Simplified Dilute Acetic Acid Based Extraction Procedure for Fractionation and Analysis of Wheat Flour Protein by Size Exclusion HPLC and Flow Field-Flow Fractionation

A simple, highly efficient and reproducible two-step extraction procedure using dilute acetic acid without (AN) and then with sonication (AS) has been developed for the fractionation of wheat flour protein. Approximately 97% of total protein was extracted from a Canadian hard red spring wheat flour; an additional 1.2% protein could be recovered by further extraction with 1% DDT and 50% 1-propanol (AR). Size-exclusion HPLC (SE-HPLC) and flow field-flow fractionation (flow FFF) showed that the AN extract, which accounted for most of the total extractable protein (AN + AS + AR), consisted primarily of monomeric protein. The AS extract was composed primarily of polymeric proteins. Flow FFF showed that AN polymeric protein, including that eluting at the SE-HPLC void volume, showed smaller Stokes diameters than AS polymeric protein. Flow FFF profiles of AS SE-HPLC subfractions showed that the void volume subfraction contained monomeric and small polymeric protein in addition to large polymeric protein, indicating formation of larger complexes through interaction between some or all of the components. AN and AS extracts, as well as SE-HPLC and flow FFF fractions thereof, showed a fairly wide range of values among 12 Canadian hard red and white spring wheat cultivars. The proportion of total protein in the AS extract and in the larger sized polymeric protein fractions from SE-HPLC and flow FFF were highly positively correlated to farinograph mixing time.