Mrs medium

Lactobacillus paracasei DTA81 (Guerra et al. 2018 (link)) was routinely grown using MRS medium (Sigma, MO) at 37°C for 24 h. For in‐vivo assays, overnight cultures were centrifuged at 5000 g for 5 min, washed two times with sterile PBS (NaCl 8·0 g l⁻¹, KCl 0·2 g l⁻¹, Na₂HPO₄ 1·44 g l⁻¹, KH₂PO₄ 0·24 g l⁻¹, pH 7·4) and resuspended in skim milk (10%) to a final concentration of about 10¹⁰ CFU per ml.

The bacterial culture media for the fermentation were prepared in two ways. The first was to culture L. gasseri and L. plantarum in MRS medium (69966, Sigma-Aldrich, St. Louis, MO, USA) for 48 h at 37 °C and then use both the bacteria and the culture medium (strains with culture medium). The second was to use only the medium from which the bacteria were removed through centrifugation (4 °C, 8935× g, 10 min) after the same 48 h of culturing (only culture medium). For fermentation, 2 g of the strains with the culture medium and only the culture medium were mixed with 20 g of HIO, and then fermented in a shaking incubator (37 °C to 200 rpm). Fermentation was carried out in a glass tube for 0–35 d, and after completion, the oil and layered medium were removed with a micropipette. The HIO after fermentation was evaluated for the content of free glycerol and free fatty acids, which are products of lipid decomposition. Measurements were performed according to the protocols enclosed in the Free Glycerol Kit (ab65337, abcam, Cambridge, UK) and Free Fatty Acid Quantitation Kit (MAK044, Sigma-Aldrich, St. Louis, MO, USA), respectively.

Bifidobacterium infantis BCRC14602, Bifidobacterium adolescentis BCRC14606, Bifidobacterium bifidum BCRC14615, Bifidobacterium longum BCRC14634, Bifidobacterium breve BCRC11846, Lactobacillus rhamnosus GG BCRC16000, Lactobacillus delbrueckii subsp. bulgaricus BCRC10696, Lactobacillus plantarum BCRC11697, Lactobacillus acidophilus BCRC14079, Streptococcus salivarius subsp. thermophiles BCRC14085 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu city, Taiwan). All LAB strains were grown in MRS medium (Sigma-Aldrich, MI, USA). For storage, stock cultures were kept in 20% glycerol at -80°C. Viable cells were grown in MRS medium at 37°C for 20 hours as inoculum and sub-cultured twice a month [16 (link)]. The standard growth curve was measured at 600 nm using a Multiskan GO microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Lactobacillus paracasei BCRC14023, Lactococcus lactis BCRC12315, Lactobacillus helveticus BCRC14092, Lactobacillus rhamnosus BCRC10940, Lactobacillus johnsonii BCRC17474, Lactobacillus brevis BCRC12247, Lactobacillus delbrueckii BCRC12195, Lactobacillus gasseri BCRC14619, Lactobacillus reuteri BCRC14625, Lactobacillus helveticus BCRC12936, Lactobacillus delbrueckii BCRC14009, Bifidobacterium infantisI BCRC14633, Bifidobacterium longum BCRC14602, Bifidobacterium adolescentis BCRC14606, Bifidobacterium bifidum BCRC14615, Bifidobacterium longum BCRC14634, Bifidobacterium breve BCRC11846, Lactobacillus rhamnosus BCRC16000, Lactobacillus delbrueckii BCRC10696, Lactobacillus plantarum BCRC11697, Lactobacillus acidophilus BCRC14079, Streptococcus thermophilus BCRC14085, Lactobacillus casei BCRC10697 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu city, Taiwan) and cultured in MRS medium for routine use (Sigma-Aldrich, St. Louis, MO, USA). Bacteria were stored in MRS broth with 20% glycerol at −80 °C for long-term storage. For the activation of bacteria, all strains were thawed at room temperature and then inoculated 1% of bacteria (v/v) into the MRS broth and cultured at a constant temperature at 37 °C for 48 h and sub-cultured twice a week.

Selective media were used for the determination of intestinal bacteria: MacConkey selective medium (Sigma-Aldrich, Munich, Germany) for Entrobacteriaceae, MRS medium (Sigma-Aldrich, Munich, Germany) for Lactobacillus, agar with kanamycin, esculin and sodium azide (Sigma-Aldrich, Munich, Germany) for Enteroccocus and Garch’s medium (Sigma-Aldrich, Munich, Germany) for Bifidobacterium. The number of cfu/mL was assessed by the direct plating method. For Escherichia coli and Enterococcus faecium, aerobic conditions, and for Lactobacillus plantarum a double flood method, were applied, while for Bifidobacterium bifidum the incubation of the agar plates was performed in an anaerobic atmosphere composed of 20% CO₂ and 80% N₂, at 37 °C for 48 h in a HEPA CLASS 100 Thermo Electron incubator (Thermo Electron Corporation, Marietta, OH, USA).