Pepsin

To further investigate how well different strains of Lactobacillus plantarum may survive in digestive system, a simulated gastric juice (SGJ) and simulated small intestinal juice (SSIJ) was developed to mimic the digestive fluids produced in the stomach and small intestine, respectively. The SGJ comprised a saline solution with a pH of 2.50 and a concentration of 27 mg/mL of pepsin (Solarbio, Beijing, China). The SSIJ was prepared using pancreatin (Solarbio, Beijing, China) solution at 0.27 mg/mL in a 0.50 percent saline solution at pH 8.00. The cells of L. plantarum strains that had already been cultured were collected and suspended in SGJ at a concentration of 10⁸ CFU/mL and incubated at 37°C for three hours. After incubation, 0.5 mL of the SGJ samples were mixed with 4.5 mL of the SSIJ to mimic the environment of the small intestine and the mixture was further incubated at 37°C for an additional 3 h.
Cells survival was assessed by inoculating MRS agar plates with the cultures acquired from the simulated gastrointestinal transit tests and counting viable cells as described by Fei et al. (2018) (link). Each experiment was repeated three time to ensure accurate and consistent results.
The following equation was used to determine the survival of the strains in simulated gastric and small intestine environments.

An in vitro artificially simulated gastrointestinal juices model was applied, following the previously reported methods, with minor modifications (Li et al., 2020 (link)). The simulated gastric juice was prepared by adding 10 g/l pepsin (Solarbio, Beijing, China) to 16.4 ml of sterile 0.1 mol/l HCL, filtered through a membrane with 0.22 μm pore, and adjusted to pH 2.0, 3.0, and 4.0 using sterile 1 mol/l NaOH. The simulated intestinal juice was prepared by adding 10.0 g/l trypsin (Solarbio, Beijing, China) and 6.8 g of KH₂PO₄ to 500 ml of sterile ddH₂O. The pH was adjusted to pH 6.8 using 1 mol/l NaOH and filtered through a membrane with 0.22 μm pore. One milliliter of L. salivarius S01 (approximately 10⁷–10⁸) suspension was inoculated into 5 ml of simulated gastric juice at pH 2.0, 3.0, and 4.0, and incubated for 3 h at 37°C. One milliliter of L. salivarius S01 (approximately 10⁷–10⁸) suspension was also inoculated into 5 ml of simulated intestinal juice for 4 h. Then, the bacterial solutions were cultured on MRS agar for 24 h to determine the tolerance of selected strains to simulated gastrointestinal juice: Survival rate (%) = lg N1 / lg N2 × 100%, where N2 is the total viable counts of the selected strains at 0 h and N1 is the total viable counts after exposure to the simulated gastrointestinal juice for different time periods.

Individuals of Pacific herring (Clupea pallasii) were purchased from a seafood market in Qingdao, China. Whole fish were transported on ice to the laboratory. Upon arrival, the fish were washed, and the fish meat was collected, minced, and freezed. Five proteases (papain, flavourzyme, pepsin, trypsin, and neutrase) were obtained from Solarbio Co. (Beijing, China). An ultrafiltration (UF) system and UF membranes were buy from Laungy Co., Ltd. (Shanghai, China). Hepatocellular carcinoma (HepG2) cells were obtained from Qingdao University (Shandong, China). Trifluoroacetic acid (TFA), acetonitrile, formic acid and methanol were of guaranteed reagent and purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals and solvents were of analytical reagent, and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Pepsin, trypsin, bile salt, Pyrogallol, L(+)-ascorbic acid, Ferrous sulfate, potassium ferricyanide and trichloroacetic acid were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). DMEM medium and special modified DMEM medium were obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China). RNA Isolation Kit was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Fluorescent quantitation and a Reverse Transcription kit were obtained from United States Biological (Shanghai, China). ELISA kit was purchased from Chenglin Biotechnology Co., Ltd. (Beijing, China). DPP-IV was purchased from Sigma Chemical (St. Louis, MO, USA). All other chemicals and reagents were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).

AgNPs (⌀5 and 50 nm) were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an City, China). Triton™ X100, sodium lauryl sulfate and soy peptone were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Agar, pepsin, and Masson’s trichrome staining reagent were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Tryptone was purchased from OXOID (Shanghai, China), calcein was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China), and Cell Counting Kit-8 (CCK8) was obtained from APExBIO Technology, LLC (USA). Trypsin was purchased from Sigma–Aldrich (USA). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent was purchased from GlpBio (USA).