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Production and Purification of Whey Protein Glycate Conjugated with Low Molecular Mass Dextrans
An increasing number of people are suffering from food protein allergies, which has become a growing concern around the world. One possible helpful approach could be to glycate food proteins via Maillard reaction, which may block the IgE binding epitopes of the allergen that are responsible for eliciting an immune response. The goal of this research was to optimize processing conditions for creating and purifying whey protein isolate (WPI) glycate with dextran (DX) of 3 different molecular masses. Purification involved a combination of isoelectric precipitation and ion exchange chromatography. Glycates were characterized using size–exclusion chromatography coupled with multi–angle laser–light scattering (SEC–MALLS), glycoprotein analysis using SDS–PAGE and periodic acid Schiff’s glycoprotein staining test with fluorescence laser densitometry. The optimal conjugation condition chosen from these experiments were 10% WPI–30% Dextran (DX), pH 6.5, 62ºC for 24h for DX of molecular mass = 10 and 3.5 kDa, and 50ºC for 12h for 1 kDa DX. The optimal purification process was performed by ion–exchange chromatography: for G10 (glyate with 10 kDa DX) we used pH 2 running buffer, followed by 0.55M NaCl elution buffer; for G3.5 and G1 (glycate with 3.5 and 1 kDa DX, respectively) we used pH 3 running buffer, followed by 0.52 and 0.5M NaCl running buffer for G3.5 and for G1, respectively. The resulting protein–DX molar ratios were estimated as 1:1.6 and 1:1.8 for G10 and G3.5 with a purity of 91% and 88%, respectively. Future work will focus on examining the allergenicity of the different molecular masses of WPI–DX glycates, using blood sera from cow’s milk protein allergic patients.
Keywords: Dextran, Whey protein isolate, Maillard reaction