i followed two different protocols but my resistant starch sample does not dissolve easily in 90%DMSO and forms swollen granules due to which doesn't bind with iodine and shows lesser binding as compared to native .Can someone suggest me the best protocol for analyzing iodine binding ability and fatty acid binding ability of resistant starches?
We used this for In Vitro resistant Starch Assay
All test foods, except for the peas, were defatted before enzymatic starch digestion using petroleum ether (25 ml/g x 3, 15 min, ambient temperature). Fat-extracted samples (200 mg) were heated (3 min) to 750C with water (9 ml) to inactivate endogenous enzymes in the unprocessed products and cooled to room temperature before the pH was adjusted to 1.5-1.7 using IN HCl. Pepsin (5 mg) (E.C. 3.4.23.1, catalogue number P-6887, Sigma) was added and the samples were incubated (60 min, 370C) with shaking. Samples were neutralized with IM NaOH; phosphate buffer (pH 6.9, 10 ml of O.1M phosphate) and pancreatin (40 mg) (catalogue number P-7545, Sigma) were added and the samples were incubated (370C) again, this time for 1-6 hr, with shaking. Enzyme activity was terminated by rapidly heating the sample to boiling (Faulks and Bailey 1990). Undigested starch was determined in the pellet obtained by centrifugation (1,520 g, 10 min); the pellet was washed twice with 80% ethanol (5 ml). Starch was measured in duplicate in each of the three in vitro assays for each food after autoclaving, as described above.
Hello Samiya, I guess your RS is type 4, and to a better guess, might be the cross-linked ones.
I worked with type 2 and 3 RS and they were easily dissolved in DMSO 90%. But from the Woo and Seib 2002 It's been reported that RS4 could not be dissolved in DMSO.
I suggest you to use Megazyme kit for amylose determination.
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