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Figure S1. (a) Tricine-SDS-PAGE, (b) native PAGE and (c) reverse zymography of purified soybean trypsin inhibitor (STI). M, protein marker; 1, purified STI.

Figure S2. Detection of the effect of thermal stability. Soybean trypsin inhibitor (STI) was incubated with boiling and high pressure steaming (HPS) for different time intervals. After incubation, samples were applied to SDS-PAGE. STI treated with boiling (a) and HPS (b). M, protein marker. Incubation time was indicated above each lane.

Figure S3. Residual inhibitory activity of soybean trypsin inhibitor (STI) with boiling (a) and high pressure steaming (HPS) (b) treatments. The remaining inhibitory activity was determined in buffer A. Different concentrations of STI were preincubated at different temperatures [(□) 10 μg mL−1; (▲)2.5 μg mL−1; (●) 1.0 μg mL−1; (M) 0.5 μg mL−1]. Vertical bars represented ± standard error.

Figure S4. Effect of simulated gastric fluid (SGF) digestion on soybean trypsin inhibitor (STI). STI was untreated (a), boiling (b) and high pressure steaming (HPS) (c). Enzymatic digestion was performed, followed by tricine-SDS-PAGE. After electrophoresis, proteins were visualised by staining with Coomassie Brilliant Blue R-250 (CBB). In the control experiments (con), proteinase was replaced with buffer A, M, protein marker.

Figure S5. Western blot detection of the degradation of soybean trypsin inhibitor (STI) in simulated gastric fluid (SGF). M, protein marker; STI was untreated (a); treated with boiling (b); treated with high pressure steaming (HPS) (c). The proteins were separated by tricine-SDS-PAGE and transferred to nitrocellulose, followed by an immunological detection with rabbit anti-STI polyclonal antibody to detect the IgG binding of digested samples.

Figure S6. Residual inhibitory activity of soybean trypsin inhibitor (STI) after boiling and high pressure steaming (HPS) treatments. The concentration of STI was 0.25 mg mL−1. Control was untreated; boiling was at 100 °C for 30 min; HPS was at 121 °C for 30 min. STI was incubated with different ratio of porcine pepsin for 1 h, and then, its residual inhibitory activity was determined. Vertical bars represented ± standard error. Columns with the same letter was not significantly different (P < 0.05).

Figure S7. Effect of the interaction between soybean trypsin inhibitor (STI) and trypsin. STI was untreated (a); STI treated with boiling (b); STI treated with high pressure steaming (HPS) (c). Soybean trypsin inhibitor (STI) was incubated with trypsin at room temperature for 1 h and then 4× Laemmli buffer was added and incubated for 30 min at room temperature followed by checking with SDS-PAGE at 4 °C. In control experiment (con), proteinase was replaced with 20 mm Tris–HCl, pH 8.0; M, protein marker; T, trypsin.

Figure S8. Mobility of soybean trypsin inhibitor (STI) heated at different temperatures. M, protein marker.

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