic, acetic, propionic, or butyric acid (from either Merck or Sigma-Aldrich), were added to freshly relaunched overnight cultures diluted down to an optical density (OD) at 600 nm of 0.1. The prototroph sfu1Δ mutant (SN668) and its isogenic control (SN425) were kindly provided by Suzanne Noble (Noble et al. 2010 (link)).

The cements were condensed gently in cylindrical Plexiglas vessels of internal diameter 1.5 mm and length 10 mm corresponding to an exposed surface area of 1.77 mm² and volume of 17.67 mm³ (n = 18 per cement group). Three buffer solutions were prepared: 20 mM glycine (Merck Millipore, Darmstadt, Germany) buffered to a pH of 10.4, 20 mM HEPES (Fluka, Sigma Aldrich, Diegem, Belgium) buffered to a pH of 7.4 and 20 mM butyric acid (Merck Millipore, Darmstadt, Germany) buffered to a pH of 4.4. Cylindrical Plexiglass vessels were randomly allocated to each group of the pH buffer solutions (n = 6 per cement group). Each Plexiglass vessel was placed in 10 mL of respective pH medium in a PE flask immediately after their setting time. The sealed flasks were stored at 37 °C on a shaker. At 3 h, 1, 3 and 7 days, the Plexiglass vessels were moved to new flasks with fresh buffer solution and the liquid in which they were previously kept was subjected to calcium analysis.

Fecal samples (n = 5 per group) were stored frozen at the end of the experiment. A fixed amount was weighed (0.5 g) and suspended in ultra-pure water (2.5 mL) in a 15 mL tube, vortexed briefly, and then a total of 7.5 mL 1.33% HCl/ethanol solution was added. The samples were homogenized for 2 min and centrifuged at 17,968 g for 10 min at room temperature. Supernatant was promptly transferred to a 2 mL sample vial. This step was repeated three times. A quantity of the pooled extract containing acetate, propionate and butyrate were transferred into a 2 mL glass vial and loaded onto an Agilent 7890 gas chromatograph (GC) system with automatic loader/injector (Agilent Technologies, Santa Clara, CA, United States). Pure fatty acid standards (cod. 71251 Acetic Acid, cod. 94425 Propionic Acid, cod. 19215 Butyric Acid Merck Life Science, Milano, Italy) were also prepared to prepare a calibration curve and each sample was analyzed three times on the same day (De Caro et al., 2020 (link)).

Acetic acid (≥99.99 % purity), propionic acid (≥99.5 % purity), isobutyric acid (≥99.5 % purity), butyric acid (>99 % purity), isovaleric acid (>99 % purity), 2-methylbutyric acid (>99 % purity), and valeric acid (≥99.8 % purity) were purchased from Merck (Gillingham, UK). Acetic acid‑d₄ (≥99.5 % purity, >95 % isotopic enrichment) was purchased from Alfa Aesar (Heysham, UK), isobutyric acid-d3 (98.7 % purity, >95 % isotopic enrichment) was purchased from QMX (Thaxted, UK), propionic acid-d3 (98 % purity, 99.6 % isotopic enrichment), butyric acid-d7 (98 % purity, 99.3 % isotopic enrichment), isovaleric acid-d9 (98 % purity, 97.9 % isotopic enrichment), 2-methylbutyric acid-d3 (99 % purity, 99.3 % isotopic enrichment) and valeric acid-d9 (98 % purity, 98.6 % isotopic enrichment) were purchased from Toronto Research Chemicals (Toronto, Canada). Methyl tertbutyl ether (MTBE) (99.9 % purity) was purchased from Acros Organics (Loughborough, UK). LC-MS grade water and hydrochloric acid (1 M) were purchased from VWR Chemicals (Lutterworth, UK). All SCFAs included within the assay are detailed in Fig. 1.Fig. 1

Chemical structure and terminology for the seven straight- and branched-chain short-chain fatty acids (SCFAs) measured within the assay.

Acetic acid (>99.7%), propionic acid (>99.5%), butyric acid (>99%), NH₄Cl, and NaCl were purchased from Sigma-Aldrich (Burlington, MA, US) and KCl of reagent grade was provided from Merck (Darmstadt, Germany). The water used in the preparation of all synthetic solutions was ultrapure (Milli-Q at 25°C).

High purity SCFA standards for gas chromatography (GC) analysis, including acetic acid, propionic acid and butyric acid, were purchased from Sigma Aldrich Chemical Co. (St Louis, MO, USA). l-Cysteine hydrochloride monohydrate, bile salts, tryptone, yeast extract, glucose and other chemicals were obtained from Sangon Biotech (Shanghai, China). Media used in this study included: brain heart infusion (BHI) media (Solarbio, Beijing, China), gut microbiota medium (GMM) without agar (Goodman et al. 2011 (link)), fastidious anaerobe broth (FAB, Solarbio, Beijing, China), bacterial growth media (BGM) (Mcdonald et al. 2013 (link)). All media were prepared following the manufacturer’s instructions.