Trypsin

Whey protein concentrate (WPC) and soy protein isolate (Soy) were obtained from Wheyco GmbH, (Hamburg, Germany) and Solae LLC, (St. Louis, MO, USA), respectively. Subtilisin (EC 3.4.21.62) and trypsin (EC 3.4.21.4), kindly donated by Novozymes (Bagsvaerd, Denmark), were the enzymes employed in hydrolysis. The refined fish oil Omevital 18/12 TG Gold was purchased from BASF Personal Care and Nutrition GmbH (Illertissen, Germany) with a minimum content of omega-3 fatty acids of 35% (18% of EPA and 12% of DHA).

Fresh eggs were purchased from Hubei Shendan Health Food Co. (Xiaogan, China). Alcalase, Neutrase, Protamex, papain and trypsin were purchased from Novozymes, Denmark. HPLC grade alcohol and n-butanol were purchased from Comet and Aladdin. All other chemical reagents are analytically pure and were purchased from Sinopharm Chemical Reagent Co. Commercial kits for determining the activities of serum ALT, AST, and hepatic reduced glutathione (GSH) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Liver alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), and malondialdehyde (MDA) activity assay kits were purchased from Suzhou Comin Biotechnology Co. (Suzhou, China).

Ctenopharyngodon idella scales were purchased from a local supermarket (Wuhan, China), washed, dried, and stored at room temperature until use. Novozymes (Beijing, China) Investment Co., Ltd. was the source of the alcalase, papain, trypsin, neutral protease, and pepsin protease and bovine serum albumin (BSA). Sephadex G-50 and ion exchange (sulfopropyl-Sephadex C-25) were provided by Yuanye Chemical Scientific Co. (Shanghai, China). All chemicals employed were reagent grade or higher.

WG (79% protein) was purchased from Beijing Rui Mai Jia He Co., Ltd. (Beijing, China). Amylase, Alcalase 2.4 L and Trypsin were purchased from Novozymes (Beijing, China). All other chemicals and solvents were of analytical grade.

Hydrolysates of whey and soy protein were produced using subtilisin (EC 3.4.21.62) and trypsin (EC 3.4.21.4) (Novozymes, Denmark) as enzymes. To evaluate the effect of degree of hydrolysis on the emulsifying properties, five hydrolysates of a different degree of hydrolysis (2%, 4%, 6%, 10% and 14%.) were produced with each enzyme. Hydrolysis was carried out in a jacketed stirred tank reactor that maintained a temperature at 50 °C during operation. When the protein solution (40 g/L) reached 50 °C, enzyme was added to the reactor at an enzyme/substrate ratio of 0.5%. The pH was maintained constant at pH 8 by adding NaOH 1M. The reaction was monitored by the pH-stat method [28 (link)], which relates in real time the consumption of base with the number of peptide bonds cleaved according to Equation (1): $DH=\frac{B\cdot N_b}{\alpha \cdot m_p\cdot h_{TOT}}\cdot 100$
where B (mL) is the amount of base consumed, N_b (eq/L) is the normality of the base, α is the average degree of dissociation of the α-NH₂ groups released during hydrolysis, m_p (g) is the mass of protein in the substrate and h_TOT (meq/g) is the number of equivalents of peptides bonds per gram of protein. After reaching the desired DH, the reaction was stopped by thermal deactivation of the enzyme at 90 °C for 5 min. Afterward, the samples were freeze-dried for further use.

Silver carp skin was supplied by Hubei Zhongke Agriculture Co., Ltd. (Jingzhou, China). Alcalase, papain and trypsin were purchased from Novozymes (Beijing, China). Collagen type I from rat tail tendon, pepsin from porcine gastric mucosa and pancreatin from porcine were purchased from Sigma‐Aldrich (Shanghai, China). Foetal bovine serum (FBS), Dulbecco modified Eagle's minimal essential medium (DMEM), penicillin, streptomycin, non‐essential amino acids (NEAA) and Hank's buffered saline solution (HBSS) were purchased from Gibco (Life Technologies, Grand Island, NY, USA). The BCA protein assay kit was purchased from Beijing Solarbio Science and Technology Co., ltd. (Beijing, China). Mouse cytokine array kits were obtained from R&D systems (Minneapolis, MN, USA). Commercial kits used for determination of TGF‐β₁, VEGF, PF4, GMP‐140, β‐thromboglobulin (β‐TG) and serotonin (5‐HT) were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). The mouse antibodies against TGF‐β₁, type I collagen and GAPDH were purchased from Abcam (Cambridge, UK). Other chemicals used were analytical grade or better.