Flavourzyme

Full‐fat soybean flakes were kindly provided by the Lanshan Group Corporation (Liaocheng, China). The full‐fat soybean flakes contained 21.5% oil, 42.5% protein, 32.8% carbohydrate, 3.2% ash, and 11.5% moisture (on dry weight basis). Flavourzyme (an enzyme cocktail of endopeptidases and exopeptidases) and Alcalase 2.4L (an endoproteinase from Bacillus licheniformis) were purchased from Novozymes (Beijing, China). Protex 7L (a neutral metalloendopeptidase from Bacillus amyloliquefaciens) and Protex 6L (an alkaline serine endopeptidase from B. licheniformis) were purchased from Genencor International (Rochester, NY, USA). Glucose, fructose, galactose, sucrose, raffinose, and stachyose standards as well as benzoyl‐DL‐arginine‐p‐nitroanalide hydrochloride (BAPA) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Molecular weight standards containing thyroglobulin (670 kDa), bovine globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa) were purchased from Bio‐Rad Laboratories (Richmond, CA, USA). All other reagents were of analytical grade unless otherwise specified.

Soy PI was purchased from YUWANG ECO. (Shandong, China) and wheat PI and pea PI were purchased from Roquette (Lestrem, France). Plant PIH were prepared by the hydrolysis of plant PI using three proteases: bromelain (EC 3.4.22.32, Chappion Biotechnology, Chiayi, Taiwan), Neutrase (EC 3.4.24.28, Novozymes, Bagsvaerd, Denmark) and Flavourzyme (EC 3.4.11.1, Novozymes). Briefly, plant PI solution (100 mg plant PI /mL) was prepared by dissolving plant PI (1 kg) in distilled water (10 L) at 95 °C for a period of 1 h. Subsequently, three proteases including bromelain (1,000 CDU/mL), Neutrase (0.0024 AU-N/mL) and Flavourzyme (3.3 LAPU/mL) were added to the plant PI solution. The protease-containing plant PI solution was incubated at 45 °C for 24 h. Then, the hydrolyzed plant PI solution was heated at 95 °C for 1 h to inactivate the protease. The plant PI solution was then centrifuged at 9000× g at 4 °C for 10 min. Filtering the supernatant using a filter membrane (No. 1, pore size: 6 µm, Advantec Co. Ltd., Tokyo, Japan). Note that the proteases (bromelain, Neutrase^® and Flavourzyme^®) were denatured during the heating and filtration process and were therefore not included in the plant PIH. Finally, the plant PIH solution was freeze-dried to obtain a powder that was kept in an airtight container at 25 °C before use.

CN (purity > 85%), ALA (purity > 85%), BLG (purity > 85%), o-phthalaldehyde, o-phenylenediamine dihydrochloride, dithiothreitol, and gelatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase (catalytic activity: 105,466 U/g), Protamex (catalytic activity: 144,524 U/g), and Flavourzyme (catalytic activity: 20,786 U/g) were obtained from Novozymes (Bagsvaerd, Denmark).

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.

All solvents and reagents were purchased from Merck Life Science (Milano, Italy) and Fluorochem (Milano, Italy) and were used without further purification. Soy protein isolate (SPI) type 90 was kindly provided by ABS FOOD s.r.l. (Peraga di Vigonza, Italy). The chemical composition was ≥90.0% proteins, ≤7.0% moisture, ≤1.0% fat, ≤6.0% ash, and 1% total fiber (dry matter basis as reported by the producer). Proteolytic enzymes were kindly supplied by Novozymes^® (Bagsværd, Denmark): the endopeptidase Alcalase^® 2.4 L FG (enzyme activity: 2.4 AU g⁻¹) derived from Bacillus licheniformis and the mixture of endopeptidase and exopeptidase Flavourzyme^® (enzyme activity: 1000 LAPU g⁻¹) derived from Aspergillus oryzae.