Raman spectroscopic determination of extent of deamidation of food proteins

Abstract

Soy protein isolates, egg white protein and whey protein isolates are utilized widely in the food industry as functional ingredients. Deamidation has been reported to improve the functional properties of these three protein products and other food proteins. The extent of deamidation in acid-treated proteins is measured commonly by an ammonia electrode. However, this method is tedious and time-consuming. Our objective is to develop a new analytical technique based on Raman spectroscopy to measure the extent of deamidation of food proteins.The 3 proteins were deamidated to varying degrees by treatment with hydrochloric acid, and the degrees of deamidation were determined by analyzing ammonia generated by an ammonia electrode. Raman spectra were recorded by a Renishaw Raman Imaging Microscope equipped with a 514 nm Argon ion laser. The laser was focused on the solid protein samples placed on microscopic slides. Deamidation of the 3 proteins led to the appearance of a new 1780 cm^-1 band in the Raman spectra, attributed to the liberation of carboxyl groups from glutamine and asparagine residues. The ratio of the new band to a 1003 cm^-1 phenylalanine stretch band (used as an internal standard) was plotted against the extent of deamidation determined by ammonia electrode. A linear fit was obtained with a correlation coefficient (R²) of 0.9914 for soy protein isolates, 0.9982 for whey protein isolates and 0.9813 for egg white protein. The data clearly demonstrate a strong linear relationship between the 1780 cm^-1 band intensity and level of deamidation. The Raman spectroscopic technique provides an alternative method for determining level of deamidation of food proteins with several advantages. The procedure is simple, fast, non-destructive, and requires almost no sample preparation making it convenient for routine analysis. Without the need for any chemical reagents, the proposed method is safe and environmentally friendly.