Resistant starch is the starch not hydrolyzed by pancreatic a-amylase. Hydrolysis products, solubilized in 80% ethanol, are discarded. Resistant starch, present in the pellet, is solubilized in 2N KOH then hydrolyzed into glucose with an amylo-glucosidase. Glucose is then quantified with a glucose oxidase/peroxidase analysis kit.
Tris maleate buffer 0.1M pH 5.25 containing CaCl2 4 mM and sodium azide (0.02%).
A: 0.2M tris maleic acid (24.2 g of tris hydroxymethylaminomethane and 23.2 g of maleic acid in 1 L) B: 0.2M NaOH
(250 ml of A + 48.5 mL of B) then 588 mg CaCl2, 2H2O and 200 mg NaN3 diluted with water to 1000 mL final volume Pancreatic a-amylase Sigma ref A-3176 (500U) 10 mg/mL of tris maleate buffer Acetic acid 0.5M, CaCl2, 2H2O 4 mM
28.6 mL pure acetic acid and 4 ml CaCl2, 2H2O 1M (1.47 g in 10 mL
H2O) diluted with water to 1000 mL final volume "S
Solution of saturated benzoic acid
Glucose standard 2.5 mg/mL (2.5 g of anhydrous glucose dehydrated t?
under vacuum) in 1L with 50% saturated benzoic acid solution Potassium hydroxide 4M (224.4 g/L)
Amyloglucosidase Novo Nordisk Bioindustries AMG 400L, 400 AGU/ |
Sol. 1: 6 AGU/mL. Dilute the enzyme solution: 0.15 mL + 9.85 mL I
Sol. 2: 14 AGU/mL. Dilute the enzyme solution: 0.14 mL + 3.86 mL i,
GOD-PAP kit Merckotest ref Merck 14365
Determine total starch content of the sample
Weigh the amount of sample containing 50 mg of starch in centrifuga-tion tubes of 100 mL.
Add 5.8 mL of tris maleate buffer and 4 mL of enzymatic solution (10 mg of pancreatic a-amylase/mL of buffer) and 0.2 mL amyloglucosi-dase 6AGU/mL (Sol. 1)
Mix and incubate in a shaking bath at 37°C during 16 h. Add 40 mL absolute ethanol, mix and leave 1 h at ambiant temperature. Centrifuge 10 min at 3000 g.
Discard the supernatant and rinse the pellet with 10 mL 80% ethanol, centrifuge and discard the supernatant (twice). Collect the pellet with 10 mL H2O. Leave in a shaking bath during 30 min. Cool in ice.
Add 10 mL KOH 4M and shake under magnetic shaking at 0°C during 30 min.
Transfer 1 mL in 10 mL acetic acid solution 0.5M containing CaCl2 and add 0.2 mL amyloglucosidase 14 AGU/mL (Sol. 2). Incubate 30 min in a bath at 70°C then 10 min at 100°C. Cool
Neutralize with 0.6 mL KOH 4M. Centrifuge 10 min at 4000^
Collect 0.1 mL of supernatant in 2 mL of GOD-PAP reactant. Shake, leave 20 min at 37°C and read the absorbance at 510 nm. In parallel, prepare 2 glucose standards and 2 enzyme blanks in the same conditions: 0.5 mL of glucose standard or 0.5 mL H2O diluted in a same volume of 4M KOH are transferred to tubes containing 10 mM acetic acid solution, incubate 30 min at 70°C then 10 min at 100°C, cool, neutralize, centrifuge and then analyze the glucose as previously described.
• The samples are analyzed in duplicates. Calculation
Resistant starch % of the dry matter = Ae*Vt*C*D*0.9/(Ast*We)
Ae = Absorbance of the sample corrected from the absorbance of the enzyme blank
Vt = Volume of the test (10) D = Dilution factor
C = Concentration in mg/mL of glucose standard (2.5)
Champ et al.
Ast = Absorbance of the standard corrected from the absorbance of the enzyme blank and diluted like the sample
We = Weight of dry sample in mg
0.9 is the correction factor glucose ^ starch
Fresh samples like canned lentils or beans or cooked pasta have to be passed through a mincer (Plate of 9 mm diam. holes) before weighing (equivalent of 50 mg starch).
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