Extraction and quantification of exopolysaccharides

The extracellular polysaccharides in biofilms were determined using the phenol-sulfuric acid method (Cao et al., 2021). V. parahaemolyticus was treated with PCA at 37°C for 24 h. The cells were centrifuged (2,500 × g for 15 min), and the supernatant was separately mixed with a threefold volume of 95% (v/v) ethanol and precipitated at 4°C for 24 h. The mixture was then centrifuged to collect the precipitates (2,500 × g for 15 min). Then, 5% phenol and 90% H₂SO₄ were added to the precipitate, mixed, and placed in the dark for 1 h at 25°C. The final mixture was centrifuged at 9600 × g for 10 min, and the absorbance of the supernatant at 490 nm was measured.