Laqua f 71

To estimate the effect of acidification of BHI broth by growing streptococci, BHI broth containing HEPES buffer (0.1 M, pH 7.2; Invitrogen) and phosphate buffer (0.1 M, pH 7.2) were prepared. Exponential phase cultures of streptococci (2 × 10⁹ cfu) were incubated with IAV (ca. 1 × 10⁶ pfu) in these BHI broths (0.5 ml) at 37°C in a 5% CO₂ atmosphere. After incubation for 3 h, bacterial growth was stopped by adding penicillin and streptomycin, and the mixture was centrifuged to remove the bacteria. The IAV titer of the supernatants was determined using a plaque assay. The final pH of cultures in the stationary phase of growing streptococci in BHI broth was directly measured using pH meter (LAQUA F-71; HORIBA, Kyoto, Japan) after incubation at 37°C in a 5% CO₂ atmosphere for 18 h.
The effect of acidic pH on IAV inactivation was studied using BHI broth containing sodium acetate (NaOAc) buffer, whose pH was adjusted to 4.0, 4.5, 5.0, or 5.5. IAV in these BHI broth was incubated at 37°C for 3 h, and the viral titer was determined using a plaque assay.

The culture pH, dissolved carbon dioxide concentration (DCDC), and biomass (Supplementary Table S1, Figures 2, 3) were measured. To measure the pH, 50 mL of the supernatant of the BG11 medium or BX1.5 culture was harvested, and the pH was measured (LAQUA F-71: Horiba Co., Ltd., Kyoto, Japan). To measure DCDC, 50 mL of the BG11 medium or BX1.5 culture supernatant was harvested and measured using a portable carbon dioxide gas concentration metre (Model CGP-31, DKK-TOA Co., Ltd., Tokyo, Japan). The 50 mL of BX1.5 culture were harvested, centrifuged (8,000 × g, 10 min, 10°C), and the cell biomass containing the BX1.5 extracellular polysaccharide (bxEPS) was stored at −80°C until use. The cell matter was freeze-dried as DCM (dried cell matter with bxEPS) in a vacuum freeze dryer (FZ-Compact; Labconco Co., Ltd., Kansas City, United States of America) for 72 h. The weight of DCM (DMW) was measured using an electron precision balance (MS603S; METTLER TOLEDO, Tokyo, Japan). Of note, since the supernatant and cells cannot be separated by centrifugation due to the abundant bxEPS production, 20 mL of the cell culture was directly transferred to a 50 mL tube, frozen at −80°C, and lyophilised as the DCM sample.