Sigma p 7000

A 2-step (i.e., gastric and pancreatic phases) static in vitro starch digestion procedure was used [22 (link)]. Briefly, samples were inserted in 50 mL tubes containing glass balls and pre-treated with a 0.05 M HCl solution (5 mL) containing pepsin (5 mg/mL; Sigma P-7000, Sigma-Aldrich^® Co., Milan, Italy) for 30 min at 37 °C. The pH was then adjusted to 5.2 by adding 0.1 M sodium acetate buffer before the addition of 5 mL of an enzyme mixture with an amylase activity of about 7000 U/mL given by pancreatin (about 7500 FIP-U/g; Merck 7130, Merck KGaA, Darmstadt, Germany), amyloglucosidase (about 300 U/mL; Sigma A-7095, Sigma-Aldrich^® Co., Milan, Italy), and invertase (about 300 U/g; Sigma I-4504, Sigma-Aldrich^® Co., Milan, Italy) [22 (link)]. Aliquots (0.5 mL) were taken from each tube at 0 (before the addition of the enzyme mixture simulating the pancreatic phase), 30, 60, 120, and 180 min after the addition; absolute ethanol was added, and the amount of released glucose was determined colorimetrically (GODPOD 4058, Giesse Diagnostic snc, Rome, Italy). A blank was also included. A factor of 0.9 was used to convert mono to polysaccharide. The in vitro predicted glycemic index (pGI) was calculated as reported by Giuberti et al. (2015) [22 (link)]. For each treatment, samples were analyzed in triplicate.

Extra in situ rumen incubation was performed with the same method described above, only that samples were incubated for 16 h. Intestinal digestibility of rumen undegradable protein (RUP) was determined by using three-step in-vitro method described by Calsamiglia and Stern [12 (link)]. Briefly, residues at 16 h in situ rumen incubation time were collected, rinsed until the water was clear, dried at 55 °C for 48 h, and grounded to pass through a 1-mm screen Wiley mill. Then, 1 g of residues were weighted, placed into nylon bag, and incubated in HC1 solution (pH = 1.9) containing 1 g of pepsin (sigma, P-7000, Sigma Chemical, St. Louis, MO, USA) at 39 °C for 1 h. The samples from the five different blocks were set as repetitions, and all tests were run in triplicates. After incubation, the bags were rinsed under cool tap water until all the buffer were removed. Then, the bags were placed into culture bottle with 2 L of preheated trypsin solution (pH = 7.75, KH₂PO₄ buffer solution) (sigma P-7545, Sigma Chemical, St. Louis, MO, USA) containing 50 μg/L of thymol and 3 g of trypsin per liter, and shake cultured at 39 °C for 24 h. After that, the sample bags were rinsed again under tap water until the water was clear, and subsequently dried at 55 °C for 48 h before the measurement for N content.

To initiate gastric digestion in a conical flask, 5 g of feed (6 replicates) was added to 12.5 mL G-buffer with pepsin (0.4 g) from porcine gastric mucosa (EC 3.4.23.1, Sigma P7000, >250 units/mg; Sigma-Aldrich, St. Louis, MO, USA) at 39°C for 4 h in a shaking water bath at 150 rpm. The reaction was stopped by adding 0.625 g NaHCO₃ and adjusting the pH to 6.8 with 0.2 N HCl or NaOH. In the intestinal phase digestion, 200 mL of prewarmed (39°C) I-buffer was added to the conical flask and mixed well. Next, 0.5 g pancreatin from porcine pancreas (8×USP, Sigma P7547; Sigma-Aldrich, USA) in 50 mL I-buffer was slowly added to the flask in the shaking water bath using a syringe. The intestinal phase digestion proceeded at 39°C for the following 16 h in a shaking water bath at 150 rpm. A check valve was set on the top of the flask to release the accumulated gas during the long reaction time. After the digestion process, the flask was moved to an ice bath to stop the reaction and transfer the content into a 500 mL centrifuge tube. The residue was collected by centrifugation at 8,000×g for 20 min (Model 7000; KOUBOTA, Tokyo, Japan). The precipitate was resolved in 5 mL of distilled water then frozen at −80°C overnight. All collected samples were dried by lyophilization and stored at 4°C as the fermentation substrate after weighing.