Vitamin k1

Tween-80 (Kemiou Chemical reagent, Tianjin, China), vitamin K1 (Solarbio Biotechnology, Beijing, China), and protohemin (Meilun Biotech, Dalian, China) were used in anaerobic culture. The culture medium, named General Anaerobic Medium (GAM) (Hopebiol Biotechnology, Qingdao, China), was composed of peptone 5.0 g, proteose peptone 5.0 g, enzymatically digested soybean meal 3.0 g, serum powder 10.0 g, beef extract 2.2 g, yeast extract 2.5 g, liver infusion 1.2 g, soluble starch 5.0 g, dextrose 0.5 g, sodium chloride 3.0 g, monopotassium phosphate 2.5 g, L-tryptophan 0.2 g, L-arginine 1.0 g, sodium thioglycollate 0.3 g, and L-cysteine monohydrochloride 0.3 g, and water was added up to 1000 mL. pH 7.3 was adjusted, sterilized at 121 °C for 20 min, and stored at 4°C for culture. The culture solution (KH₂PO₄ 4.5 g, Na₂HPO₄ 6 g, Tween-80 0.5 g, agar 1 g, added distilled water to 1000 ml) was mixed with GAM 3:7 (V/V) to obtain the transfer solution.

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).

Fn (Fn standard strain ATCC 25586, donated by the University of Louisville, USA) was cultured in a brain–heart infusion medium (Solarbio) containing 5% sterile defibrinated sheep blood (Solarbio), 1% haem chloride (Solarbio), and 0.1% vitamin K1 (Solarbio) at 37 °C in an anaerobic workstation (COY) containing 85% N₂, 10% H₂, and 5% CO₂. The purity of Fn was detected by gram staining and culture on Columbia blood agar plates [12 (link)]. The Fn bacterial solution (OD600 = 1–2) with better viability was selected. When the cell confluence was 60–70%, Fn was added into the cell culture medium at an MOI of 10. Different times of infection (24 or 48 h) were used. A follow-up experiment was performed.