Fig. 3

Evaluation of stability and activity of KvarIa after in vitro gastric digestion assay. A Evaluation of residual activity by agar diffusion assay. Protein samples were incubated at 37 °C, 200 rpm in gastric digestion buffer (pepsin:protein ratio 1:40). Aliquots of reaction (50 µl) were removed at different time points (0.5, 5, 10, 20, 30, and 60 min) and digestion was stopped by the addition of 0.5 M ammonium bicarbonate to inactivate pepsin. The pH of samples with coated KvarIa was adjusted to 8 to get Eudragit coat dissolved. The dilutions of all samples by ratio 1:2 were made in distilled water and 5 µL drops of diluted samples were applied on K.quasipneumoniae DSM28212 MHA plates for soft agar overlay assay. B SDS-PAGE of KvarIa after simulated gastric digestion. The 16% TRIS-Tricine gels; unstained marker—PageRuler™ Unstained Low Range Protein Ladder (ThermoFisher), 10 µl of each sample loaded per line. Pepsin appears as the stable band between 30 and 42 kDa markers (green arrow). The red arrow indicates the full-size KvarIa. Exposure of KvarIa to pepsin (1:40 pepsin:lysin wt:wt) in acidic SGF results in the rapid breakdown of the protein to lower MW degradation products. Sampling times are shown in minutes