P7000

A modified 3-step in vitro procedure described by Gargallo et al. [29 (link)] was used to determine intestinal digestibility of rumen undegradable protein (IDP). Briefly, dried duplicate undegradable residues from the 16 h in situ ruminal incubation were pooled and ground to pass through a 1-mm sieve. Six sub-samples of dried sample residues (500 mg each) were then weighed into Ankom F57 filter bags and heat-sealed. Twenty-four sample bags were incubated in each incubation jar of a Daisy^II incubator (ANKOM Technology, Fairport, NY, USA) containing 2 L of 0.1 M pre-warmed HCl solution (pH = 1.9) and 1 g L⁻¹ of pepsin (P-7000, Sigma, St. Louis, MO, USA), with constant rotation at 39 °C for 1 h. After incubation and washing, sample bags were reintroduced into the same incubation jars containing 2 L of pre-warmed pancreatin (0.5 M KH₂PO₄ buffer, pH = 7.75, 50 mg/kg of thymol, and 3 g/L of pancreatin (P-7545, Sigma)) and incubated with constant rotation at 39 °C for 24 h. After removal, bags were rinsed with tap water until the water ran clear, dried at 55 °C for 48 h, and reweighed. Undegradable residues from ruminal and intestinal incubations were analyzed for nutrient concentration.

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.

A pepsin and trypsin resistant gliadin (PT-gliadin) was prepared according to the method previously described with minor modifications [12 (link)]. Each 50 g Sigma gliadin (G-3375; Sigma, St. Louis, MO) or 50 g wheat flour was dissolved in 500 ml 0.2 N HCl for 2 h at 37 °C with 1 g pepsin (P-7000; 800–2,500 units/mg protein, Sigma, St. Louis, MO). The resultant peptic digest was further digested by addition of 1 g trypsin (P-8096, activity, 4x USP specifications; Sigma, St. Louis, MO), after pH adjusted to 7.4 using 2 M NaOH. The solution was stirred vigorously at 37 °C for 4 h, boiled to inactivate enzymes for 30 min, lyophilized, and then stored at −20 °C until used. PT-gliadin was freshly suspended in a sterile phosphate buffered saline (PBS, 0.15 M NaCl, 0.0027 M KCl, 0.0081 M disodium phosphate and 0.0015 M monopotassium phosphate, pH 7.2) to a final concentration of 1 mg/ml. PD-casein (BactoTryptone, Sparks, MD) was used as a negative control in the experiment.

The in vitro gastrointestinal digestion was based on the protocol as detailed by Minekus et al. [24 (link)]. Simulated salivary fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared following the scheme previously reported [24 (link)]. In particular, the method (scaled up for 500 µL of liquid sample) included an oral phase composed of SSF at pH 7.0, with salivary α-amylase (75 U/mL; from human saliva Type IX-A, Sigma-Aldrich Co. Milan, Italy). The oral step was run at 37 °C for 2 min. Then, the oral bolus samples were mixed (ratio 1:1) with the SGF at pH 3.0 containing porcine pepsin (2000 U/mL; P7000; Sigma-Aldrich Co. Milan, Italy). The gastric phase was carried out for 120 min at 37 °C. Gastric chyme was mixed (1:1) with the SIF at pH 7.0 containing pancreatin (100 U/mL; P1750; Sigma-Aldrich Co. Milan, Italy) and bile salts (10 mM; B8631; Sigma-Aldrich Co. Milan, Italy). The intestinal phase was carried out for 120 min at 37 °C. During the in vitro digestion, appropriate amounts of HCl (1 M) and NaOH (1 M) were added for pH adjustment. At selected time points (i.e., gastric and pancreatic end-phases), corresponding digestion sample tubes for each EVOO sample were cooled on ice to stop the reaction. The experiment was performed in triplicate (n = 3) and all the in vitro digestion steps were carried out in amber bottles in the dark.

The gastric digestion was obtained using pepsin from porcine gastric mucosa ≥250 units/mg (Sigma #P7000), and the intestinal digestion was obtained using pancreatin from porcine pancreas 4USP (Sigma #P1750), bile extract porcine (Sigma #B8631), and a 10 KDa dialysis membrane (SnakeSkin^® Dialysis Tubing, Thermo Scientific, Paisely, UK). Sodium bicarbonate was purchased from Normapur Prolabo, France. Sodium hydroxide, hydrochloric acid 6 M, potassium chloride and sodium chloride were used in the digestion and were purchased from Sigma Aldrich.

In vitro starch digestibility was measured according to the study of Goh et al. [16 (link)]. The cooked noodles (50 mg) homogenized in distilled water were mixed with 10 mL of HCl-KCl buffer (pH 1.5) and 0.2 mL of pepsin solution (0.1 g/mL) (P7000, from porcine gastric mucosa, 250 U mg⁻¹, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) in a shaking water bath at 40 °C for 1 h. Then, sodium acetate buffer (0.5 mol/L, pH 6.9) was added into the mixture and filled up to 25 mL, followed by the reaction with 5 mL α-amylase (2.6 IU) (10,080, from hog pancreas, 50 U mg⁻¹, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) in the water bath at 37 °C. The solution (1 mL) was taken out at 0, 15, 30, 45, 60, 90, 120 and 180 min, respectively, and the reaction was stopped in a 100 °C water bath for 5 min. The content of reducing sugar in the obtained solution was determined by DNS method, with glucose as the standard. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) were defined as the fractions digested within 15 min, between 15 and 120 min and undigested after 120 min, respectively. The contents of RDS, SDS and RS were calculated according to reported equations [17 (link)].