L cysteine hydrochloride

Bifidobacterium bifidum G9-1, Akkermansia muciniphila ATCC BAA835 ^T, and Lactobacillus reuteri DSM 20016 ^T were obtained from the Culture Collection of Biofermin R&D Center (Kobe, Japan), ATCC (Manassas, VA, USA) and DSMZ (Braunschweig, Germany), respectively.
Bifidobacterium bifidum G9-1 and Lactobacillus reuteri DSM 20016 T were cultured for 18 h at 37°C in GAM broth (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 0.7% glucose and 0.1% Tween-80. Akkermansia muciniphila ATCC BAA835 was cultured for 18 h at 37°C in BHI broth (Kanto Kagaku Co., Ltd., Tokyo, Japan) supplemented with 0.5% L-cysteine hydrochloride (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan). Before administration, bacteria were washed twice with phosphate-buffered saline (PBS), and the pellets obtained after low-speed centrifugation were stored at −80°C until use.

Glucose, K₂HPO₄, Polysorbate 80 (Tween 80; for biochemistry grade), sodium ascorbate (Wako special grade), l-cysteine hydrochloride, CsCl, H₂O₂, and SrCl₂, were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). These reagents without grade description were of guaranteed grade. Casein peptone and yeast extract were from Nihon Pharmaceutical Co. Ltd. (Osaka, Japan). MRS medium and beef extract were from Becton, Dickinson and Co. (Franklin Lakes, NJ, USA). Bis–Tris was obtained from MP Biomedicals (Santa Ana, CA, USA).