Identification of the wheat seed protein CM3 as a highly active emulsifier using a novel functional screen

Lyophilized albumin protein fractions were prepared from flour of four varieties of wheat: Triticum aestivum cvs. Mercia and Riband, Triticum aestivum var. spelta, and Triticum turgidum var. durum (Kamut). The dry powders were redissolved in sodium phosphate buffers at pH 3.0, 6.5, or 8.0 and at ionic strengths of 0.1 or 1.0 M to a concentration of 0.1% (w/v). Emulsions formed by sonication of protein solutions with n-hexadecane were aged at room temperature and separated into aqueous, interstitial, and interfacial phases. The distinct emulsion components were lyophilized and analyzed by RP-HPLC. A protein was observed to be preferentially located in the interfacial component and subsequently purified from a total albumin fraction and identified by N-terminal sequencing as CM3, an α-amylase inhibitor subunit. Measurement of the equilibrium surface tension of CM3 as a function of protein concentration demonstrated that it was at least as active as bovine β-lactoglobulin, an established protein emulsifier. Furthermore, measurement of the surface dilational elastic modulus at an air/water interface demonstrated the formation of a viscoelastic film, while fluorescence and FT-IR spectroscopic measurements on adsorbed and nonadsorbed CM3 suggest that the secondary structure is essentially unchanged upon adsorption to an oil/water interface. It is concluded that functional screening is a valid approach to identify novel protein emulsifiers in complex mixtures.