Neutral protease

Camellia seeds were purchased from Quzhou City, Zhejiang Province. Angiotensin I-converting enzyme (ACE) from rabbit lungs (0.1 U) (Table A1), ACE substrate hippuryl-histidyl-leucine (HHL) and hippuric acid (HA) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neutral protease (50,000 U/g), alkaline protease (200,000 U/g), papain (800,000 U/g), trypsin (250,000 U/g), and Sephadex G-25 were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade.

Wheat and maize starches were purchased from He Nan Enmiao Food Co., Ltd. (Zhengzhou, China) sweet potato starch was purchased from the Beijing Gusong Economic and Trade Company (Beijing, China), mung bean starch was purchased from Hengshui Fuqiao Starch Co., Ltd. (Hengshui, China) and cassava starch was purchased from Guangxi Napoheshan Starch Co., Ltd. (Baise, China). Bacillus subtilis thermostable and mid-temperature α-amylase (12000 U/mL), microbial lipase (20,000 U/g) and neutral protease (50,000 U/g) were all produced by Beijing Solarbio Science & Technology Co. Ltd. (Beijing, China). Sodium hydroxide, sodium chloride and hydrochloric acid were obtained from Tianjin Fengchuan Chemical Reagents Co., Ltd. (Tianjin, China). Sodium chloride (Analytical reagent with 99.5% NaCl) was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. (Tianjin, China). Pullulanase (CAS No.: 9075-68-7, 1000 U/mL) was obtained from Beijing Solarbio Science & Technology Co., Ltd. The dialysis bags (viskase MD44-14, Avg. flat width 44 mm (diameter 28 mm), MWCO 14,000 Da) were produced by the Union Carbide Corporation (Danbury, Connecticut, United States). Tris, glycine, disodium EDTA, urea, guanidine hydrochloride and DTNB reagents were all obtained from Beijing Soleibo Science & Technology Co. Ltd. The P120H Ultrasonic Cleaner was produced by Elma Schmidbauer GmbH in Germany.

Cecum samples were first freed from residual fat tissue, Peyer’s patches, feces and then cut into smaller pieces and incubated in Hanks’ Balanced Salt Solution with 2% FCS, 5 mM of EDTA and 2 mM of dithiothreitol for 30 min at 37°C and vortexed. The inter-epithelial lymphocytes (IEL) fraction was dissected by filtering over a 70 μm cell strainer. To recover the lamina propria lymphocytes (LPL) fraction, IEL-depleted intestine pieces were washed in Hanks’ Balanced Salt Solution supplemented with 2% FCS and enzymatically digested for 45 min at 37°C with Collagenase type IV (Solarbio), Neutral protease and DNase I (Solarbio) in 1640 medium. Single-cell suspensions were generated by filtering over a 70-μm cell strainer. The IELs and LPLs were purified by density centrifugation on a 67% and 44% percoll gradient (Cytiva).

ATP (Amresco, USA), hypoxanthine (Sigma, USA), lactalbumin hydrolysate (Difco, USA), hemoglobin from bovine blood (Sigma) and bovine hemoglobin hydrolysate were added to the basic medium and the effects as nutrients were evaluated. The additives were dissolved in the basic medium and sterilized through 0.22 μm membrane filters and their final concentrations were 0.2 mg/ml, 5×10^-7 M, 2 mg/ml, 2 mg/ml and 2 mg/ml, respectively.
To produce bovine hemoglobin hydrolysate, 2 g crystalline bovine hemoglobin was dissolved in 45 ml deionized water. Subsequently, 0.133 g neutral protease (Solarbio, China) was added to the solution to reach a desired concentration (4,000 U/g) for hydrolysis. Digestion proceeded at pH 7.5, 45°C for 10 h. The pH of solution was maintained at 7.5 by addition of 2 M NaOH, as needed, every 2 h. The reaction was stopped by incubation at 90°C for 30 min. Tubes were centrifuged at 4°C at 1,500 g for 15 min. The supernatant was transferred into 50 ml tubes and snap frozen in liquid nitrogen. Hemoglobin hydrolysate was vacuum-dried overnight in a vacuum concentrator (Labconco, USA). Pellets were collected and stored at room temperature. The proteolysis of the bovine hemoglobin was analyzed on SDS-PAGE gels (12%) stained with Coomassie Blue.

Alcalase (2.4 AU/g) was obtained from Novozymes Biotech (Copenhagen, Denmark). Papain (8 × 10⁵ U/g), neutral protease (6 × 10⁴ U/g) and Flavourzyme (1.5 × 10⁴ U/g) were obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). 2.2-diphenyl-1-picrylhydrazyl (DPPH) and N, N, N’, N’-tetramethylethylenediamine (TEMED) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Copper sulphate, potassium sulphate, boracic acid, sulfuric acid, acrylamide, methylene diacrylamide, tris-(hydroxymethyl)-aminomethane (Tris), and lauryl sodium sulfate (SDS) were obtained from Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Protein molecular weight marker (Low) was obtained from Takara Bio (Dalian) Co., Ltd. (Dalian, China). Coomassie brilliant blue R250, Coomassie brilliant blue G250, methyl red, methylene blue, ammonium persulfate and methanal and sodium hydroxide were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade.