Effect of heat treatments on foaming and physico-chemical properties of bovine and camel sodium caseinate.

The objective of this study was to examine the foaming properties of sodium caseinates (Na-cas) extracted from bovine and camel fresh milks after heating at 70, 80, 90 and 100°C for 30 min at laboratory scale. Experimental results indicated that greater foam was obtained with camel Na-cas than with bovine Na-cas at all heating temperatures due to the higher ß-casein content in camel caseins (~53.4% of total proteins, RP-HPLC results). Increasing heat-treatment temperature to 100°C significantly enhanced the foaming properties by raising surface hydrophobicity and decreasing electronegative charge as well as interfacial tension values upon heating. This study concluded that camel Na-cas has important foaming properties in agricultural and food industries.

Heating during agitation of ß2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ß2-Microglobulin (ß2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ß2m in patients are observed at neutral pH, formation of ß2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that ß2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the ß2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.

Self-Assembled Modified Soy Protein/Dextran Nanogel Induced by Ultrasonication as a Delivery Vehicle for Riboflavin.

A simple and green approach was developed to produce a novel nanogel via self-assembly of modified soy protein and dextran, to efficiently deliver riboflavin. First, modified soy protein was prepared by heating denaturation at 60 °C for 30 min or Alcalase hydrolysis for 40 min. Second, modified soy protein was mixed with dextran and ultrasonicated for 70 min so as to assemble nanogels. The modified soy protein-dextran nanogels were characterized by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and ζ-potential studies to confirm the formation of NGs. Transmission electron microscopy (TEM) revealed the NGs to be spherical with core-shell structures, in the range of 32-40 nm size. The nanogels were stable against various environmental conditions. Furthermore, the particle size of the nanogels hardly changed with the incorporation of riboflavin. The encapsulation efficiency of nanogels was found to be up to 65.9% at a riboflavin concentration of 250 µg/mL. The nanogels exhibited a faster release in simulated intestine fluid (SIF) compared with simulated gastric fluid (SGF). From the results obtained it can be concluded that modified soy protein-dextran nanogels can be considered a promising carrier for drugs and other bioactive molecule delivery purposes.

Quality evaluation of human serum albumin prepared by heat denaturation in Iran: an experience for developing countries.

Blood and plasma are unique resources and access to these products save life. In this study, albumin demand and surplus plasma makes it possible to use local experiences in plasma industry for preparation of albumin so plasma was heated after stabilization; afterward denatured proteins were precipitated and separated by continuous centrifuge system. The supernatant contained albumin was filtrated, diafiltrated, ultrafiltrated, formulated and pasteurized. Albumin preparation in pilot scale with heat denaturation was performed for the first time in Iran. This method using surplus plasma is recommended for all countries that have no access to plasma fractionation industry. Therefore with more attention it has potential for use in the production of safe plasma derived products and thereby it can be used as a safe product in clinic.

Effect of Different Crosslinkers on Denatured Dentin Collagen's Biostability, MMP Inhibition and Mechanical Properties.

OBJECTIVE: Sound, natural dentin collagen can be stabilized against enzymatic degradation through exogenous crosslinking treatment for durable bonding; however, the effect on denatured dentin (DD) collagen is unknown. Hence, the ability of different crosslinkers to enhance/restore the properties of DD collagen was assessed. METHODS: Demineralized natural and DD collagen films (7 mm × 7 mm × 7 µm) and beams (0.8 mm × 0.8 mm × 7 mm) were prepared. DD collagen was experimentally produced by heat or acid exposure, which was then assessed by various techniques. All specimens were then treated with 1 wt% of chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide (EDC/NHS) and two structurally different flavonoids-theaflavins (TF) from black tea and type-A proanthocyanidins from cranberry juice (CR) for either 30 s or 1 h. The controls were untreated. Dentin films were assessed for chemical interaction and cross-linking effect by FTIR, biostability against exogenous collagenase by weight loss (WL) and hydroxyproline release (HYP), and endogenous matrix metalloproteinases (MMPs) activity by confocal laser microscopy. Dentin beams were evaluated for tensile properties. Data were analyzed using ANOVA and Tukey's test (α = 0.05). RESULTS: Compared with natural collagen, DD collagen showed pronounced structural changes, altered biostability and decreased mechanical properties, which were then improved to various degrees that were dependent on the crosslinkers used, with EDC/NHS being the least effective. Surprisingly, the well-known MMP inhibitor EDC/NHS showed negligible effect on or even increased MMP activity in DD collagen. As compared with control, cross-linking induced by TF and CR significantly increased collagen biostability (reduced WL and HYP release, p < 0.05), MMP inhibition (p < 0.001) and mechanical properties (p < 0.05), regardless of denaturation. CONCLUSIONS: DD collagen cannot or can only minimally be stabilized via EDC/NHS crosslinking; however, the challenging substrate of DD collagen can be enhanced or restored using the promising flavonoids TF and CR.

Assessing the Aggregation Propensity of Single-Domain Antibodies upon Heat-Denaturation Employing the ΔTm Shift.

Nano differential scanning fluorimetry is used to quantify protein thermostability and has substantially expanded the spectrum of convenient biophysical parameters used to characterize proteins. Here, this technique is used to measure the ΔTm shift for single-domain antibodies (sdAbs), which represents a comprehensive metric for the aggregation propensity of sdAbs upon heat-denaturation. By relating two melting curves at different protein concentrations, the ΔTm shift described in this protocol is ideally suited for high-throughput measurements to guide protein engineering, formulation development, and developability assessment of sdAbs.

Kinetics of heat-induced denaturation of proteins in model infant milk formulas as a function of whey protein composition.

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow's milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, ß-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when ß-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of ß-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing ß-lactoglobulin.

RP-HPLC method for simultaneous quantification of free and total thiol groups in native and heat aggregated whey proteins.

Disulfide formation of whey proteins during heat treatment via thiol oxidation is important with regard to techno-functional properties. Due to the formation of other oxidation products than disulfides, the decrease in free thiol concentration is not proportional to the disulfide formation. Thus, in order to evaluate thiol reactivity and disulfide concentration both parameters are required. Currently applied methods focus mainly on the loss of free thiols using the spectrophotometric Ellman's assay. Next to that, we improved an existing RP-HPLC assay using the thiol reagent 4,4'-Dithiodipyridine (DTDP) to quantify free thiols as well as total (free thiols and disulfide bonds) thiols of native and heat-treated whey proteins. Thereby, the sample preparation technique, the sample handling, and the analysis technique were optimized. Thus, the paper provides a simple RP-HPLC method for quantification of thiol oxidation reactions to determine heat-induced changes in the structure of whey proteins. In addition, the method should be applicable to other protein systems due to the method validation by proteins of different amounts of free and total thiols in their structure.•Simple RP-HPLC method for quantification of free and total thiols using 4,4'-Dithiodipyridine (DTDP).•High recovery rates for free and total thiols.•High stability within 24 h.

Anti-progressive Effects of a Series of Glycinyl and Alaninyl Triazolyl-oxazolidinones on Kelly Neuroblastoma Cell Line.

BACKGROUND/AIM: Neuroblastoma (NB), the most common extracranial malignant childhood tumor accounts for about 15% of cancer-related deaths in children. Despite the intensive treatment of patients with high-risk scarification of NB, clinical outcomes indicate tumor recurrence greater than 50% and late severe adverse effects. Oxazolidinones are 5-membered heterocyclic compounds with antibacterial activity against resistant bacterial strains. Structural modifications around the oxazolidinone moiety have resulted in derivatives with anti-cancer properties against proliferation, motility, and invasion of breast cancer cells. This study aimed to examine the anti-cancer potential of novel oxazolidinones against a model of a neuroblastoma cell line. MATERIALS AND METHODS: Newly synthesized and characterized triazolyl-oxazolidinone derivatives were incubated with neuroblastoma Kelly cells. The anti-proliferation and anti-progression effects of the compounds were evaluated by MTT, and adhesion with migration assays. RESULTS: The 5-nitrofuroyl glycinyl-oxazolidinone containing 4-methyltriazolyl group demonstrated the most potent activity with an IC50=6.52 µM. Furthermore, the D-isomer of 5-nitrothiophenecarbonyl alaninyl containing derivative reduced the adhesion to fibronectin by 56.34%, while the D-isomer of 5-nitrofuroyl alaninyl derivative reduced the migration of Kelly cells by 29.14%. CONCLUSION: The presence of the 4-methyltriazolyl moiety seems to enhance the anti-proliferative property of triazolyl-oxazolidinone derivatives, as demonstrated by PH-145. There is little or no effect of the stereochemistry of the alanine side-chain on the antiproliferative effect, as demonstrated by the 5-nitrofuroyl D- and L-alaninyl containing derivatives with similar IC50 values. The observed differences in the inhibition of adhesion and migration by the oxazolidinones on Kelly cells provide a new therapeutic approach that needs further investigation.

Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing.

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80-110°C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110°C at pH values corresponding to the general pH values of foods (pH 5-7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion.

Heat denaturation of the antibody, a multi-domain protein.

The antibody is one of the most well-studied multi-domain proteins because of its abundance and physiological importance. In this article, we describe the effect of the complex, multi-domain structure of the antibody on its denaturation by heat. Natural antibodies are composed of 6 to 70 immunoglobulin fold domains, and are irreversibly denatured at high temperatures. Although the separated single immunoglobulin fold domain can be refolded after heat denaturation, denaturation of pairs of such domains is irreversible. Each antibody subclass exhibits a distinct heat tolerance, and IgE is especially known to be heat-labile. IgE starts unfolding at a lower temperature compared to other antibodies, because of the low stability of its CH3 domain. Each immunoglobulin domain starts unfolding at different temperatures. For instance, the CH3 domain of IgG unfolds at a higher temperature than its CH2 domain. Thus, the antibody has a mixture of folded and unfolded structures at a certain temperature. Co-existence of these folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate which causes the inactivation of the antibody.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of ß-lactoglobulin.

The effect of high pressure homogenization (HPH) on the structure of ß-lactoglobulin (ß-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from ß-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified ß-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cß-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cß-lg = 1 wt%. At this protein concentration, the prior denaturation of ß-lg by HPH did not modify the droplet size of nanoemulsions prepared with these ß-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra.

A newly developed method of analysis of difference-UV spectra was successfully implemented in the study of the effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate. It was found that whey proteins exhibit their highest stability against heat denaturation at pH 3.75. At very low pH values, i.e. 2.5, they exhibited considerable cold denaturation, while after heating at this pH value, the supplementary heat denaturation rate was lower compared to that at neutral pH. The highest heat denaturation rates were observed at pH values higher than neutral. High power sonication on whey proteins, previously heated at 90°C for 30min, resulted in a rather small reduction of the fraction of the heat denatured protein aggregates. Finally, when ethanol was used as a cosolvent in the concentration range 20-50%, a sharp increase in the degree of denaturation, compared to the native protein solution, was observed.

Studying the denaturation of bovine serum albumin by a novel approach of difference-UV analysis.

A novel approach in the analysis of difference-UV spectrophotometric data for determining the heat denaturation degree of bovine serum albumin (BSA) was assessed. Five different parameters of difference-UV spectra were obtained by subtracting spectra of unheated and denatured protein solutions at different temperature-time combinations. BSA was found to exhibit a maximum degree of heat denaturation of about 17% compared to the complete unfolding caused by 6M guanidine hydrochloride. This low degree of heat denaturation is probably caused by the aggregation of the initially unfolded protein molecules. The kinetic analysis exhibited discontinuities in the Arrhenius plots, distinguishing the unfolding and aggregation phases of the denaturation process, whereas such a discrimination could not be obtained by differential scanning calorimetry analyses. The proposed method is accurate, fast, simple and sensitive enough to detect changes in the protein heat denaturation even at short temperature-time intervals.

Binding of Escherichia coli Does Not Protect Tulane Virus from Heat-Inactivation Regardless the Expression of HBGA-Like Molecules.

Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases.

On the water-holding of myofibrils: Effect of sarcoplasmic protein denaturation.

The role of heat-denatured sarcoplasmic proteins in water-holding is not well understood. Here we propose a new hypothesis that in PSE-like conditions denatured sarcoplasmic proteins aggregate within and outside myofilaments, improving the water-holding of denatured myofibrils. The process is compartmentalized: 1) within the filaments the denatured sarcoplasmic proteins shrink the lattice space and water is expelled; and 2) between the myofibrils and in the extracellular space, the coagulated sarcoplasmic proteins trap the expelled water from interfilamental space. The effect of sarcoplasmic proteins on the water-holding of myofibrils following incubation for 1h at 21 to 44°C was investigated. Our results were consistent with the new hypothesis. Myofibrils without sarcoplasm had the poorest water-holding. With increasing amount of denatured sarcoplasmic proteins, the water-holding of heat-denatured myofibrils improved proportionally. X-ray diffraction was used to measure the lattice space between the filaments. Precipitated sarcoplasmic proteins shrank (P<0.001) the lattice spacing by 6.3% at 44°C.

Heat-Induced Soluble Protein Aggregates from Mixed Pea Globulins and ß-Lactoglobulin.

The present work investigates the formation of protein aggregates (85 °C, 60 min incubation) upon heat treatment of ß-lactoglobulin (ßlg)-pea globulins (Glob) mixtures at pH 7.2 and 5 mM NaCl from laboratory-prepared protein isolates. Various ßlg/Glob weight ratios were applied, for a total protein concentration of 2 wt % in admixture. Different analytical methods were used to determine the aggregation behavior of "mixed" aggregates, that is, surface hydrophobicity and also sulfhydryl content, protein interactions by means of SDS-PAGE electrophoresis, and molecule size distribution by DLS and gel filtration. The production of "mixed" thermal aggregates would involve both the formation of new disulfide bonds and noncovalent interactions between the denatured ßlg and Glob subunits. The majority of "mixed" soluble aggregates displayed higher molecular weight and smaller diameter than those for Glob heated in isolation. The development of pea-whey protein "mixed" aggregates may help to design new ingredients for the control of innovative food textures.

Correlation analysis for heat denaturation of Trp-cage miniprotein with explicit solvent.

Energetics was analyzed for Trp-cage miniprotein in water to elucidate the solvation effect in heat denaturation. The solvation free energy was computed for a set of protein structures at room and high temperatures with all-atom molecular dynamics simulation combined with the solution theory in the energy representation, and its correlations were investigated against the intramolecular (structural) energy of the protein and the average interaction energy of the protein with the solvent water. It was observed both at room and high temperatures that the solvation free energy is anticorrelated to the structural energy and varies in parallel to the electrostatic component of the protein-water interaction energy without correlations to the van der Waals and excluded-volume components. When the set of folded structures sampled at room temperature was compared with the set of unfolded ones at high temperature, it was found that the preference order of the two sets is in correspondence to the van der Waals and excluded-volume components in the sum of the protein intramolecular and protein-water intermolecular interactions and is not distinguished by the electrostatic component.

Harnessing denatured protein for controllable bipolar doping of a monolayer graphene.

In this work, we demonstrated tunable p- and/or n-type doping of chemical vapor deposition-grown graphene with the use of protein bovine serum albumin (BSA) as a dopant. BSA undergoes protonation or deprotonation reaction subject to solution pH, thereby acting as either an electron donor or an electron acceptor on the graphene surface layered with denatured BSA through π-stacking interaction. This direct annealing of graphene with denatured BSA of amphoteric nature rendered facilitated fabrication of a p- and/or n-type graphene transistor by modulating pH-dependent net charges of the single dopant. Following AFM confirmation of the BSA/graphene interface assembly, the carrier transport properties of BSA-doped graphene transistors were assessed by I-V measurement and Raman spectra to show effective charge modulation of the graphene enabled by BSA doping at various pH conditions. The protein-mediated bipolar doping of graphene demonstrated in our work is simple, scalable, and straightforward; the proposed scheme is therefore expected to provide a useful alternative for fabricating graphene transistors of novel properties and promote their implementation in practice.