Soybean trypsin inhibitor (STI) was obtained from simulated soybean whey wastewater through a sustainable method consisting of isoelectric precipitation, ammonium sulfate salting out, and gel filtration chromatography, and the effect of temperature, pH, and pepsin on the stability of STI was also discussed. The results showed that the recovery rate of the trypsin inhibitory activity was 89.47%, the purity and the specific activity of STI were 71.11%, and 1442.5 TIU/mg in the conditions of pH 4.0 and 40% ammonium sulfate saturation. The soybean Kunitz trypsin inhibitor (KTI) and soybean Bowman–Brik trypsin inhibitor (BBI) were obtained via gel filtration chromatography, and their specific activity levels were 1733.5 TIU/mg and 2588.3 TIU/mg, respectively. The STI displayed good stability over a wide temperature and pH range. The STI, KTI, and BBI were all resistant to pepsin hydrolysis, and their ability was ranked as BBI > STI > KTI. These findings will provide a theoretical basis for recycling STI from soybean whey wastewater and promoting better active compound utilization.

Supramolecular structures obtained from protein–polysaccharide association may be applied to encapsulate bioactive compounds or to improve the physical stability and texture properties of colloid–based products. In this study, the interaction of 0.1 wt% soybean trypsin inhibitor (STI) with different concentrations of chitosan (CS) in aqueous solutions was investigated under different pH by the analysis of state diagram, turbidity, zeta potential, spectroscopy, and microstructure; the protective effect of STI–CS complex coacervates on STI stability in simulated gastric juice was also discussed. The results suggested that interactions between STI and CS could form soluble/insoluble complexes mainly through hydrophobic interactions (pH 4.0) or electrostatic interactions (pH 6.0). The CD spectra showed that the secondary structure of STI did not change significantly when CS with the same charge was mixed with STI, and the secondary structure of STI was slightly changed when CS with the opposite charge was mixed with STI. Simulated gastric digestion experiments showed that the complex formed by non-covalent bonding had a protective effect on the active protein. This study provides information about the effect of different CS concentrations and pH values on the formation of complexes of CS and STI in an aqueous solution and provides theoretical references for the construction of supramolecular-structured carrier substances based on CS and STI.

The aim of this work was to prepare insulin-loaded nanoparticles using soybean trypsin inhibitor (STI) and chitosan (CS) as a potential coating. The nanoparticles were prepared by complex coacervation, and characterized for their particle size, polydispersity index (PDI), and encapsulation efficiency. In addition, the insulin release and enzymatic degradation of nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were evaluated. The results showed that the optimal conditions for preparing insulin-loaded soybean trypsin inhibitor-chitosan (INs-STI-CS) nanoparticles were as follows: CS concentration of 2.0 mg/mL, STI concentration of 1.0 mg/mL, and pH 6.0. The INs-STI-CS nanoparticles prepared at this condition had a high insulin encapsulation efficiency of 85.07%, the particle diameter size was 350 ± 5 nm, and the PDI value was 0.13. The results of the in vitro evaluation of simulated gastrointestinal digestion showed that the prepared nanoparticles could improve the stability of insulin in the gastrointestinal tract. Compared with free insulin, the insulin loaded in INs-STI-CS nanoparticles was retained at 27.71% after 10 h of digestion in the intestinal tract, while free insulin was completely digested. These findings will provide a theoretical basis for improving the stability of oral insulin in the gastrointestinal tract.