Trypsin

The gastrointestinal transit tolerance of L. casei was measured according to the method of Maragkoudakis et al.[29 (link)]. Pepsin (1:10,000, Sangon Biotech Ltd., China) was suspended in 0.5% w/v sterile saline at 3 g/l and the pH adjusted to 3.0 to simulate gastric juices. In this simulated gastric juice, the bacterial incubation time was 180 min. Trypsin (1:250, Sinopharm Chemical Reagent Ltd., China) and bile salt (LP0055, Thermo Fisher Scientific, UK) were dissolved in 0.5% w/v sterile saline at a concentration of 1 g/l and 3 g/l, respectively, then adjusted to pH 8.0 to simulate small intestinal juices. In the simulated small intestinal juices, the bacterial incubation time was 240 min. The viable counts of L. casei were determined by plate counting at 0 and 420 min (180 min plus 240 min). The gastrointestinal transit tolerance of L. casei strains was determined by comparing the number of viable cells at different time points.

Takifugu flavidus were purchased from Zhangzhou City of Fujian Province, China. The skin was shelled, husked, and used for further experimentation. Alcalase, pepsin, and trypsin were purchased from Sinopharm Group (Beijing, China); angiotensin-I-converting enzyme, hippuryl-l-histidyl-L-leucine (HHL), hippuric acid, and CNBr-activated Sepharose 4B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Captopril was procured from MedChem Express (Monmouth Junction, NJ, USA). All other chemicals/reagents used were of analytical grade or HPLC grade.

Subtilisin (EC 3.4.4.16, 6  ×  10⁴ U/g), bromelain (EC 3.4.22.4, 5  ×  10⁵ U/g) and papain (EC 3.4.22.2, 8  ×  10⁵ U/g) were all purchased from China, Beijing Solebel Co., Ltd. Trypsin (EC 3.4.21.4, 5  ×  10⁴ U/g) and Na₂CO₃ were purchased from Shanghai, China, Sinopharm Chemical Reagent Co., Ltd. Eight-gram samples of Bombyx mori raw silk (SOHO Biomaterials Science and Technology Co., Ltd., Suzhou, China) were placed in 400 mL of solutions containing subtilisin, Trypsin, bromelain and papain. According to the protease commodity information and based on previous research, optimal degumming conditions were used for each of the four enzymes [23 (link),30 (link),31 (link)], as shown in Table 1. The pH values were adjusted with sodium dihydrogen phosphate–citric acid buffer. The degummed silks were immediately placed into boiling water to inactivate the enzymes and then thoroughly washed with distilled water. The above steps were repeated once. The traditional Na₂CO₃-degumming method was used as a control. In brief, 8 g of raw silk was boiled three times for 30 min each in 400 mL of 0.5 g/L Na₂CO₃ aqueous solution and then thoroughly rinsed with deionized water. Next, all degummed silk fibers were air-dried in an oven at 60 ± 2 °C. The degummed silk fibers were marked as subtilisin-silk, Trypsin-silk, bromelain-silk, papain-silk and Na₂CO₃-silk.

CCK-8 (Cat # 96992) was purchased from Sigma (St. Louis, MS, USA). Fetal bovine serum (FBS) was purchased from Ausbian (Sydney, Australia). DMEM (10-013–CVR) was purchased from Corning Inc. (Corning, NY, USA). Trypsin (Cat # T4665) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). D-Hanks Hanks was obtained from Shanghai GeneChem Co. Ltd. (Shanghai, China). 4-hydroytamoxifen (4-OH-TAM) was purchased from Sigma (St. Louis, MS, USA). TRIZOL RNA Isolation Kit (Catalog #: 12183555) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). M-MLV (M1705), dNTPs (U1240) and RNase inhibitor (N2115) were obtained from Promega (Madison, WI, USA). Oligo dT (B0205) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Bulge-LoopTM miRNA qPCR Primer Sets were synthesized by Ibibio (Guangzhou, Guangdong, China). Reverse and forward primers were synthesized by Shanghai Genechem Co., LTD (Shanghai, China). SYBR Master Mixture (DRR041B) was obtained from TAKARA (Daliang, Liaoning, China). Reagents for reverse transcription were purchased from Axygen (Union City, CA, USA).