Pepsin p7000

HPLC grade methanol, ethanol, hexane, acetonitrile and methyl tert-butyl ether, and sodium hydroxide and hydrochloric acid were purchased from Fisher Scientific (Mississauga, ON, Canada), and Suprapur formic acid from VWR (Mississauga, ON, Canada). High-purity lutein 00012453 and zeaxanthin 00026504 standards were purchased from ChromaDex (Irvine, CA, USA). Ferulic acid was purchased from Sigma (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Digestive enzymes, including α-amylase from human saliva A0521-2.5KU, pepsin P7000, and pancreatin 1760-25G, were purchased from Sigma Aldrich. Porcine bile extract SC214601 was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The enzyme inhibitors AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride] and PMSF (phenylmethylsulfonyl fluoride) were purchased from Millipore Sigma (Oakville, ON, Canada) and VWR Canada, respectively. The nano pure water was obtained from the Milli-Q integral water purification system (Millipore Ltd., Etobicoke, ON, Canada).

The survival of selected LAB through simulated gastrointestinal juices was examined according to a previous report with slight modification (Jamyuang et al., 2019 (link)). After two generations of cultivation, the LAB strain was inoculated with 1% (v/v) inoculum in MRS liquid medium and incubated for 18 h at 37°C. Cells were harvested by centrifugation (8000 rpm at 4°C for 5 min) and cell number was adjusted to 10⁹ CFU/mL by adding PBS solution. The obtained bacterial suspensions (1 mL) were inoculated to 9 mL of filter sterilized simulated gastric juice solution [3 g/L pepsin (p7000, sigma), pH 2.0] at 37°C for 60 min. The gastric juice solution was removed by centrifugation (8000 rpm, 5 min) and subsequently re-suspended in 9 mL of filter sterilized simulated small intestinal juice solution [1 g/L trypsin (T105532, Aladdin), 0.3% bile salt, pH 8.0]. Samples were incubated statically at 37°C for 120 min. Then, the intestinal juice was removed by centrifugation (8000 rpm at 4°C for 5 min). The cell pellet was suspended in a sterile 0.85% NaCl (w/v) solution. Survival cells were counted on MRS plates. The survival rate (%) was calculated by the following equation:
where N₀ and N_t represent viable bacterial cells before and after growth in the simulated gastrointestinal tract, respectively.

β-glucuronidase (G0751), ethyl acetate, hexane, isopropyl alcohol, methanol, Na₂-ethylenediaminetetraacetic acid (E5134) and pepsin (P7000) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). L-Ascorbic acid and NaOH were obtained from Avantor Performance Materials Poland S.A. (Gliwice, Poland). HCl and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Naringenin (N5893) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Millipore Milli-Q system (Millipore Co., Millford, MA, USA) was used to produce ultrapure water.