Enzymatic protein crosslinking in wheat-based foods.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This research investigated the use of the enzymes transglutaminase (TGA) and glucose oxidase in bread and croissants, in order to enhance their functional properties and baking perfmmance. The means by which these enzymes are thought to act is by the crosslinking of specific proteins of wheat flour. Whereas glucose oxidase reportedly improves baking quality through the addition of disulfide crosslinks to gluten proteins, TGA catalyses the formation of E-(y-glutamyl-lysine) crosslinks.
SDS-PAGE and HPLC enabled the identification of the changes caused by the crosslinking reactions. Crosslinking of the wheat proteins by TGA occurred during dough mixing, with no further effects apparent with prooving or with increased enzyme concentration. Long-term frozen storage of croissant doughs maintained the initial effect of adding TGA to the croissant formulation.
The proteins most dramatically affected by TGA were the large, insoluble glutenins, the proteins responsible for the strength and elasticity of dough. The gliadin proteins were apparently unchanged by TGA in situ, although in in vitro studies the gliadins were crosslinked by TGA, leading to the novel conclusion that the gliadins appear to be inaccessible for crosslinking while within the polymeric gluten complex. The albumins and globulins were also shown to be crosslinked by TGA and it appears that this contributed to the reported improvements in crumb strength and texture.
Studies were then conducted using glucose oxidase as the crosslinking agent in bread and croissants. Molecular studies showed the effect of disulfide crosslinking on the glutenins to be far less than with TGA, thus explaining the lack of any observed increase in product volume. However, improvements to crumb texture could be explained by the crosslinking of the albumins and globulins by glucose oxidase. The occurrence of non-disulfide crosslinks was demonstrated in specific protein fractions from glucose oxidase-treated doughs, in contrast to the literature. Thus glucose oxidase was found to crosslink the albumins and globulins with both disulfide and non-disulfide crosslinks, and the glutenins with some non-disulfide crosslinks.
Measurements of lysine upon crosslinking was tested as a means to measure rate of the TGA reaction. An existing assay, which measures the formation of hydroxamate by TGA, was developed for use in dough. A new method was developed for the measurement of TGA activity using computer image analysis software. Three model proteins, bovine serum albumin (BSA), ribonuclease (RNAse A) and K-casein were tested for crosslinking rates by TGA and the measured rates were related to literature reports. This method was then used to measure the crosslinking rate of a high molecular weight subunit of glutenin, by TGA.
Significant insights were gained from this research regarding the crosslinking of wheat proteins. These findings are likely to have major implications for the development of enzymatic improvers in baked products.