Bile extract

The upper GI digestion procedure was adapted from Tzounis et al. (47 (link)) and Gaisawat et al. (53 (link)). Oral digestion was performed by addition of α-amylase (2 mL 0.47 g/mL; A3176, Sigma Aldrich, St. Louis, MO, USA) with 5 min incubation at 37^°C and pH 7.0, followed by adjusting the pH to 2 and adding pepsin solution (2 mL 0.8 g/mL; P7125, Sigma Aldrich, St. Louis, MO, USA) to simulate gastric digestion at 37^°C for 1.5 h. Subsequently, the pH was adjusted to eight using NaOH (1N, Sigma Aldrich, St. Louis, MO, USA) and then 30 mL pancreatic juice [NaHCO₃ (12 g/L), bile extract (6 g/L) and pancreatin (0.9 g/L), Sigma Aldrich, St. Louis, MO, USA] were added and incubated at 37^°C for 2 h to mimic small intestinal digestion.

Hydrogen peroxide (H₂O₂) was purchased from Bendosen Laboratory Chemicals (Selangor, Malaysia). Pepsin, pancreatin, bile extract, sodium biocarbonate (NaHCO₃), fluorescein sodium salt, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), dichloro-dihydro-fluorescein diacetate (DCFH-DA), RPMI 1640 medium, fetal bovine serum, antibiotic, potassium persulphate (K₂S₂O₈), 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) reagent, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder were purchased from Sigma-Aldrich (St. Louis, MO, USA), while other cell culture materials were purchased from BD Biosciences (NJ, USA).

Reagents for extraction and chromatographic grade solvents were purchased from Thermo Fisher Scientific (Pittsburgh, PA). Fetal bovine serum (FBS) was purchased from Gemini Bio-Products (West Sacramento, CA). Fungizone, penicillin-streptomycin, L-glutamine and non-essential amino acids were purchased from Life Technologies Corporation (Grand Island, NY). DMEM medium with 25 mmol/L glucose, HEPES buffer, bile extract, porcine amylase, pepsin, lipase and pancreatin, apo-8’-carotenal, all-trans-β-carotene (E-βC; > 98% pure), αC (98% pure) and αTC were purchased from Sigma-Aldrich (St. Louis, MO). Phytoene and phytofluene were kindly provided by Dr. Ken Riedl, Nutrient & Phytochemical Analytic Shared Resource (NPASR) at The Ohio State University. All other reagents and supplies were purchased from ThermoFisher Scientific unless otherwise specified.

Pea seeds (Pisum sativum var. Bajka) were obtained from Company of Horticulture Seeds and Nursery in Ożarów Mazowiecki, Poland. HHL (hippuryl-L-histidyl-L-leucine), pepstatin A, PMSF (phenylmethanesulfonyl fluoride), α-amylase from hog pancreas (50 U/mg, 10080, Sigma), pepsin from porcine gastric mucosa (250 U/mg, P7000, Sigma), pancreatin from porcine pancreas (P7545, Sigma), enalapril, bile extract, and TNBS (2,4,6-trinitrobenzenesulfonic acid) were purchased from Sigma-Aldrich Company, USA, and Amino Acid Standard from Pierce, USA. Any other chemicals were of analytical grade.

The survival of the lactic acid bacteria to simulated gastro-intestinal (GI) tract conditions was investigated as described previously [17 (link)]. Briefly, each lyophilized strain (2 g, 10⁹ CFU/gr) was rehydrated in 100 ml of demineralized water for 15 min at room temperature. The rest of the experiment was performed at 37 °C in a water bath. The stomach was simulated by adding 1 ml of porcine pepsin solution (7 mg/ml porcine gastric mucosa p7000, Sigma) and decreasing the pH in four steps of 15 min to 4.8, 4.5, 3.5 and 2.5. After 75 min, the entry into the proximal duodenum was mimicked by increasing the pH to 6.5 by adding 0.1 N NaOH and, after 90 min, 10 ml of porcine bile extract solution (80 mg/ml of bile extract, Sigma) and 2 ml of porcine pancreatin solution (50 mg/ml pancreatin, Sigma) were added. After 3 h, bile salts were deactivated by adding 11.5 mM of calcium chloride. The pH was maintained at 6.5 until 6 h, which was the end of the experiment. Samples for the total cell count analysis were taken at the different time points, diluted in phosphate buffered saline and plated in several dilutions on MRS agar plates. Plates were incubated for 48–72 h at 37 °C and thereafter the colony forming units (CFU/ml) were counted. The experiments have been performed in triplicate.

Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) α-amylase, pancreatin, pepsin, bile extract, Folin-Ciocalteau reagent, linoleic acid, ammonium thiocyanate, and haemoglobin were purchased from Sigma-Aldrich Company (Poznan, Poland). All others chemicals were of analytical grade.

Ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)), α-amylase, pancreatin, pepsin, xanthine oxidase, lipoxygenase (from Glycine max, type 1_B), bile extract, Folin-Ciocalteau reagent, linoleic acid and ammonium thiocyanate were purchased from Sigma-Aldrich company (Poznan, Poland). All others chemicals were of analytical grade.