Effect of fluorescence dyes on wet gluten structure studied with fluorescence and FT-Raman spectroscopies

Fluorescence spectroscopy coupled with FT-Raman spectroscopy were used to determine effect of two fluorescent dyes (fluorescein and rhodamine B) on the structure of wet gluten (hydrated gluten). Additionally, PCA analysis was applied to find correlations between fluorescence and Raman parameters. The fluorescent dyes, regarded as model representatives of phenolic compounds, were added to model dough in six concentrations (0.05%–0.5%). Wet gluten samples and starch slurries (as a residue after washing out gluten) were excited by two wavelengths – one assigned to tryptophan, and the second to the particular dye. Fluorescence and UV-VIS analysis of starch slurries show that the dyes can be completely bound to gluten proteins because of their absence in the slurries. Analysis of the spectroscopic results indicate that fluorescein and rhodamine B interact with gluten proteins in different ways depending on the type of functional groups present in the dye structure. The fluorescein molecules, due to the presence of hydroxyl and carboxyl groups may incorporate into gluten network by forming hydrogen and/or covalent bonds, whereas rhodamine B with four methyl groups may be enclosed in hydrophobic pockets formed by gluten polypeptide chains. These differences can be related to the type of functional groups present in the dye structure (fluorescein has one hydroxyl group, rhodamine B has four methyl groups). PCA analysis showed correlations between dye fluorescent parameters (maximum wavelength and band intensity) and some structural parameters (presence or absence of bands assigned to α-helices, β-turns and SS bonds conformations). The PCA results also suggest that mechanism of interactions gluten – dye depends on type of functional groups present in the dye molecule.