Anaeropack system

The following four strains of B. longum used in in vitro assimilation tests were obtained from stock cultures maintained at the Morinaga Milk Industry Co., Ltd., Zama, Japan: MCC00300, MCC00055, MCC00198, and MCC00231. B. longum subsp. longum JCM7052 and JCM1217 were obtained from the Japan Collection of Microorganisms (Riken Bioresource Center, Ibaraki, Japan). The strains were precultured at 37°C under anaerobic conditions in MRS broth containing 0.05% l-cysteine hydrochloride using an AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan). The precultured cells were inoculated into MRS medium containing 1.0% gum arabic AGP or α-d-Galp-(1→3)-l-Ara-free gum arabic AGP as the sole carbon source and then cultured for 48 h at 37°C under anaerobic conditions. The absorbance at 600 nm of each culture medium was measured after 8, 12, 23, and 48 h. The experiment was performed in duplicates. To analyze the residual l-arabinose on α-d-Galp-(1→3)-l-Ara-free gum arabic AGP after culture, the polysaccharide fraction was separated using ethanol precipitation. The precipitate was dissolved in water and then incubated with 0.021 μM BlArafE in 40 μL of 50 mM sodium acetate buffer at 37°C for 16 h. The reaction products were analyzed by TLC, as mentioned above.

Probiotic strains used were obtained from commercial sources and are described in Table 1. Bacillus subtilis strain HU58 was cultivated aerobically at 37°C for 24 h on Miller LB agar plates (Miller, 1972 ) containing (per L): Tryptone (10 g), Yeast Extract (5 g), NaCl (5 g), and agar (15 g). Anaerobic bacteria Bifidobacterium longum strain BB536 and Lactobacillus acidophilus strain DDS-1 were cultivated on plates made from MRS broth (BD Difco) supplemented with 0.05% (final concentration) L-cysteine and agar (15 g/L). Anaerobic strains were cultivated at 37°C for 48 h in the Anaeropack system (Mitsubishi Gas Chemical America, New York, NY, United States).

We created HG/Hypo conditions by adding 33.3 mM glucose to the endothelial cell medium with 1% FBS, and the Anaeropack system (Cat# D-07, Mitsubishi Gas Chemical Co, Japan) was used to create a hypoxia condition. Next, HUVECs in a complete endothelial cell medium with 5.5 mM glucose without the Anaeropack system were cultured as the control group (normal glucose, NG). After HUVECs were cultured for 2 days under NG and HG/Hypo conditions, MoS₂ NDs (50 µg/mL) or PBS were added to the culture medium for 12 h for subsequent experiments. HUVECs were incubated with Baf A1 (1 nM) (Cat# HY-100558, MedChemExpress, USA), 3-MA (Cat#HY-19132, MedChemExpress, USA) and SQ22536 (250 µM) (Cat# S8283, Selleck, USA) to inhibit autophagy and cAMP level, respectively.

Conidia of each strain were inoculated onto GMM agar plates and incubated for 72 h at 37 °C in an anaeropack system (Mitsubishi Gas Chemical, Tokyo, Japan). In this system, the concentration of oxygen was controlled within the range of approximately 1–3% (hypoxia). Control plates were incubated at 37 °C under standard oxygen conditions (21% O₂) for 72 h.

Isolation, characterization, and identification (based on 16SrRNA-specific PCR) of P. histicola has been described previously (5 (link)). Briefly, P. histicola was grown at 37°C for 3 days in trypticase soy broth (TSB) (Hardy Diagnostics Santa Maria, USA) in an anaerobic jar with an AnaeroPack system (Mitsubishi Gas Chemical America) (5 (link)).