Assay Procedure for Resistant Starch Determination The assay procedure for resistant starch was adapted from

AOAC Method 2002.2 and AACC method. Measurement of Resistant Starch Hydrolysis and solubilisation of non-resistant starch An accurately weighed 100 + 5 mg of sample was placed in a 16 x 125 mm tube. A 4.0 ml of pancreatic α – amylase containing 3 U/ml amyloglucosidase was then added to each tube and mixed. The tubes were then attached horizontally in a shaking water bath aligned in the direction of the motion. Samples were incubated at 37 °C with continuous shaking for exactly 16 hours. After incubation, the tubes were removed from the water bath and the excess surface water was removed using a paper towel. The contents were treated with 4.0 ml 99 % ethanol with vigorous stirring on a vortex mixer. The tubes were centrifuged at approximately 3,000rpm for 10 minutes. After centrifugation, the supernatant was carefully decanted into collecting flask and the residue was re-suspended by adding 2 ml of 50% ethanol with vigorous stirring on a vortex mixer. Another 6ml of 50% ethanol was added to resuspend the residue and again subjected to centrifugation at 3,000 rpm for 10 minutes. The supernatant was carefully decanted and the suspension and centrifugation process was repeated once more. The supernatant was again decanted after the final suspension and centrifugation. The tubes were inverted on absorbent paper to drain excess liquid. The collected

0 ml of distilled water was added and again mixed using a vortex mixer. The reaction was stopped by placing the tubes in an ice cold water bath and then cooled to room temperature. From that extract. The final volume of the sample was approximately 10.0 ml of aliquot (in triplicate) was obtained and to which 2.resistant Starch (Solubilised Starch) .8 was added to each tube with stirring followed by immediate addition of 0. The tubes were covered with marbles upon heating in a boiling water bath for 10 minutes. The resistant starch was dissolved with 2M KOH with stirring for approximately 20 minutes in an ice/water bath.supernatant was used for the analysis of non-resistant starch while the residue was use for the resistant starch measurement. 3. The absorbance was then obtained by reading the sample at 500 nm. Since the samples have < 10 % RS content.2 M sodium acetate buffer. Measurement of Resistant starch The residue obtained from the hydrolysis and solubilisation of non-resistant starch contained the non-digestible starch or the resistant starch of the sample. pH 3.3 ml without dilution. 1. After cooling. The tubes were placed in a water bath at 50°C for incubation in 30 minutes with intermittent mixing on a vortex mixer. Then.0 ml of 3.1 ml of 3300 U/ml amyloglucosidase. Measurement of Non.5-dinitrosalysilic acid solution was added and mixed well.0 ml of 1. an 8. direct centrifugation at 3000 rpm for 10 minutes was done.

3. the so-called reducing sugars. The volume was adjusted to 100 ml with water in a volumetric flask and was then mixed well.5-dinitrosalicylic acid) Method DNS method is a test for the presence of free carbonyl group (C=O). glucose and the ketone functional group in fructose. DNS (3. for example. which itself is not necessary for the color reaction. Simultaneously.5-nitrosalicylic acid under alkaline conditions: oxidation aldehyde group reduction 3.5dinitrosalicylic acid (DNS) is reduced to 3-amino.5-dinitrosalicylic acid carboxyl group 3-amino. . is added in the reagent to absorb the dissolved oxygen. This involves the oxidation of the aldehyde functional group present in. A 1.0 ml aliquot of the solution (in triplicate) was obtained and glucose concentration was determined using the DNS method described in the previous paragraph.Non-resistant starch was measured by combining the supernatant solutions obtained on centrifugation of the initial incubation with supernatants obtained from the subsequent two 50% ethanol washings during the hydrolysis and solubilisation of nonresistant starch. sulfite.5-nitrosalicylic acid Because dissolved oxygen can interfere with glucose oxidation.

The presence of glucose in the sample was confirmed by red brown color of the solution after addition of 3. Reaction of DNS with glucose.non-resistant (solubilised) starch and total starch content (%.5dinitrosalicylic acid.5-dinitrosalicylic acid.5-dinitrosalicylic acid) was reduced to 3amino-5-nitrosalicylic acid while glucose was oxidized to gluconic acid. Calculations Calculation of resistant starch .The above reaction scheme shows that one mole of sugar will react with one mole of 3. DNS (3.on a dry weight basis) in test samples as follows: . Absorbance was read at 500 nm with the absorption of the –NH2 group. Figure 8.

Resistant Starch (g/100g sample)(samples containing < 10% RS): ( ) Non-Resistant (Solubilised) Starch (g/100g sample): ( ) Total Starch = Resistant Starch + Non-Resistant Starch .

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