Response surface methodology and optimization of hydrolysis conditions for the in vitro calcium-chelating and hypoglycemic activities of casein protein hydrolysates prepared using microbial proteases

This study represents the first investigation into food-derived peptides with anti-diabetic and calcium-chelating activities. This work aimed to assess the potential of casein, a promising protein source, for generating bioactive peptides with dual functionalities using combined enzymes, introducing an innovative exploration in research based on our current knowledge. Eleven proteases were employed to prepare casein hydrolysates. Protease A “Amano” 2SD demonstrated the highest degree of hydrolysis (DH = 24.69 ± 1.74), resulting in hydrolysates with enhanced calcium-chelating properties, and PROTIN SD-NY10 exhibited potent anti-diabetic activity with a degree of hydrolysis of (DH 13.84 ± 1.67). A combination of Protin SD-NY10 and Protease A “Amano” 2SD was selected to produce casein peptides with both calcium-chelating and anti-diabetic properties. Single factor experiments and response surface methodology (RSM) using a factorial design were employed to optimize hydrolysis conditions. The results of single factor optimization revealed that a protease ratio of 4:2, hydrolysis time of 6 h, protease concentration of 0.5%, substrate concentration of 10%, hydrolysis temperature of 50 ℃ to 55 ℃, pH values of 6.5 to 7.0, yielded maximum values for both functionalities. Hydrolysis time, pH, and enzyme concentration significantly influenced the production of calcium-chelating peptides. Using RSM, optimal conditions were determined: hydrolysis time 4.3 h, enzyme concentration 0.75%, and pH 7.0, with a calcium-chelation rate of 93.28%, which was quite similar to the predicted value. Ultrafiltration of hydrolysates revealed that the < 1KDa peptide fraction at a concentration of 10 mg/ml displayed higher calcium-chelation capacity (60.84%) and α-glucosidase inhibitory activity (38.54%). This study highlights the potential of casein to generate bifunctional hydrolysates with calcium-chelating and anti-diabetic activity, supporting its application as a novel, natural ingredient for functional food product development and a potential functional food supplement that can be used for medical purposes.