Porcine pancreatic lipase

Gallic acid, rat intestinal acetone powder, porcine pancreatic α-amylase, 4-methylumbelliferone, glucose oxidase kits and 3,5-dinitrosalicylic acid p-nitrophenylbutylrate (p-NPB), oleic acid, phosphatidylcholine, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, porcine cholesterol esterase, porcine pancreatic lipase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cholesterol test kits were purchased from HUMAN GmbH Co. (Wiesbaden, Germany). Total bile acid kit was purchased from Bio-Quant Co. (San Diego, CA, USA). All other chemical reagents used in this study were of analytical grade.

The (‘Pera’ variety) orange waste extracts and the commercial porcine pancreatic lipase (Sigma-Aldrich^®) were used in the hydrolysis of crude soybean oils and soybean oil wastes using 5% lipase extract over the reaction medium volume. Crude oil- and waste-reagent blanks were prepared [21 ]. The hydrolysis rate was calculated by taking into consideration the NaOH volume used in the titration (mL), multiplied by the concentration and molar mass of the base. The resulting value was then divided by the oil mass (g) and multiplied by the oil saponification index; then, the result was multiplied by 100 in order to be expressed as percentage.
The analyses were performed in duplicate, whereas the hydrolysis data analyzed through acid titration were compared through analysis of variance (ANOVA) and subjected to Tukey test, at 5% probability level, in the Assistat 7.7 beta software. The formed FAE (fatty acid esters) were analyzed through gas chromatography as described below.

The (‘Pera’ variety) orange waste extracts and the commercial porcine pancreatic lipase (Sigma-Aldrich^®) were used in the alcoholysis of the crude soybean oil and soybean oil waste, using 5% lipase extract over the reaction medium volume. The system was incubated for 72 hs [22 ]. Crude oil- and waste-reagent blanks were prepared. The analyses were performed in duplicate; the identification and quantification of FAE (fatty acid ethyl esters) were performed as described below.

The peptide AVFQHNCQE was synthesized by CASLO Aps. (Kongens Lyngby, Denmark) and its purity was 98.94%. Captopril was provided by Santa Cruz Biotechnology (Dallas, TX, United States). Pepsin, bile salts mixture, trypsin, α-chymotrypsin, porcine pancreatic lipase, colipase, trifluoroacetic acid (TFA), formic acid (FA) LC-MS grade, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Madrid, Spain). Acetonitrile and methanol LC-MS grade were purchased from Merck (Darmstadt, Germany). The synthetic isotopically labeled peptide IFV*TGQDYNDK (V* = Val-13C5,15N) was obtained from Biomatik (Ontario, Canada) and used as internal standard (IS) for MS experiments. Chicken foot hydrolysate Hpp11 was obtained by our group following the procedure described by Bravo et al. [22 ,24 (link)]. All other chemical solvents used were of analytical grade.

Egg L-α-phosphatidylcholine (PC, lecithin grade 1, 99% purity) was obtained from Lipid Products (South Nutfield, Surrey, UK). Porcine gastric mucosa pepsin, bovine α-chymotrypsin, pancreatic α-amylase, porcine colipase, porcine pancreatic lipase and bile salts were obtained from Sigma (Poole, Dorset, UK). Lipase for the gastric phase of digestion was a gastric lipase analogue of fungal origin (F-AP15) from Amano Enzyme Inc. (Nagoya, Japan). All flavonoid and other phytochemical standards were obtained from either Sigma-Aldrich (Poole, UK) or Extrasynthese (Genay, France). All solvents were HPLC grade, water was ultra-pure grade, and other chemicals were of AR quality.

Chitosan (MW: ~2100 kDa and DDA: 85%, determined using gel permeation chromatography and ¹H-NMR, respectively) was purchased from Marine Bio Resources Co., Ltd., Samutsakhon, Thailand. Ascorbic acid and H₂O₂ (50%, v/v) were acquired from Loba Chemie Pvt. Ltd., Mumbai, India. GAL, FER, CAF, CAT, and EGCG were obtained from Xi’an Julong Bio-Tech Co., Ltd. (Xi’an, China). Further, α-amylase, α-glucosidase, porcine pancreatic lipase, rat intestinal acetone powder, acarbose, orlistat, 4-nitrophenyl α-D-glucopyranoside (PNP-glycoside), and 4-methylumbelliferyl oleate (4-MU) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemicals used for AO activities were also acquired from Sigma-Aldrich (St. Louis, MO, USA). Microbial media were bought from HiMedia Laboratories, Mumbai, India.

FX was extracted and refined from brown algae, as described previously [48 (link)], and prepared and identified in our laboratory (purity ≥ 50%) [49 (link),50 (link)]. Analytically pure sodium borohydride, lithium aluminum hydride, sodium hydroxide, and sodium bicarbonate were obtained from National Pharmaceutical Holding Chemical Reagent Co., Ltd. (Shanghai, China). All enzymes, such as porcine pancreatic lipase, Pseudomonas cholesterol esterase, and Candida rugosa lipase, were obtained from Sigma-Aldrich (St Louis, MO, USA). Lipase (Brand: Kramer) was obtained from Ziyi Reagent Factory (Shanghai, China). The protein ladder was acquired from Thermo Fisher (Waltham, MA, USA). Escherichia coli strain BL21 (DE3) was preserved in our laboratory. HPLC-grade methanol was obtained from Merck KGaA (Darmstadt, Germany). Ultrapure water was produced using a Millipore Milli-Q system (Millipore Corp., Billerica, MA, USA). The chemical reagents used in this research were of analytical grade and purchased from Sigma-Aldrich (Saint Louis, MO, USA).