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The consumption of whole-grain food—including pasta—has been increasing steadily. In the case of whole-grain pasta, given the many different producers, it seems important to have some objective parameters to define its overall quality. In this study, commercial whole-grain pasta samples representative of the Italian market have been characterized from both molecular and electronic-senses (electronic nose and electronic tongue) standpoint in order to provide a survey of the properties of different commercial samples. Only 1 pasta product showed very low levels of heat damage markers (furosine and pyrraline), suggesting that this sample underwent to low temperature dry treatment. In all samples, the furosine content was directly correlated to protein structural indices, since protein structure compactness increased with increasing levels of heat damage markers. Electronic senses were able to discriminate among pasta samples according to the intensity of heat treatment during the drying step. Pasta sample with low furosine content was discriminated by umami taste and by sensors responding to aliphatic and inorganic compounds. Data obtained with this multidisciplinary approach are meant to provide hints for identifying useful indices for pasta quality. Practical Application As observed for semolina pasta, objective parameters based on heat-damage were best suited to define the overall quality of wholegrain pasta, almost independently of compositional differences among commercial samples. Drying treatments of different intensity also had an impact on instrumental sensory traits that may provide a reliable alternative to analytical determination of chemical markers of heat damage in all cases where there is a need for avoiding time-consuming procedures.

The development of innovative legume-enriched rice products is a promising way to exploit rice varieties with a low sensory grade. In this work, a multidisciplinary approach was applied to the characterization of extruded breakfast cereals prepared from African-grown Oryza glaberrima (cv. Viwonor) or Oryza sativa (cv. Jasmine 85) enriched with 30% cowpea flour, obtained from sprouted or nonsprouted cowpea. Regardless of the rice species, addition of sprouted cowpea flour conferred a peculiar volatiles profile, rich in sour, bitter, and astringent taste. Protein structural indices provided molecular insights about the macroscopic differences among samples. Extruded products from O. glaberrima were characterized by lower expansion rates with respect to those obtained from O. sativa, regardless of the type of cowpea flour. Sprouting time had a positive influence on the hardness of extruded glaberrima-based products, facilitating formation of a more compact matrix, but it did not influence sativa-based produc...

An attempt was made to evaluate gluten structural changes in refined and whole wheat pasta from hard white winter wheat to elucidate the impact of whole wheat components on the formation and structure of the gluten network in pasta. Attenuated total reflectance–FTIR spectroscopy was used to track gluten secondary structure through most of the major steps in pasta processing: raw material, mixing, drying, and cooking. Protein solubility, accessible thiols, and SDS-PAGE data were also collected to provide additional information on the nature of protein interactions and network composition. Few secondary structural differences were observed between refined and whole wheat flours from hard white wheat. However, mixing induced a significant shift to β-sheet structures in refined dough that was not equally matched by whole wheat dough. Drying under both high temperature, short time (HT) and low temperature, long time (LT) conditions resulted in a reversion to structural distributions similar to those for flour ...

The network-forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS-accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol-blocking agent N-ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high-molecu...

Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron-uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at “catalytic” NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific “iron-uptake-competent” conformations of apoRd on the NP surface. This bio-mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor-triggered refolding, particularly when over-expressed under limited cofactor accessibility. Proteins 2014; 82:3154–3162. © 2014 Wiley Periodicals, Inc.

Structural changes ensuing from the non-covalent absorption of bovine beta-lactoglobulin (BLG) on the surface of polystyrene nanoparticles were investigated by using spectroscopic approaches, by assessing the reactivity of specific residues, and by limited proteolysis/mass spectrometry. Also, the immunoreactivity of absorbed and free BLG was compared. All these approaches indicated substantial rearrangements of the protein structure in the absorbed state, in spite of the reported structural rigidity of BLG. Changes made evident by experimental measurements were confirmed by computational approaches. These indicate that adsorption-related changes are most marked in the area between the main C-terminal alpha helix and the beta-barrel, and lead to full exposure of the thiol on Cys121, consistent with experimental measurements. In the computational model of bound BLG, both Trp61 and Trp19 also move away from their neighboring quenchers and become solvent-exposed, as indicated by fluorescence measurement. Upon binding, the beta-barrel also loosens, with a substantial increase in immunoreactivity and with noticeable changes in the trypsinolytic pattern. The possible general significance of the structural changes reported here for non-covalently adsorbed BLG is discussed with respect to recognition events involving surface-bound proteins, as are aspects related to the carrier function(s) of BLG, and to its use as a common ingredient in many food systems. Proteins 2014; 82:1272–1282. © 2013 Wiley Periodicals, Inc.

A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

The ability of high hydrostatic pressure processing to promote changes in both the structural properties of fiber and the interaction of fiber with water were addressed. Both coarse and fine bran from milling of common wheat were considered. Treatment-induced morphological changes were most pronounced in fine bran, whereas treatment of coarse bran resulted in the largest change in water-holding capacity. The significance of the process-induced changes is discussed in terms of their practical relevance in the production of fiber-enriched foods.

Active cellulose-based packaging materials were prepared by binding lysozyme to paper modified with anionic polyelectrolytes. Polyelectrolytes improve the paper binding capacity towards the positively charged lysozyme and play a protective role towards lysozyme activity during the paper-making process. The charge density of paper was increased by incorporating carboxymethylcellulose (CMC) or polygalacturonic acid (PGA). The presence of either CMC or PGA greatly increased the amount of bound lysozyme, and the stability of its binding towards buffers or towards non-ionic chaotropes which disrupt the three dimensional structure in macromolecules (urea, 8 M). Binding of lysozyme to modified papers was sensitive to anionic detergents (sodium dodecyl sulphate, 1%) and to non-chaotrope salts (NaCl, 0.5M). These data provide information about the nature of the interactions between the protein and the various types of paper, and provide insights on how to preserve the activity of lysozyme-loaded paper. The polymers used for lysozyme immobilization were found to affect only marginally the structural and functional properties of the antimicrobial protein, and to facilitate the recovery of structural features of the protein after heat treatment. Presence of a negative polyelectrolyte (and of CMC in particular) improves the thermal stability of the protein, making it resistant to thermal inactivation, even under the conditions used for drying processes without compromising the paper's mechanical properties. The activity measurements showed that paper-bound lysozyme retains its lytic (and therefore, antimicrobial) activity against the cell walls of Micrococcus lysodeikticus, the target microrganism used as standard for lysozyme bioassays. Copyright © 2009 John Wiley & Sons, Ltd.

Bovine β-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 °C at neutral pH. At these temperatures β-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, β-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of β-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of β-lactoglobulin hydrolysis were assessed by using various β-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.

Purpose : This study was meant to characterize the serum proteome in a small geographical cluster of sporadic ALS subjects originating from a restricted geographical area and sharing the same environmental exposure, in a broader context of evaluating the relevance of environmental factors to disease onset, status, and progression. Experimental design : An Artificial Neural Network based software was used to compare the relative abundance of proteins identified as different (by means of bi-dimensional electrophoresis and mass spectrometry) in the serum proteome of patients and age-matched healthy controls. Results : The patient's group was characterized by altered levels of acute phase reactants and of proteins involved in lipid homeostasis, along with over-representation of the APOE*4 allele. Conclusions and clinical relevance : Characterization of the serum proteome in a small cluster of sporadic ALS patients, originating from a geographically restricted area with a high prevalence of the disease and evaluation of the results with software based on artificial neural networks, highlights the association of the relative abundance of some proteins (most notably, acute phase reactants and lipid homeostasis proteins) with the disease presence and status. This article is protected by copyright. All rights reserved

The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfurtransferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH–cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 degrees C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ << Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 degrees C for Fe2+ Rd and 83 degrees C for Fe3+ Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+ Rd, stoichiometric Fe3+/cysteine-ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.

The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.

The effect of pasteurization and repasteurization on disulfide chemical reactivity of milk proteins was tested after proteolytic digestion. Pasteurization at 75°C, but not at 80°C or repasteurization, resulted in a decrease in disulfide reactivity compared with raw milk. No differences among heated milks were observed in in vitro protein digestibility, electrophoretic behavior or surface hydrophobicity. Whey proteins extracted from milk pasteurized at 75 and 80°C had lower solubility, protein digestibility, disulfide reactivity and surface hydrophobicity than those extracted from raw milks. SDS-PAGE and surface hydrophobicity parameters (K d , F max ) of whey proteins suggested that differences in disulfide reactivity were a consequence of disulfide-mediated selective aggregation of the whey proteins.