Pepsase

The exogenous carbohydrases used in the present study were manufactured via microbial engineering, that were kindly provided by Shandong Lonct Enzymes Co., Ltd. (Linyi, Shandong, China). The activities of the glucoamylase, pullulanase, maltase, thermostable α-amylase, medium temperature α-amylase, and cold-active α-amylase were 200,000, 8,000, 1,000, 40,000, 5,000, and 20,000 U/ml, respectively. The pepsase and amylase, which were purchased from Sigma Aldrich Co. (St. Louis, MO, USA), and trypsase and chymase, which were purchased from Amresco Co. (Houston, TX, USA), were used in the simulated small intestinal fluid. The water used in the present study was deionized with conductivity less than 0.5 us/cm and pH between 6.8 and 7.2. And the in vitro digestion experiments were conducted in the third generation of simulated monogastric animal digestion system (SDS-Ⅲ, Zhongben Intelligent Technology Development Co., Ltd., Changsha, Hunan, China). The preparations of the digestive fluid, as well as the machine settings were followed by Zhao’s descriptions [14 (link)].
The composition of corn-soybean based diets for broilers at 1 to 3 wk was followed arbor acres plus standard, which was showed in Table 1.

Skipjack tuna (K. pelamis) muscle was provided by Ningbo Today Food Co., Ltd (China). Sephadex G-25 resins and NO assay kit (Nitrate reductase approach, A012-1) were purchased from Nanjing Jiancheng Bioengineering Institute (China). ET-1 ELISA Kit (HM10108) was purchased from Shanghai Qiaodu Biotechnology Co. Ltd. (China).Trypsase (≥20,000 U/g), pepsase (≥500,000 U/g), papain (≥400,000 U/g), ACE (A6778) and N-[3-(2-Furyl) acryloyl]-Phe-Gly-Gly (FAPGG, F713) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (China). Alcalase (B8360, 2.0 × 10⁵ U/g), 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; M8180), Penicillin-Streptomycin Liquid (P1400), dimethylsulfoxide (DMSO), fetal bovine serum (FBS; 11011-8611), HEPES buffer (H8090), norepinephrine (NE; IN0530), and Cap (C7510) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (China). ACEi Peptides of (TMAP1-TMAP6) with purity higher than 98% were synthesized in Shanghai Apeptide Co. Ltd. (China).

KGM (Mn = 6.9 × 10⁵ Da) was purchased from Chengdu Root Industry Co., Ltd. (Chengdu, China). Dextran sulfate sodium (DSS), Cur, dexamethasone, lipopolysaccharide (LPS), interleukin-4 (IL-4), β-mannase, and pepsase were purchased from Sigma-Aldrich. Trifluoroacetic anhydride (TFAA), 1,8-diazabicyclo (5,4,0)-7-undecene (DBU), polyethylene glycol succinate (TPGS), and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Atlanta, GA). The Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotech (Jiangsu, China). SGF was prepared by adding 0.1 N HCl, 2 g NaCl, and 3.5 g pepsase to 1 L deionized water. SIF was prepared by adding 102 mg KH₂PO₄ and 155 mg Na₂HPO₄·2H₂O to 1 L SGF; subsequently, 0.01 M NaOH solution was used to adjust pH to 7.4, and then 4 mg β-mannase was added. Unless stated otherwise, the other chemicals were of analytical grade and were used without further treatment.