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.
Sigma a 7095
Manufactured by Merck Group
Sourced in Italy
Sigma A-7095 is a laboratory instrument designed for gas chromatography (GC) applications. It functions as a dual-channel flame ionization detector (FID) to facilitate the separation, identification, and quantification of chemical compounds in complex mixtures. The device offers consistent and reliable performance in analytical procedures.
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3 protocols using sigma a 7095
1
In Vitro Starch Digestion Assay
2
In vitro Starch Digestion Kinetics
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After a simulated mastication step using a meat mincer, cooked samples (i.e., 2 g) were subjected to a gastric digestion phase in a 0.05 M HCl solution (pH = 2) containing pepsin (5 mg/mL; Sigma P 7000; Sigma-Aldrich Co., Milan, Italy) for 30 min at 37 °C under agitation [24 (link)]. Then, the pH was adjusted to 5.2 by adding 0.1 M sodium acetate buffer, and the pancreatic phase at 37 °C was simulated through the addition of pancreatin (7500 FIP-U/g; Merck 7130, Merck KGaA, Darmstadt, Germany), amyloglucosidase (300 U/mL; Sigma A-7095, Sigma-Aldrich Co., Milan, Italy) and invertase (300 U/g; Sigma I-4504, Sigma-Aldrich Co., Milan, Italy) [24 (link),26 (link)]. Aliquots (2 mL) were taken every 30 min up to 180 min of the pancreatic phase, mixed with absolute ethanol, and the amount of the released glucose was determined (GODPOD 4058, Giesse Diagnostic snc, Rome, Italy). The percentage of digested starch was calculated using a factor of 0.9. The starch hydrolysis index (HI) was derived from the area under the starch hydrolysis curve (0–180 min) with white wheat bread (total starch content of 72.3 g/100 g DM), as a reference [24 (link),26 (link)]. Analyses were run in triplicate.
3
Enzymatic Starch Digestibility Analysis
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An enzyme solution was freshly prepared by adding porcine pancreatic α-amylase (Sigma A-3176, Sigma-Aldrich, UK; 16 U/mg) (1.5 g) in a sodium acetate buffer (pH 5.2) (10 mL). The mixture was incubated at 37°C for 10 min and centrifuged (1500 g) for 10 min. The supernatant was transferred into a beaker and mixed with amylo-glucosidase (Sigma A-7095, Sigma-Aldrich, UK; 300 U/mL) at 8:1 (v/v) (Mutungi et al., 2011) . The modified rice flour (100 mg) was suspended in a 0.1 M sodium acetate buffer (21 mL) and incubated at 37°C with continuous shaking (200 strokes/min) for 15 min. The freshly prepared enzyme solution (1.6 mL) was added to the suspension, mixed for 1 min and incubated at 37 °C in a shaking water bath (200 strokes/min). After incubation for 20 and 120 min, an aliquot (0.2 mL) was taken and added into absolute ethanol (4 mL), mixed well and centrifuged (5000 g) for 10 min. The supernatant was then collected for RDS and SDS determination. The glucose content was measured by adding a glucose oxidase-peroxidase assay kit (GOPOD, Megazyme International, Ireland) into the aliquot and incubated at 50°C for 20 and 120 min. The aliquot was then measured in a spectrophotometer at 510 nm absorbance. Starch fractions that were digested (measured as % glucose) within 20 min and 20-120 min were calculated as RDS and SDS (Lin et al., 2009; Englyst et al., 1992) .
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