Raman spectroscopic study of deamidated food proteins

Abstract

Three food protein products, soy and whey protein isolates and spray-dried egg white powder, were deamidated to various levels by 0.04 N hydrochloric acid. The extents of deamidation were determined using an ammonia electrode. Raman spectra of the modified proteins were obtained and analyzed. A new CO stretching vibration band was observed at 1780 cm⁻¹, attributed to γ-carboxyl groups of aspartic and glutamic acids. Calibration curves were constructed by plotting the intensity ratio of the 1780 cm⁻¹ band to the 1003 cm⁻¹ phenylalanine stretching band (as an internal standard) against the extent of deamidation. Linear fits were obtained with high correlation coefficients r > 0.987. Effect of deamidation on the conformation of these proteins was also studied by monitoring changes in Raman spectral characteristics, including a transition from ordered to disordered structures, exposure of tryptophan residues from a buried, hydrophobic microenvironment, and probable conformational changes of the aliphatic residues.

Introduction

Food proteins can be chemically modified to improve specific functional properties for effective use as food ingredients. Chemical modification is also a potential route for increasing the world’s protein food supply by making the products better suited for human and animal nutritional utilization (Feeney & Whitaker, 1985). Deamidation is an attractive way to improve the functional properties of food proteins by liberating carboxyl groups and thereby increasing negative charge and hydration. Deamidation is a specifically attractive modification for plant storage proteins which are rich in amidated glutamic and aspartic acid residues. Soy proteins, for example, have been reported to display significant improvement in functional properties, even at low levels of deamidation (Matsudomi, Sasaki, Kato, & Kobayashi, 1985). Improvement in solubility and some functional properties of soy okara protein by acid deamidation has been reported (Chan & Ma, 1999). Deamidation also led to marked improvement in solubility, emulsifying properties and water- and fat-binding capacities in oat protein isolates (Ma & Khanzada, 1987). Moreover, the deamidation of wheat protein isolate has more recently been investigated in an attempt to increase the quality and acceptability of a food product by improving stability and mouthfeel through improved functional properties (Webb, Naeem, & Schmidt, 2002).

Acid deamidation has advantages over other deamidation methods, including less severe conditions than heat-induced deamidation, without the toxicity problem as in base deamidation, and is more cost-effective than is enzymatic deamidation. However, acid deamidation can lead to the splitting of peptide bonds, and mild conditions are needed in order to minimize peptide bond hydrolysis. Deamidation leads to increases in the number of negative charges in proteins which may result in drastic changes in conformation. It has been reported that the helical content of wheat gluten decreased curvilinearly with decreases in level of deamidation, and it was suggested that the conformational changes might be caused by increased electrostatic repulsion and decreased hydrogen bonding (Matsudomi, Kato, & Kobayashi, 1982).

The level of changes in functional properties and conformation in deamidated food proteins is generally dependent on the degree of modification. Therefore, it is important to measure the extent of deamidation accurately in order to ensure that desirable functional properties are attained in the products. The extent of modification in acid-deamidated proteins is commonly measured by an ammonia electrode (Meyerson, McMurtrey, & Davis, 1978). However, this procedure is tedious and time-consuming. It would be desirable to develop an analytical method for solid samples, providing a convenient, non-destructive, and rapid determination of the extent of deamidation in modified proteins. Raman spectroscopy has recently been used in our laboratory to determine the degree of acetylation, succinylation and amidation in three food proteins (Wong et al., 2007, Zhao et al., 2004a, Zhao et al., 2004b). Advantages of this technique are that Raman spectroscopy can analyze samples in both liquid and solid states, at ambient temperature and pressure, as well as, in many cases, without destroying the samples (Li-Chan, 1996). In addition, Raman spectroscopy can provide qualitative information on food proteins (Choi and Ma, 2007, Ellepola et al., 2006, Li-Chan, 1996, Li-Chan et al., 1994, Li-Chan and Qin, 1998, Lippert et al., 1976, Meng et al., 2003, Tu, 1986). Since the main purpose of deamidation of food proteins is to improve functional properties resulting from altered protein conformation, it is important to study the effect of deamidation on protein conformation.

In the present investigation, Raman spectroscopy was used to determine the extent of deamidation in modified soy protein isolates, whey protein isolates, and spray-dried egg white. Conformational changes of these proteins under the influence of deamidation were studied by examining the Raman spectral data.