Structuring and texturing gluten‑free pasta: egg albumen or whey proteins?

The effects of adding egg albumen or whey proteins to pasta made from parboiled rice flour (PR) were investigated. Pasta quality was evaluated in terms of color, furosine content, and cooking properties (water absorption, cooking loss, and consistency at the optimal cooking time). The surface heterogeneity of the cooked and uncooked materials was studied, and some starch properties (pasting properties and starch susceptibility to \u3b1-amylase hydrolysis) were assessed, along with the features of the protein network as determined by conditional solubility studies and with ultrastructural features of the cooked products. Egg albumen improved pasta appearance and gave a product with low cooking loss, firmer, and nutritionally more valuable than the other ones. In albumen-enriched pasta, small starch granules appear homogeneously surrounded by a protein network. In the uncooked product, the protein network is stabilized mostly by hydrophobic interactions, but additional disulfide interprotein bonds form upon cooking. Thus, addition of 15 % liquid albumen to PR results in significant improvement of the textural and structural features of rice-based gluten-free pasta

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction

Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules-that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product-was observed at creaming temperatures higher than 22\uc2\ub0C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37\uc2\ub0C before creaming at 8\uc2\ub0C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants

The role of pH on the thermodynamics and kinetics of muscle biochemistry: An in vivo study by 31P-MRS in patients with myo-phosphorylase deficiency

AbstractIn this study we assessed ΔG′ATP hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG′ATP of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme

Stabilization of the "open" conformer of apoIscU on the surface of polystyrene nanobeads accelerates assembly of a 2Fe2S structure

AbstractThe scaffold protein IscU is involved in the assembly/transfer of FeS clusters. IscU exists in both open and closed conformation. The clusterless open conformation of IscU adheres to the hydrophobic surface of polystyrene nanobeads, as observed for other proteins. Increased accessibility of the ligand cysteines in bound IscU facilitates assembly of a 2Fe2S cluster, and the cluster-bearing structured form of IscU does not interact with the nanobeads, thus ensuring turnover. The dependence of accelerated cluster assembly on the nanobeads concentration pointed to steric and crowding effects as for promoting cluster formation, and confirms the requirement for structural flexibility of IscU

Stabilization of the “open” conformer of apoIscU on the surface of polystyrene nanobeads accelerates assembly of a 2Fe2S structure

The scaffold protein IscU is involved in the assembly/transfer of FeS clusters. IscU exists in both open and closed conformation. The clusterless open conformation of IscU adheres to the hydrophobic surface of polystyrene nanobeads, as observed for other proteins. Increased accessibility of the ligand cysteines in bound IscU facilitates assembly of a 2Fe2S cluster, and the cluster-bearing structured form of IscU does not interact with the nanobeads, thus ensuring turnover. The dependence of accelerated cluster assembly on the nanobeads concentration pointed to steric and crowding effects as for promoting cluster formation, and confirms the requirement for structural flexibility of IscU

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thincut meat: preliminary assessment

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm 122) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 \ub0C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g 121). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases

Glycosylation Tunes Neuroserpin Physiological and Pathological Properties

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism

Abstract: Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes -imino acids generated by L- or D-amino acid oxidases with a preference for those deriving from Ala > Leu = L-Met > L-Gln, whereas it was poorly active on L-Phe and L-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms