Surface staining of wheat starch granules with remazolbrilliant blue R dye and their extraction with aqueous sodium dodecyl sulfate and mercaptoethanol

Wheat starch granules were stained with Remazolbrilliant blue R (RBB) dye. The RBB-stained starch granules (RBB-starch) were successively extracted with 1% sodium dodecyl sulfate (SDS) solution containing 1% 2-mercaptoethanol (2-ME) at room temperature for 1 day (extraction 1), 5 days (extraction 2), and 15 days (extraction 3). The stained molecules were solubilized first, an indication that the RBB dye was bound to the granule surface. After each extraction with 1% SDS solution containing 1% 2-ME, granules were examined by scanning electron microscopy. The appearance of the residual starch structure changed greatly between extraction 2 and 3. After extraction 3, only a shell-like structure was present. X-ray diffraction analysis of this sample indicated an absence of crystallinity. The SDS extracts were subjected to Sepharose CL-2B gel-filtration chromatography. Two blue peaks were observed for extraction 1 : one high and one low molecular size. The low molecular weight peak decreased for extraction 2 and was absent for extraction 3. The high molecular weight blue peak did not disappear, although the ratio of unstained carbohydrate to RBB-stained carbohydrate increased with time. The low molecular weight blue peak was further subjected to the Sephadex G-50 gel-filtration chromatography, and the resulting single peak was analyzed for sugars, proteins and peptides, and lipids. Only lipids were found. The main TLC spot was RBB stained and indicated lysophosphatidylglycerol by Dittmer and periodate spray reagents.