Alcalase

Lutein (purity > 90%), alcalase (enzyme activity > 200 U/mg), bile salts, and molecular weight standards including bacitracin, aprotinin, myohemoglobin, bovine serum albumin, and thyroglobulin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Soybeans were bought from Fengyuan Zhongye Co., Ltd. (Lianyungang, China). Pepsin (enzyme activity > 2500 U/mg), trypsin (enzyme activity > 2500 U/mg), 8-aniline-1-naphthalensulfonic acid (ANS), and trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma-Aldrich Co., (St. Louis, MO, USA). All other reagents and solvents were of at least analytical grade.

The water used in this study was deionized (18 MΩ·cm) from Water Purification System. Sephadex G-100, DEAE-52, 2,2-diphenyl-1-picrylhydrazyl, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, 2,4,6-tri(2-pyridyl)-s-triazine, 6-hydroxy-2,5,7,8-tetramethlchroman-2-carboxylic acid, casein, glacial acetic acid, Tris-base, alcalase, trypsin, papain, neutrase, pepsin, and ammonium persulfate were purchased from Yuanye Biotech Co. Shanghai, China. Absolute ethanol (analytical grade), acetic acid, acetone, ferric chloride, ferrous sulphate, glucose, methanol (analytical grade), perchloric acid, phenol (redistilled reagent grade), potassium per-sulphate, potassium sulphate, sodium acetate, and sodium hydroxide were purchased from DaMao Chemical Co. Tianjin, China. Boric acid, copper sulfate, hydrochloric acid, methylene blue indicator, methyl red indicator, petroleum ether, sulfuric acid, and sodium thiosulfate were purchased from Guangzhou Chemical Co. (Guangzhou, China). Bovine serum albumin, Tris-HCl buffer, SDS, tetramethylethylenediamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA) RIPA lysis buffer, acrylamide/bis-acrylamide mix, ladder protein, were purchased from Thermo Fisher Scientific Co. (Shanghai, China). All other chemicals were of analytical grade.

Defatted soy flour with low protein denaturation was obtained from Pingdingshan Jinjing Biological Technology Co., Ltd. (Zhengzhou, Henan, China). Alcalase (200 U mg⁻¹) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). C3G (purity ≥ 98%) was purchased from Nanjing Spring and Autumn Biotech Co., Ltd. (Nanjing, Jiangsu, China). 1-anilinonaphthalene-8-sulfonic acid (ANS) was obtained from Aladdin Co., Ltd. (Shanghai, China). 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) was obtained from Fluorochem Co., Ltd. (Hadfield, Derbyshire, UK), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Molecular weight standards including cytochrome C, aprotinin, bacitracin, Gly−Gly−Tyr−Arg, Gly−Gly−Gly, carbonic anhydrase, bovine serum albumin, alcohol dehydrogenase, β-amylase and apoferritin were all purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). All other reagents were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise noted.