Ain 93m

Four-week-old male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Kanagawa, Japan). The animals were housed individually in wire-mesh cages kept in an air-conditioned room with a 12 h light-dark cycle (lighting from 7:00 a.m. to 7:00 p.m.) at a temperature of 23 ± 1 °C, a relative humidity of 55 ± 5%, and a ventilation rate of about 15 times per hour. The rats were quarantined for 1 week and were allowed free access to a commercial pelleted diet (AIN-93M, Oriental Yeast Co., Ltd., Tokyo, Japan) or Se-deficient diet (AIN-93M base, Oriental Yeast Co., Ltd.). The normal diet and Se-deficient diets used in this study contained 0.22 and 0.06 mg/kg of Se, respectively. Purified water supplied by an ultra-pure water supply system (Millipore Corporation, Darmstadt, Germany) or BSO-containing purified water were given for drinking ad libitum using water bottles.
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, JAPAN TOBACCO INC. (approval code 17084 (approved on 26 July 2017) and approval code 18091(approved on 24 July 2018)). This study was conducted in accordance with the Japanese Law for the Humane Treatment and Management of Animals (Law No. 105, as revised in 2013, issued in 1 October 1973).

Eighteen-week-old SAMP8 mice were divided into seven groups: standard diet–fed group (AIN-93M, Oriental Yeast Co. Ltd., Tokyo, Japan) (control), 5% (w/w) BWF-, BOF-, TWF-, WF-, and RF-containing diet–fed groups, and 0.05% (w/w) rutin-containing diet–fed group (n = 10 in each group), as shown in Table 2. The contents of protein, lipid, carbohydrate, and dietary fiber provided by BWF, BOF, TWF, WF, or RF were calculated to balance the ingredients provided in AIN-93M diet. SAMR1 mice (n = 8) were used as the normal aging control and fed the AIN-93M diet. Mice were allowed free access to food and tap water. Food intake and body weight were recorded every week. At 42–43 weeks of age, all mice were subjected to the Barnes maze and passive avoidance tests to assess their cognitive performance. All mice were euthanized at 44 weeks of age after the last memory test, and the brain and cecal contents were collected. For Western blotting, the hippocampal tissues of mice brain were frozen in liquid nitrogen. For immunostaining, the brain tissues were fixed in paraformaldehyde and subsequently embedded in paraffin.

BALB/c and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan) and maintained under specific pathogen‐free conditions in the animal facility of Kitasato University. The mice were housed in individual cages under a 12‐h/12‐h light‐dark cycle at 23 ± 5°C throughout the experimental period. The mice were divided into two groups (control and collagen) and fed a diet based on AIN‐93M (powdered form; Oriental Yeast; Tokyo, Japan), which contained only casein as a protein source. The control feed contained 18% casein, and the collagen‐peptide feed contained 14% casein and 3.08% collagen peptides, substituting on an equal nitrogen basis. To equalize protein administration among the animals, food intake was moderately restricted to approximately 90% of free‐feeding weight. Mice (age 4 weeks) were fed 3.0 g/day on days 1–3, 3.5 g/day on days 4–7, 4.0 g/day on days 8–17, and 4.5 g/day after day 18. Water was provided ad libitum. Mice with free access to a standard diet and water were used in the experiments of in vitro T‐cell differentiation and signal transduction. All animal protocols were approved by the Animal Care and Use Committee of Kitasato University (No. 1711).

Wild-type 6-week-old male mice from the BALB/c background (Tokyo Laboratory Animals Science Co., Japan) were used. All mice were housed in a 12:12 light:darkness cycle (Zeitgeber time (ZT) 0 = 8 AM, ZT 12 = 8 PM). Before feeding experiments, the mice were fed AIN93M (Oriental Yeast Co., Japan) for 1 week. After that, the mice were fed inulin- or cellulose-supplemented diets for two weeks in the morning or evening. To prepare the fiber-supplemented feed, AIN93M powder was mixed with 5% inulin or 5% cellulose, and then pellets were formed with tap water. Fecal samples were collected for 16S rRNA gene sequencing and single-cell sequencing, and cecum samples were harvested before and after the 2-week fiber feeding for metabolomic analysis. All samples were stored at − 80 °C.

The Si-based agent was produced from Si powder (Osaka Titanium technologies Co., Ltd. Si 4N Powder, < 300 µm). The powder was sifted and Si powder of sizes less than 45 µm was obtained. Then, Si nanopowder was produced by use of the beads milling method, as previously described^{12 (link)}. We prepared custom diets: 2.5% Si-based agent containing AIN93M, and only AIN93M as the control (Oriental Yeast Co., Ltd., Tokyo, Japan). Each diet was administered one week before the start of DSS administration.

AIN-93 M (Oriental Yeast Co., Ltd., Tokyo, Japan) was used as the normal diet. A special diet containing 1.0 wt% Si-based agent in AIN-93 M was made as described previously^{16 (link),20 (link)–22 (link)}. The Si-based agent reacts with water to continuously generate a high amount of hydrogen in the alkaline intestinal environment. The diet also contains pH-adjusting agents, mainly citric acid, which prevent the Si-based agent from reacting with air moisture during feeding.