L cysteine hydrochloride

Laminarin (Eisenia bicylis source) (Phaeophyceae) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sodium alginate and fucoidan (Undaria pinnatifida source) (Phaeophyceae) were brought from Sigma-Aldrich (Saint Louis, United States). P. haitanensis polysaccharides (PHP) was prepared using a method previously reported by our research team (Qiu et al., 2020 (link); Yu et al., 2023 (link)). In brief, the decolorized and deproteinized P. haitanensis powder was boiled in a water bath at 90°C for 2 h after adding 10 times volume distilled water (w/v). Centrifugation was used to extract the mixture’s supernatant, which was then combined with three liters of 95% ethanol. Following centrifugation for isolation, the precipitate was dissolved in water, frozen in a −20°C freezer, and subjected to freeze-drying to obtain the PHP. Standard dextrans with molecular weight from 4.66 kDa to 496 kDa were purchased from Aladdin (Shanghai, China). _D-glucose, _L-cysteine hydrochloride, hemin, vitamin K₁ were procured from Solarbio Science & Technology (Beijing, China). Brain heart infusion (BHI) was brought from Hopebio (Qingdao, China). All other chemicals of analytical grade were acquired from XiLong Scientific Co., Ltd. (Shantou, China).

The collected GCF samples were vortex shaken and isolated using disposable loops (Biologix, Shandong, China) on 10% brain-heart infusion (BHI) sheep blood plates (Solarbio, Beijing, China) containing 5 μg/mL hemoglobin chloride, 1 μg/mL vitamin K1 (VK1), 0.4 mg/mL L-cysteine hydrochloride and 5 mg/mL yeast extract (Solarbio, Beijing, China). The incubation took place at 37°C with 80% humidity under anaerobic conditions (10% CO2, 10% H2, and 80% N2) within an anaerobic culture vessel (YY-s Plus, LongFuJia Biotechnology, Beijing, China) for a culture period of 7–15 days. Single colonies were meticulously chosen and subjected to purification once more. The resulting single colonies were inoculated with a 5% fetal bovine serum (FBS) BHI liquid medium containing hemoglobin chloride and VK1 to be enriched and stored at −80°C. American Type Culture Collection (ATCC) 51700 P. gulae was purchased as a quality control bacterium.