3D-front-face fluorescence spectroscopy and independent components analysis: A new way to monitor bread dough development.

Following bread dough development can be a hard task as no reliable method exists to give the optimal mixing time. Dough development is linked to the evolution of gluten proteins, carbohydrates and lipids which can result in modifications in the spectral properties of the various fluorophores naturally present in the system. In this paper, we propose to use 3-D-front-face-fluorescence (3D-FFF) spectroscopy in the 250-550nm domain to follow the dough development as influenced by formulation (addition or not of glucose, glucose oxidase and ferulic acid in the dough recipe) and mixing time (2, 4, 6 and 8min). In all the 32 dough samples as well as in flour, three regions of maximum fluorescence intensities have been observed at 320nm after excitation at 295nm (Region 1), at 420nm after excitation at 360nm (Region 2) and 450nm after excitation at 390nm (Region 3). The principal components analysis (PCA) of the evolution of these maxima shows that the formulations with and without ferulic acid are clearly separated since the presence of ferulic acid induces a decrease of fluorescence in Region 1 and an increase in Regions 2 and 3. In addition, a kinetic effect of the mixing time can be observed (decrease of fluorescence in the Regions 1 and 2) mainly in the absence of ferulic acid. The analysis of variance (ANOVA) on these maximum values statistically confirms these observations. Independent components analysis (ICA) is also applied to the complete 3-D-FFF spectra in order to extract interpretable signals from spectral data which reflect the complex contribution of several fluorophores as influenced by their environment. In all cases, 3 signals can be clearly separated matching the 3 regions of maximal fluorescence. The signals corresponding to regions 1 and 2 can be ascribed to proteins and ferulic acid respectively, whereas the fluorophores associated with the 3rd signal (corresponding to region 3) remain unidentified. Good correlations are obtained between the IC score values of the 3 signals and the fluorescence intensities in Region 1, Region 2 and Region 3. Ferulic acid addition increases fluorescence in Region 2 and decreases fluorescence in Region 1, probably via a reabsorption of the protein fluorescence by ferulic acid. These phenomena are less pronounced when glucose oxidase is present. The enzymatic oxidation of ferulic acid by the glucose oxidase-peroxidase association could explain some of these effects.

The bread dough stability improving effect of pyranose oxidase from trametes multicolor and glucose oxidase from Aspergillus niger: unraveling the molecular mechanism.

Glucose oxidase (GO) and pyranose oxidase (P2O) improve dough stability and bread quality. We here studied whether their mode of action resides in cross-linking of proteins and/or arabinoxylan (AX) molecules through the production of H2O2. Evidence for both was deduced from a decrease in extractability of protein and AX from dough made with P2O, GO, or H2O2, using sodium dodecyl sulfate containing buffer and water, respectively. The addition of H2O2, P2O, or GO to a glutathione solution sharply decreased its sulfhydryl (SH) content. P2O or GO can trigger protein cross-linking through the formation of disulfide (SS) bonds. As a result thereof, SH/SS interchange reactions between low molecular mass SH containing compounds and gluten proteins can be hampered. Furthermore, a decrease in the level of monomeric ferulic acid (FA) esterified to AX in dough points to a role of FA bridges in cross-linking of AX molecules. Our results indicate that the molecular mechanism of dough and bread improvement by P2O and GO resides in cross-linking of gluten proteins and AX by formation of H2O2. They furthermore show that the extent of cross-linking upon addition of P2O or GO strongly depends on the concentration (and production rate) of H2O2.

Analysis and modeling of the ferulic acid oxidation by a glucose oxidase-peroxidase association. Comparison with a hexose oxidase-peroxidase association.

A commercial glucose oxidase (GOX) from Aspergillus niger was partially characterized. The enzyme exhibited a two-step transfer mechanism, and the kinetic constants toward glucose and oxygen were determined. Under conditions similar to dough making (glucose concentration and pH), GOX does not exhibit maximum activity. A hexose oxidase (HOX) from Chondrus crispus was partially characterized as well. The HOX activity is not far from the optimum in the kneading conditions (pH and glucose concentration). A peroxidase (POD) purified from wheat germ was used to oxidize ferulic acid in the presence of GOX or HOX. Hydrogen peroxide produced during the glucose oxidation activates the wheat germ POD. Ferulic acid oxidation in solutions containing different ratios of POD + GOX or HOX + POD was followed by UV spectrophotometry. For the same dosage, the HOX-POD system is the most efficient for peroxidase activation. Using absorbance data and kinetic constants of GOX and POD, a mathematical model describing the release or consumption of the different reactants (hydrogen peroxide, oxygen, and ferulic acid) in the medium was developed, and experimental data correlated well with calculated values. The results obtained will be applied to investigate the effect of GOX and HOX activities on the rheological properties of dough.

Separation and identification by gel filtration and high-performance liquid chromatography with UV or electrochemical detection of the disulphides produced from cysteine and glutathione oxidation.

Methods for quantification of oxidised and reduced forms of glutathione (GSSG and GSH) and cysteine (CSSC and CSH) and the disulphide glutathione-cysteine (GSSC) resulting from the oxidation of the mixture of CSH and GSH are performed by RP-HPLC with coulometric and UV detection after separation of these compounds by size-exclusion fast protein liquid chromatography. The fractionation of the disulphides (GSSG, GSSC and CSSC) was achieved by size exclusion using a Superdex peptide column coupled with an UV detection at 254 nm. The conditions of separation of these compounds by RP-HPLC were optimised using the response surface methodology. Optimal peak resolution and retention times were obtained on a C18 YMC ODS AQ column with 20 mM of ammonium phosphate at pH 2.5 and 2% of acetonitrile in the elution phase. In these experimental conditions, CSH, CSSC, GSH and GSSG were eluted within 20 min. Coulometric detection enabled a sensitivity 100 times higher for the disulphides than the UV detection at 220 nm. These methods were applied to follow the consumption of thiols and the disulphide formation by three oxidising systems, sulphydryl oxidase, glutathione dehydroascorbate oxidoreductase and potassium bromate. This study revealed that the relative proportions of the disulphides formed were similar for the three oxidising systems when the reactions are in their state of equilibrium.

Oxidation of ferulic acid or arabinose-esterified ferulic acid by wheat germ peroxidase.

The oxidation of ferulic acid (FA) or 5-O-(trans-feruloyl)-L-arabinose (EFA) by a purified wheat germ peroxidase was followed by UV spectrophotometry and high-performance liquid chromatography using an electrochemical detection. Wheat peroxidase (POD) exhibits a ping-pong bireactant mechanism forming phenoxy radicals more rapidly from FA than from EFA in routine assay conditions. When both the free and the esterified forms of FA are present, the reverse was found. This result could be due to a nonenzymatic cooxidation of FA by the phenoxy radicals of EFA leading to the formation of phenoxy radicals of FA and the EFA regeneration. Addition of ascorbic acid (AA) provokes a delay of FA consumption. AA reduced very rapidly the phenoxy radicals formed by POD back to initial phenol avoiding the formation of ferulate dimers until it was completely oxidized in dehydroascorbic acid. Conversely, cysteine addition slowed but did not delay the FA consumption. The thiol reduced a fraction of the phenoxy radicals produced by wheat POD and was oxidized into cystine, while the other part of phenoxy radicals formed ferulate dimers. These results could be of interest to understand the POD effect on the wheat dough rheological properties.