Sialic acid assay kit

Determination of sialic acid content was performed as described previously [37 (link)], with slight modifications. Briefly, the single colonies (05ZYH33 and ΔhtpsA) were picked from Colombia sheep agar plates, inoculated into THB liquid medium (containing 10% fetal bovine serum), and cultured to OD₆₀₀ = 0.3 at 37°C. The pellets were harvested by centrifugation at 8, 000 × g for 5 minutes. After washing with PBS, the resuspended pellet was centrifuged again at 8, 000 × g for 5 minutes and resuspended in 600 μL of PBS, of which 500 μL of the suspensions was disrupted by sonication. The supernatant was collected by centrifugation at 15, 000 × g for 15 minutes at 4°C. After adding 10 μL of sialidase (0.25 U), the supernatants were hydrolyzed for 1 hour at 37°C. The sialic acid concentration was detected using a Sialic Acid Assay Kit (Jian Cheng, Nanjing, China), according to the manufacturer’s instructions. The following formula was used for calculating the sialic acid concentration:
SAcontent(mg/1010cfu)=ODsample−ODblankODstandard−ODblank×Cstandard×Vstandard×MWSA×1010cfusampleNote: SA represents sialic acid; OD represents optical density value at 560 nm wavelength; C represents concentration of standard mmol/L; V represents volume of standard; MW represents molecular weight of sialic acid, 309.3 g/mol.

CPS was extracted as described previously (20 (link)). Briefly, bacteria were grown to mid-log phase in 30 mL THB at 37°C. The cell densities were measured based on OD₆₀₀. Then, the cells were pelleted by centrifugation at 3,500 × g for 15 min, washed twice with PBS, suspended in 0.8 M NaOH, and incubated for 48 h at 37°C. After being neutralized with HCl, the samples were centrifuged at 10,000 × g for 10 min and filtered to remove the insoluble material. The supernatant was concentrated using Amicon Ultra10 filters (Millipore, Bedford, MA, USA). After being washed twice with distilled water, the extracted polysaccharide was collected from the membrane by resuspension in water. The concentrations of sialic acid were determined by a sialic acid assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions.

For the preparation of sialic acid, the thiobarbituric acid method of Warren-Aminoff was used with several modifications [59 (link),60 (link)]. The bacteria were grown in TSB with 10% (v/v) FBS for 5 h at 37 °C and pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS), and adjusted to 1 × 10¹⁰ CFU/mL. Bacteria in suspension (1mL) were lyophilized and then incubated with 500 µL of hot acid (0.4 M H₂SO₄, 80 °C) for 1 h to release sialic acid through the hydrolysis performed on lyophilized bacteria. The concentrations of sialic acid were measured by a sialic acid assay kit (Nanjing Jiancheng Bioengineering Institute) according to the specifications of the manufacturer.