CMC Determination of Chobimalt

The CMC of Chobimalt (Anagrade, CH220, purity > 99%; M_w = 1035 Da, Anatrace, OH, USA) was measured using the fluorescence intensity of a fluorescence probe 8-Anilino-1-naphthalenesulfonic acid (ANS, A1028; Sigma Aldrich, St. Louis, MO, USA). Chobimalt samples (150 µL) in concentrations from 0.05 µM up to 100 µM were mixed with 5 µL ANS (from a freshly prepared 5 mM stock solution, 5 mM in 50 mM PBS). Three different experimental approaches were applied and Chobimalt was dissolved in: (i) milli-Q water (denoted as H₂O); (ii) acidic condition, pH 2.0 (adjusted using HCl, denoted as H₂O-HCl); (iii) the presence of 100 mM NaCl at pH 2.0 (H₂O-NaCl). The fluorescence intensity was measured using a 96-well plate by a Synergy MX (BioTek) spectrofluorometer. The excitation was set at 380 nm and the emission was recorded at 485 nm. The excitation and emission slits were adjusted to 9.0/9.0 nm and the top probe vertical offset was 6 nm. Each experiment was performed in triplicates; the error bars represent the average deviation for repeated measurements of three separate samples.