Neutrase

Milk powder from Hormel Foods USA was used. The milk contained 33.7% (w/w) protein and 5.3% (w/w) moisture. Commercial proteases Alcalase 2.4 L (serine endoprotease from Bacillus licheniformis), Neutrase (metallo-endoprotease from Bacillus amyloliquefaciens) and Protamex (a protease with endoprotease and exopeptidase activity from Bacillus licheniformis and Bacillus amyloliquefaciens) supplied by Novozymes (Bagsvaerd, Denmark) were used. Analytical grade quality reagents were used in all experiments.

PPI was purchased from Roquette (Lestrem, France). PPIH was prepared by hydrolyzing PPI with the proteases bromelain (Chappion Biotechnology, Chiayi, Taiwan), Neutrase (Novozymes, Bagsvaerd, Denmark), and Flavourzyme (Novozymes, Bagsvaerd, Denmark). Briefly, PPI solution (100 mg PPI/mL) was prepared by dissolving PPI (1 kg) in distilled water (10 L) at 95 °C for a period of 1 h. The proteases bromelain (1000 CDU/mL), Neutrase (0.0024 AU-N/mL), and Flavourzyme (3.3 LAPU/mL) were then added to the PPI solution. The protease-containing PPI solution was incubated at 45 °C for 24 h. The hydrolyzed PPI solution was heated to 95 °C and maintained at that temperature for 1 h to halt protease activity. The PPI solution was then centrifuged at 9000× g at 4 °C for 10 min. Filtering the supernatant using No. 1 ADVANTEC filters resulted in a clear, yellow permeate (PPIH solution, 36 mg PPIH/mL). Note that the proteases (bromelain, Neutrase^®, and Flavourzyme^®) were denatured during the heating and filtration process and were, therefore, not included in the PPIH. Finally, the PPIH solution was freeze-dried and held in an airtight container at 25 °C prior to use.

Alcalase, Flavorzyme, Neutrase, and Protamex were purchased from Novozymes (Bagsvaerd, Denmark). Bromeline and Papain were purchased from Daesong Sangsa (Seoul, Korea). All chemicals for antioxidant and anti-aging tests were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MS, USA). All other reagents and solvents used in this study were of analytical grade.

Defatted hot-pressed peanut meals were gifts of Shandong Jinsheng Grain Oil and Food Co., Ltd. (Linyi, China). Alcalase, Protamex, Papain, Flavourzyme, and Neutrase were purchased from Novozymes (Beijing, China). α-glucosidase and its substrate p-nitrophenyl-α-D-glucopyranoside (pNPG) were bought from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Other reagents were analytically pure.