Analysis of wheat storage proteins by exhaustive sequential extraction followed by RP-HPLC and nitrogen determination

Abstract

Wheat storage proteins are responsible for the viscoelastic properties of dough. Their effect on dough rheology depends on the glutenin components and the proportion of gliadins, high and low molecular weight glutenin subunits (HMW‐GS, LMW‐GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A method of sequential extraction and quantification of wheat flour proteins was developed. Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) was used to quantify gliadins and HMW‐GS and LMW‐GS. Non‐prolamin proteins and lipids were extracted with phosphate buffer and phosphate buffer containing Triton X114 respectively and 70% (v/v) ethanol was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0·05 M tetraborate pH 8·5, 2% (v/v) 2‐mercaptoethanol, 1 g litre−1 glycine, and 8 M urea was selected. It facilitated the most complete extraction and was compatible with RP‐HPLC analysis of extracts. The quantities of extracted prolamins were estimated from RP‐HPLC profiles using peak area determination. The concentration of different classes of prolamins obtained by RP‐HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars was 0·99 (coefficient of variation (CV)=7·6%) for gliadins and 0·89 (CV=9·1%) for glutenins. Quantifications of proteins by N determination and RP‐HPLC were strongly correlated: regression coefficients were 0·86 for total prolamins and gliadins, and 0·71 for glutenins with the gradient of regression of approximately 1. Therefore, RP‐HPLC could be used to quantify prolamin groups without using N determination. © 1998 SCI.

Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens

“…Crude gliadins were extracted from wheat flour (Hardi cultivar) using the sequential procedure developed by Osborne et al as adapted by Nicolas et al . Briefly, the albumin‐globulin fraction of the flour was removed by several washings with saline buffer; the flour was then suspended in 70% ethanol for 1 h at room temperature to solubilize the gliadins.…”

Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation

“…Gliadins were extracted from wheat flour (cultivar Hardi) using a sequential procedure . Briefly, the albumins/globulins (water‐/salt‐soluble proteins from wheat flour) were extracted from the flour with a saline buffer; after several washings, the flour was suspended in 70% ethanol for 1 h at room temperature; after centrifugation, the supernatant, that is the total gliadins, was collected.…”

Food allergy to wheat: identification of immunogloglin E and immunoglobulin G‐binding proteins with sequential extracts and purified proteins from wheat flour

“…Wheat proteins were extracted from 100 mg of flour (cultivar Etoile de Choisy) according to the sequential procedure of Osborne [4], adapted by Nicolas et al [33]. Albumins/globulins were extracted in 3 mL 0.05 m phosphate/0.1 m NaCl buffer pH 7.8 during 1 h at 4 °C with constant stirring.…”