Quantification of Protein "Biomarkers" in Wheat-Based Food Systems: Dealing with Process-Related Issues

Abstract

Selected food proteins may represent suitable markers for assessing either the presence/absence of specific food ingredients or the type and intensity of food processes. A fundamental step in the quantification of any protein marker is choosing a proper protocol for solubilizing the protein of interest. This step is particularly critical in the case of solid foods and when the protein analyte is prone to undergo intermolecular disulfide exchange reactions with itself or with other protein components in the system as a consequence of process-induced unfolding. In this frame, gluten-based systems represent matrices where a protein network is present and the biomarker proteins may be either linked to other components of the network or trapped into the network itself. The protein biomarkers considered here were wheat gluten toxic sequences for coeliac (QQPFP, R5), wheat germ agglutinin (WGA), and chicken egg ovalbumin (OVA). These proteins were considered here in the frame of three different cases dealing with processes different in nature and severity. Results from individual cases are commented as for: (1) the molecular basis of the observed behavior of the protein; (2) the design of procedure aimed at improving the recovery of the protein biomarker in a form suitable for reliable identification and quantification; (3) a critical analysis of the difficulties associated with the plain transfer of an analytical protocol from one product/process to another. Proper respect for the indications provided by the studies exemplified in this study may prevent coarse errors in assays and vane attempts at estimating the efficacy of a given treatment under a given set of conditions. The cases presented here also indicate that recovery of a protein analyte often does not depend in a linear fashion on the intensity of the applied treatment, so that caution must be exerted when attributing predictive value to the results of a particular study.

Keywords: biomarkers; egg; food proteins; gluten; immunochemistry; lectin; ovalbumin; pasta; whole grain.

Figure 1

Effects of temperature during high hydrostatic pressure treatment (600 MPa, 10 min) of aqueous flour suspensions (40% solids). (A): protein solubility in 60% aqueous ethanol (full symbols) or 6M urea and 10 mM DTT (open symbols). (B): immunoreactivity of the proteins solubilized by individual extractants. (C): relative immunoreactivity of solubilized proteins. Symbols in the two upper panels are slightly larger than the standard deviation from quadruplicate measurements in duplicated experiments. Routines embedded in the graphical software SigmaPlot (rev. 10, Jandel Scientific, San Rafael, CA, USA) were used for data analysis and curve fitting.

Figure 2

SDS-PAGE and Western blotting tracings for WGA extracts. The samples are identified as follows: markers (Mk); whole grain semolina (WGS); refined semolina (RS); fine millings (FM); coarse millings (CM). (A): Coomassie Blue staining; (B): Western blotting against commercial anti-WGA antibodies.