Lalatiana Rakotozafy

Effect of Mixing Conditions on the Behavior of Lipoxygenase, Peroxidase, and Catalase in Wheat Flour Doughs

ABSTRACT The effect of mixing has been tested on the extractable activities of lipoxygenase, peroxidase, and catalase from dough after 2, 5, and 20 min of mixing, and 30 min of rest period after 20 min of mixing. Different mixing conditions have been studied including temperature, atmosphere, speed, amount of water added to the dough, buffer solutions between pH 3.6 and 7.5 added to the dough, and different additives (linoleic acid, guaiacol, hydrogen peroxide, ascorbic acid, cysteine, yeast, and sodium chloride). In all the mixing conditions tested, the dough peroxidase activity remains equivalent to the initial flour activity, whereas losses in lipoxygenase and catalase activities largely varied according to mixing conditions. The results show that a self-destruction mechanism as well as physicochemical denaturation are responsible for these losses. Lipoxygenase losses seem mainly associated with the former mechanism, whereas catalase losses are highly increased in acidic conditions (physicochemical den...

Effect of Adding Exogenous Oxidative Enzymes on the Activity of Three Endogenous Oxidoreductases During Mixing of Wheat Flour Dough

ABSTRACT The behavior of different exogenous enzymes (soybean lipoxygenase [SLOX], horseradish peroxidase [HPOD], catalase from bovine liver [BCAT], and glucose oxidase [GOX] from Aspergillus niger) added to dough was studied during mixing. The effect of adding these exogenous oxidoreductases on the activity of three oxidative enzymes present in wheat flour (lipoxygenase [WLOX], peroxidase [WPOD], and catalase [WCAT]) was examined. Proper assay conditions were established to differentiate between added WLOX, WPOD, and WCAT and the corresponding activities present in wheat flour. For doughs with added SLOX, an immediate loss of extractable SLOX (≈40%) was observed which remained constant during further mixing. When compared with the control dough, addition of SLOX decreased the losses in WLOX and WCAT activities, whereas WPOD activity was unaffected. With doughs supplemented by HPOD, an immediate loss of 20% in the HPOD activity was observed which did not change after 20 min of mixing. Compared with contro...

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.

Studies on the Glutathione-Dehydroascorbate Oxidoreductase (EC 1.8.5.1) from Wheat Flour

ABSTRACT Glutathione (GSH) dehydrogenase was partially purified from wheat flour after extraction, ammonium sulfate precipitation, and ionic-exchange chromatography on diethylaminoethyl (DEAE) Sepharose CL6B. Kinetic studies showed that the optimum pH was close to 7.5. The Km values varied between 0.15 and 0.28 mM for dehydroascorbic acid (DHA) and between 1.8 and 0.62 mM for GSH when pH was varied from 5.5 to 7.5. The kinetic pattern was consistent with a sequential mechanism for the binding of GSH and DHA. NaCl is a competitive inhibitor with respect to GSH and is uncompetitive with respect to DHA, which suggests that the enzyme combines with DHA before it does with GSH. IsoDHA can replace DHA as hydrogen acceptor but with a Km of 1.2 mM. γ-Glu-cys was enzymically oxidized but much less efficiently than GSH (Vm = 47 nkat/mL and Km = 5.5 mM compared to Vm = 362 nkat/mL and Km = 1.8 mM for GSH), whereas cysteine and cys-gly were not substrates. In the presence of DHA, addition of cysteine and cys-gly to s...

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.

Extraction and physicochemical characterization of Tenebrio molitor proteins

This study focused on the extraction and physicochemical characterization of proteins from larvae and larvae meal of Tenebrio molitor. The larvae were subjected to a protein extraction process which involved a thermo-mechanical pre-treatment to produce the larvae meal. Soluble proteins from larvae and from larvae meal were subsequently extracted by solubilisation at an alkaline pH. The products obtained were then characterized and compared. The larvae and larvae meal were rich in protein (65.6% and 71.6% respectively) and displayed good essential amino acid (EAA) profiles. They contained all EAA and in sufficient quantities to meet the dietary requirements of both humans and salmon, except for a deficiency in methionine. The EAA profile of the larvae meal was also comparable to those of fish and soya meals used for feed. At pH10 and 45°C, the protein extraction yield of larvae (59.9%) was two-fold that of larvae meal (26.4%). The soluble proteins had protein contents on dry matter of 84% and 80% from larvae and larvae meal respectively. Molecular weights ranged from ≤14 to 100kDa but the two soluble proteins differed. The soluble proteins had a solubility which was highly pH-dependent, with a low solubility at pH3 to 5. Their surface charge depended on both the pH (in particular) and the NaCl concentration. The surface hydrophobicity at pH7 of soluble proteins from larvae (670.3) was higher than that of soluble proteins from larvae meal (102.5). These soluble proteins lowered the water surface tension to 42mN/m and 32mN/m for the soluble proteins from larvae and from larvae meal respectively. Chemical compounds used in this work. Glycine (PubChem CID: 750); Glycerol (PubChem CID: 753); Tris-(hydroxymethyl)aminomethane (PubChem CID: 4468930); Sodium chloride (PubChem CID: 5234); Ethanol (PubChem CID: 702); Monosodium phosphate (PubChem CID: 23672064); Disodium hydrogen phosphate (PubChem CID: 24203); 2-mercaptoethanol (PubChem CID: 1567); Hydrochloric acid (PubChem CID: 313); Bromophenol blue (PubChem CID: 8272); Sodium hydroxide (PubChem CID: 14798); Sodium dodecyl sulphate (PubChem CID: 3423265).