S220 seven compact ph

For Group b samples, 5 ml samples of medium were extracted every 2 days and filtered through 0.22 um membranes. The sterilized medium was then stored at 4°C. Changes in the pH and in the calcium concentration of the medium were detected using an S220 Seven Compact pH/ion meter (Mettler Toledo, Switzerland). We found that there was a sharp increase in pH (except in Group b3) between the first and third days. Because this may reflect the utilization of organic acids, we used high performance liquid chromatography (HPLC) to determine the organic acids present in samples (Supplementary Figure S2). Considering the disturbance of acetas in Group b1 and b2 and the expense, only Group b4 medium taken on the first and third days were analyzed in the study. Five organic acids (acetic, lactic, citric, succinic, and α-ketoglutaric acids) were used as the control sample and 100 mg/L of them were also prepared for HPLC, which was performed using a WATERS 2690 Separation Module (GenTech, United States). The column temperature was 30°C and the mobile phase consisted of basic ammonium and methanol at a ratio of 99:1. The pH was adjusted to 2.6 with phosphoric acid at a flow rate of 0.5 ml/min.

The pH of each vehicle was measured, at room temperature, using
a pH meter (Mettler Toledo S220 Seven Compact pH/Ion meter, Schwerzenbach,
Switzerland). pH measurements took place immediately after opening the soft
food/drink container or after vehicle preparation (in the case of the
formulas, squashes and Coca-Cola), and agitating the vehicle with a spatula
for 5 s.