Hyaluronic acid sodium salt from rooster comb

GBS strains were grown overnight in TSB at 30°C with 5.0% CO₂. Overnight cultures were pelleted at 4,000 rpm for 8 min, and the resulting supernatants were collected for analysis. Fifty microliters of spent supernatants was prewarmed for 5 to 10 min at 37°C with 5.0% CO₂ and then was added to 200 µl hyaluronic acid solution (1.25 mg hyaluronic acid sodium salt from rooster comb [Sigma] dissolved in 36.0 mg/ml monobasic sodium phosphate [pH 5.35] at 37°C) and incubated at 37°C with 5.0% CO₂ for 45 min. After incubation, 50 µl of 0.8 M sodium tetraborate (pH 9.1) preheated to 95°C was added, and the sample was boiled at 95°C for 3 min. Following boiling, 1.5 ml of 1.0% (wt/vol) 4-methylaminobenzaldehyde dissolved in 15.3 M acetic acid and 1.25 M HCl was added to the sample, and 200 µl was removed and read on a plate reader at 585 nm. To calculate the amount of hyaluronidase activity, standard curves of commercial hyaluronidase (hyaluronidase from bovine testes; Sigma) dissolved in TSB were created and included in conjunction with every assay. Hyaluronidase activity values from unknown samples were interpolated from the standard curve, which was fit to a 4-parameter logistic curve using GraphPad Prism 6 (La Jolla, CA). If sample reads exceeded the standard curve, the initial spent supernatant was diluted in TSB as necessary to fit within the standard curve range.