K2hpo4

Ascorbic acid (ASA), 2,6-dichloroindophenol sodium salt hydrate, chloranil, catechin hydrate, vanillin, Folin-Ciocalteu reagent, hydrocortisone, penicillin, streptomycin, gentamicin, Gallic acid, 2,2′-Azobis-amidinopropane (ABAP) and dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). NaOH, KH₂PO₄, KOH, AlCl₃, acetic acid, NaHCO₃, and K₂HPO₄ were bought from Sangon (Shanghai, China). Chlorogenic acid, Caffeic acid, 4-Coumaric acid, Ferulic acid and benzoic aicd, NaBH₄ were bought from Aladdin Co. (Shanghai, China). WME medium, Hank's balanced salt solution (HBSS), epidermal growth factor, heparin, insulin, and other cell culture reagents were purchased from Gibco Biotechnology Co. All reagents used were of analytical grade.

First, 6.96 mg of potassium hydrogen phosphate (K₂HPO₄: Sangon, Biotech, Shanghai China) and 11.76 mg of calcium chloride dehydrate(CaCl₂ 2H₂O: Sangon, Biotech, Shanghai China) were added to duplicate 10 ml CMC-ALN or CMC solutions (5 mg/ml) under stirring (500 rpm) for 10 min, respectively. Next, the solution containing CaCl₂ was added dropwise to the solution containing K₂HPO₄ under stirring (500 rpm) for 10 min to form nanocomplexes of CMC-ALN/ACP. The final concentrations of calcium and phosphate in the solution were 4 mM and 2 mM, respectively. The solution of nanocomplexes of CMC-ALN/ACP was stored in a refrigerator at 4 °C.
Prior to the remineralization treatment, 1 ml of 5% (w/v) sodium hypochlorite (NaClO) was added dropwise to a 20 ml solution of nanocomplexes of CMC-ALN/ACP or CMC/ACP under stirring (500 rpm) for 10 min to prepare HAP@ACP core-shell nanoparticles via the partial degradation of CMC-ALN or CMC. Then, 22.5 mg of Gly was added dropwise to a 20 ml solution of HAP@ACP core-shell nanoparticles to guide the nanoparticles. After 30 min, the HAP@ACP and HAP@ACP/Gly core-shell nanoparticles were characterized using TEM.

L. edodes (NACC6250, from the Jiangsu Agricultural Microbial Germplasm Resources Collection of China) was used as the wild-type (WT) strain. The WT, CK (empty-vector controls), and LEGGT-silenced, LECSL-silenced L. edodes strains were cultured at 25 °C in a solid complete yeast medium (CYM). The formulation of the CYM medium was based on the method of Zhang et al. [17 (link)]: 1% maltose (biochemical grade, Sangon, Shanghai, China), 2% glucose (biotech. grade, Sangon, Shanghai, China), 0.2% yeast extract (for microbiological grade, Sangon, Shanghai, China), 0.2% peptone (for microbiological grade, Sangon, Shanghai, China), 0.05% MgSO₄·7H₂O (biochemical grade, Sangon, Shanghai, China), and K₂HPO₄ (biochemical grade, Sangon, Shanghai, China).
After culturing for 10 days in a constant-temperature incubator at 25 °C, ten pieces of mycelial blocks were aseptically picked and used to inoculate 100 mL of CYM, then incubated in an oscillator at 25 °C and 100 rpm/min for 7 days. Subsequently, the mycelial blocks were evenly broken in aseptic conditions, and 10 mL of broken mycelium was pipetted into 100 mL of CYM liquid medium. The culture was continued for 25 days in an oscillator at 25 °C and 100 rpm/min.

P. igniarius (NACC6220) was provided by the Jiangsu Agricultural Microbial Germplasm Resources Collection of China. The fermentation experiments were performed in 250 mL flasks containing 100 mL of complete yeast medium (CYM) broth (1% maltose (biochemical grade, Sangon, Shanghai, China), 2% glucose (biotech grade, Sangon, Shanghai, China), 0.2% yeast extract (microbiological grade, Sangon, Shanghai, China), 0.2% peptone (microbiological grade, Sangon, Shanghai, China), 0.05% MgSO₄·7H₂O (biochemical grade, Sangon, Shanghai, China), and K₂HPO₄ (biochemical grade, Sangon, Shanghai, China) at 28 °C and 150 r·min⁻¹ for 8 days.

Enterotoxigenic B. fragilis (ETBF, ATCC 43860) and nontoxigenic B. fragilis (NTBF, ATCC 25285) were kindly gifted by Prof. Jing-Yuan Fang (Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China). ETBF and NTBF were cultured in brain heart infusion broth (CM1135, OXOID) supplemented with Yeast Extract (LP0021B, OXOID), K₂HPO₄ (A100705-0500, Sangon), Resazurin (R7017, Sigma), l-cysteine (C1276, Sigma), Hemin (51280, Sigma), and Vitamin K1 (A606528, Sangon Biotech) at 37°C under anaerobic condition (DG250, Don Whitley Scientific, West Yorkshire, UK).