Improved in vitro assay of resistant starch in cross-linked phosphorylated starch

In the AOAC Method 2002.02 assay for resistant starch (RS), the enzyme-resistant starch is solubilized by cold 2 M KOH, so that it can be converted enzymatically to glucose. However, cross-linked phosphorylated (CLP) starch is not soluble in cold 2 M KOH and therefore cannot be assayed directly. In this study, the enzyme-resistant starch from CLP starch was treated by 1) the directions in AOAC Method 996.11for total starch assay using hot DMSO or cold KOH; 2) two back-to-back incubations with thermal-stable α-amylase in a boiling water bath; and 3) a KOH treatment followed by two back-to-back incubations with thermal-stable α-amylase in a boiling water bath. In the last approach, the alkaline was neutralized with HCl, and the salt was removed by centrifugation. More than 95% of the RS from the CLP starch was converted to glucose. This improved method is recommended to directly determine RS in CLP starch.