Pancreatin p7545

α-Amylase from human saliva (A0521-500 UN; Type IX-A, lyophilized powder 1000–3000 U/mg protein), porcine pepsin from gastric mucosa (P6887-1G, lyophilized powder 3200–4500 U/mg protein), pancreatin (P7545, lyophilized powder, 8 X USP), and porcine bile extract (B8631, lyophilized powder) were purchased from Sigma–Aldrich (Saint-Louis, MO, USA). AEBSF- 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride was purchased from Fluka, Ref: 76307 (Sigma-Aldrich, Saint-Louis, MO, USA). All other chemicals were of analytical reagent grade and purchased from Sigma–Aldrich (Saint-Louis, MO, USA), unless stated otherwise. In all experiments, ultra-pure deionized water (18 mΩ) was used (Smart2Pure3™ Barnstead aqua purification system (Thermo Fisher Scientific, Waltham, MA, USA)). The enzyme activities were measured according to the assays detailed by Brodkorb and colleagues [10 (link)]. A mixture of seven native proteins (14.4–116 kDa), unstained for use as size standards in protein electrophoresis, were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Methanol (99.8% LC-MS) was purchased from VWR International (Barcelona, Spain). Ultra-pure water was obtained from a Milipak^® Express 40 system (Merk-Milipore, Dormstadt, Germany). Formic acid was purchased from Panreac Química (Barcelona, Spain). Sephadex LH-20, standards (isorhamnetin, quercetin, rutin, gallic acid, 4-hydroxybenzoic acid), amino acids (glycine, asparagine, glutamine, glutamic acid, proline, and tryptophan), α-amylase (10080; 79 U mg/L), pepsin (P6887; 791 U mg/L), pancreatin (P7545, 17 units TAME per mg), bile (B8381), and other reagents used for the in vitro digestion assay were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Betanin was purified from a betalain-rich concentrate extracted from commercial beetroot and betaxanthins were semi-synthesized using purified betanin [8 (link)]. Piscidic acid was purified from prickly pear peels by semi-preparative high-performance liquid chromatography (HPLC) [8 (link)]. Standards for isorhamnetin and kaempherol glycosides were kindly provided by Dr. Serna-Saldivar’s laboratory from Centro de Biotecnologia FEMSA (Escuela de Ingeniería y Ciencias, Instituto Tecnológico de Monterrey, Monterrey, Mexico), where these compounds were previously isolated from Opuntia cladodes [22 (link),23 (link)]. All isolated and semi-synthetized standards were analyzed for authenticity and purity by HPLC-ESI-MS-QTof.

Buckwheat flour was obtained from Liangchen Century Grain Co., Ltd. (Wuxi, China). Psyllium fibre was provided by Xuzhou Nature Food Co., Ltd. (Xuzhou, China). Instant dry yeast was purchased from Angel Yeast Co., Ltd. (Yichang, China). Maize oil and salt were obtained from local stores in Wuxi, China. Pepsin (P7000) and pancreatin (P7545) used for starch digestion assays were obtained from Sigma–Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Amyloglucosidase was obtained from Megazyme Inc. (Bray, Ireland). The glucose quantification kit, which employs the glucose oxidase method, was obtained from Nanjing JianCheng Bioengineering Institute (Nanjing, China).

The in vitro digestion of dietary starch was determined in quadruplicate as described by Englyst et al. with minor modifications [16 (link)]. In brief, samples (about 1 g) were incubated in a HCl solution (10 mL; 0.05 mol/L) containing 0.05 g pepsin (P-7000; Sigma-Aldrich) and 0.05 g guar gum (P-9000-30-0; Sigma-Aldrich). Tubes were incubated at 37 °C for 30 min with vibration. Then, sodium acetate solution (10 mL; 0.25 mol/L) and a mixed enzyme digestive juice [5 mL; 0.7 g pancreatin (P-7545; Sigma-Aldrich, Darmstadt, Germany), 0.05 mL amyloglucosidase, and 3 mg invertase (P-57629; Sigma-Aldrich, Darmstadt, Germany)] was added to each tube. After incubating at 37 °C for 0.25, 0.50, 1, 1.5, 2, 3, and 4 h, an aliquot of 0.5 mL was taken respectively to which absolute ethanol was added for stopping the digestion of the starch. Then the samples were centrifuged at 3000 × g for 10 min to get supernatant. The glucose content of the supernatant was measured using a commercial glucose detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The probiotic culture of B. coagulans (TISTR 1447) was sourced from the Thailand Institute of Scientific and Technological Research (TISTR) for our research conducted at Maejo University’s Food Safety and Biotechnology Lab. Gastrointestinal fluids were simulated using pepsin porcine (P6887) and pancreatin (P7545) from Sigma-Aldrich Co. in the St. Louis, MI, USA. In this research project, we utilized chemicals of analytical reagent-grade quality.