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A simple kinetic model allowed for the description of the observed decay of the oxygen content in hypoxic aqueous samples with and without headspace, in the presence of glucose oxidase (Glucox) or laccase and their substrates (glucose for Glucox and ABTS for Laccase). The experimental tests involved both the direct measurement of the oxygen content with a fluorescence-based probe and the indirect stopped-flow spectroscopic detection of colored compounds generated from suitable chromogenic reagents. The complete depletion of dissolved oxygen occurred in the no-headspace samples, whereas some residual oxygen remained in a steady state in the samples with headspace. Simple pseudo-first-order kinetics was adequate to describe the behavior of the system, as long as oxygen was the rate-limiting compound, i.e., in the presence of excess substrates. The values of the kinetic constants drawn from best-fit routines of the data from both experimental approaches were quite comparable. The oxygen residues in the samples with headspace seemed related to the low solubility of O 2 in the aqueous phase, especially if compared with the large amount of oxygen in the headspace. The extent of such residue decreased by increasing the concentration of the enzyme. The kinetic model proposed in this paper can be of help in assembling suitable sensors to be used for food safety and quality control.

Selected food proteins may represent suitable markers for assessing either the presence/absence of specific food ingredients or the type and intensity of food processes. A fundamental step in the quantification of any protein marker is choosing a proper protocol for solubilizing the protein of interest. This step is particularly critical in the case of solid foods and when the protein analyte is prone to undergo intermolecular disulfide exchange reactions with itself or with other protein components in the system as a consequence of process-induced unfolding. In this frame, gluten-based systems represent matrices where a protein network is present and the biomarker proteins may be either linked to other components of the network or trapped into the network itself. The protein biomarkers considered here were wheat gluten toxic sequences for coeliac (QQPFP, R5), wheat germ agglutinin (WGA), and chicken egg ovalbumin (OVA). These proteins were considered here in the frame of three different cases dealing with processes different in nature and severity. Results from individual cases are commented as for: (1) the molecular basis of the observed behavior of the protein; (2) the design of procedure aimed at improving the recovery of the protein biomarker in a form suitable for reliable identification and quantification; (3) a critical analysis of the difficulties associated with the plain transfer of an analytical protocol from one product/process to another. Proper respect for the indications provided by the studies exemplified in this study may prevent coarse errors in assays and vane attempts at estimating the efficacy of a given treatment under a given set of conditions. The cases presented here also indicate that recovery of a protein analyte often does not depend in a linear fashion on the intensity of the applied treatment, so that caution must be exerted when attributing predictive value to the results of a particular study.

Thermal treatments are widely applied to gluten-free (GF) flours to change their functionality. Despite the interest in using pulses in GF formulations, the effects of thermal treatment at the molecular level and their relationship with dough rheology have not been fully addressed. Raw and heat-treated red lentils were tested for starch and protein features. Interactions with water were assessed by thermogravimetric analysis and water-holding capacity. Finally, mixing properties were investigated. The thermal treatment of red lentils induced a structural modification of both starch and proteins. In the case of starch, such changes consequently affected the kinetics of gelatinization. Flour treatment increased the temperature required for gelatinization, and led to an increased viscosity during both gelatinization and retrogradation. Regarding proteins, heat treatment promoted the formation of aggregates, mainly stabilized by hydrophobic interactions between (partially) unfolded proteins. Overall, the structural modifications of starch and proteins enhanced the hydration properties of the dough, resulting in increased consistency during mixing.

Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.

To improve the current understanding of the role of stilbenoids in the management of diabetes, the inhibition of the pancreatic α-amylase by resveratrol derivatives was investigated. To approach in a systematic way, the mechanistic and structural aspects of the interaction, potential bioactive agents were prepared as single molecules, that were used for the biological evaluation of the determinants of inhibitory binding. Some dimeric stilbenoids-in particular, viniferin isomers- were found to be better than the reference drug acarbose in inhibiting the pancreatic α-amylase. Racemic mixtures of viniferins were more effective inhibitors than the respective isolated pure enantiomers at an equivalent total concentration, and displayed cooperative effects not observed with the individual enantiomers. The molecular docking analysis provided a thermodynamics-based rationale for the measured inhibitory ability and for the observed synergistic effects. Indeed, the binding of additional ligands on the surface of the alpha-amylase was found to decrease the dissociation constant of inhibitors bound to the active site of the enzyme, thus providing a mechanistic rationale for the observed inhibitory synergies.

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L -1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (k cat about 2.8 min -1 ), as well as FAD pyrophosphorolysis in a strictly Mg 2+ -dependent manner. The synthesis of FAD is inhibited by HgCl₂. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed., Competing Interests: The authors declare no conflict of interest.