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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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The current study was undertaken to provide solutions to optimize the unsaponifiable antioxidants content of bread. We report a complete description of changes in wheat carotenoids and vitamin E content from grain to bread and highlight the most important processing steps affecting their level in wheat bread. Major carotenoids losses occurred during kneading. A close correlation (r(2) = 0.97; P = 0.05) was found between carotenoid pigment losses and lipoxygenase (LOX) activity, both parameters depending on wheat genotype. The use of wheat species exhibiting high carotenoid contents and low LOX activity was shown to preserve significant carotenoid level in the bread. No relation was found between vitamin E losses during doughmaking and LOX activity. In addition, moderate kneading resulted in higher vitamin E retention in comparison with carotenoids (12% and 66% losses, respectively). It is concluded that carotenoids are more susceptible to oxidation by endogenous lipoxygenase than vitamin E during breadmaking. This study showed that bread nutritional quality, in terms of antioxidant content, could be improved by selecting suitable cereal genotypes, if this potential is preserved by milling and baking processes.

Phospholipids (PL) are minor components of wheat flour involved in baking quality and exogenous phospholipids are used as emulsifiers giving better loaf volume and crumb grain. Few biochemical data are available on the phospholipid evolution during mixing, probably because of the time-consuming methods proposed for their extraction, separation and quantification. In the present study, the extraction, separation and quantification of the main wheat flour phospholipids were carried out. Total lipids (2% dry mass of wheat flour) were extracted from flour or dough by a mixture of chloroform-methanol-water (1:1:1 (v/v)). The phospholipids were separated from the lipid extract on silica cartridge by solid-phase extraction (SPE) procedure under a 1.5-4 mmHg vacuum, at a 0.8 mL min(-1) flow rate (1 mmHg = 133.322 Pa). The recovery of the lipid extract was 100%, whereas the SPE yield for the PLs was 50%. The resulting fraction was then submitted to HPLC with evaporative light scattering detection on a Diol stationary phase allowing the separation and quantification of each class of phospholipids, in less than 16 min. The developed method allowed to quantify the phospholipid amounts from eight wheat flours as well as their evolution during mixing in the presence of phospholipase.

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.

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.

The peripheral neurotoxicity of lindane was studied in vitro on mouse phrenic-diaphragm preparation. The experiments were performed on normal and denervated preparations. Twitches evoked by direct or indirect electrical stimulations and contractions evoked by external application of cholinergic agonists were compared in the presence or absence of lindane in the bath medium. Histochemical localizations of calcium were made on fibers after chemical stimulations. The results indicated that, at the normal endplate or at extrajunctionnal sites after denervation, there was a change in the intracellular Ca2+ distribution in response to external application of concentrated cholinergic agonists. This modification caused a transient contracture and could be visualized either by 45Ca autoradiography or by Alizarin Red S intracellular precipitations. Lindane inhibited the ACh-dependent contracture (I50 less than 10(-5) M) and suppressed the histochemical localizations of calcium. These lindane effects were observed on both normal and denervated muscles. It was concluded that lindane inhibited the Ca2+ influx presented by the desensitized ACh-sensitive areas of the muscle.

Alizarin red S (ARS) is used for histological characterization of calcium. In order to improve the conditions of the use of the dye on biological materials, the chemical reaction between ARS and Ca2+ is studied in vitro. In aqueous solution ARS and Ca2+ ions precipitate to form brick-red deposits. For precipitation to occur a neutral pH (4 less than pH less than 8) is required; the stoichiometric ratio is 1:1. ARS reacts with calcium via its sulfonate and hydroxyl groups. The apparent solubility product is around 10(-7) Na+ and K+ ions do not interfere with equilibrium of the precipitation reaction but Mg2+ and soluble proteins such as bovine serum albumin react with ARS and form soluble complexes which can lead to a decrease in the ARS quantity available to react with the calcium ions.

When mouse diaphragms are incubated in vitro with [14C] inositol, radioactivity is observed at post synaptic areas in phosphatidyl-inositols and in inositol-phosphates. Synaptic desensitization suppresses the labelled inositol-phosphates. Lindane restores the labelling effect without suppressing the desensitized state.

Residual radioactivity in Rat brain structures is studied after single dose 14C Lindane administration. Short time after injection, at the onset of convulsions, white matter and myelinated structures show important retention, nucleated cells do not. This heterogeneous distribution agrees with the lipophilic behaviour of the pesticide.