Thermal Denaturation Of Proteins Research Articles

Research on improving the structural stability of surimi 3D printing through laser cooking techniques

At present, research on 3D printing technology for meat protein food is still in its early stages, and improving the molding stability and accuracy of products has always been a key challenge. Here, we take surimi protein as an example and achieve its localized thermal gelation quickly and precisely by incorporating laser sintering technology. And based on the thermal denaturation and solidification of proteins during the printing process, the structural instability problem of meat protein food 3D printing is solved. In this study, we used a CO2 laser with a power of 50 W to locally heat cure surimi during the printing process. The results show that under the conditions of 1 mm laser spot diameter, 500 mm/s scanning speed and 0.25 mm scanning interval, the shape accuracy and gel strength of surimi products with 1.5 mm layer thickness are significantly improved, and this combination parameter shows the best effect in the experiment. This study provides a theoretical basis and reference for real-time solidification in meat protein food printing.

Investigating the impact of sous vide cooking on the eating quality of spent buffalo (BUBALUS BUBALIS) meat

This study describes the impact of sous vide cooking at different temperatures and time intervals on the eating quality, specifically tenderness of two muscles, bicep femoris (BF) and semitendinosus (ST) from spent buffalo (Bubalus bubalis). Spent buffalo refers to water buffalo that are no longer considered productive following a sixth lactation cycle. Steaks from each muscle were obtained and cooked at three combinations of time and temperature, namely 55 °C-8H, 65 °C-5H, and 95 °C-45 M, respectively. Warner-Bratzler Shear Force (WBSF), cooking loss, cooking yield, color, water activity (aw), total water content (TWC), total collagen content (TCC), heat soluble collagen (HSC), myofibrillar fragmentation index (MFI), and sensory evaluation were measured. The collagen solubilization results showed that temperature and time interacted (P ≤ 0.05), reducing the toughness of the muscles. The tenderization achieved through sous vide cooking was mainly attributed to the thermal denaturation of proteins at the typically lower temperatures and extended time used, weakening of connective tissue through collagen solubilization, and water retention. More cooking loss (P ≤ 0.05) was observed at high temperature treatment of 95 °C-45 M. Meat color, TWC, MFI, and overall acceptability exhibited differences among treatments (P ≤ 0.05). An extended heat interval at lower temperatures caused initial denaturation of myofibrillar proteins, then solubilization of connective tissue proteins. Cooking treatment 55 °C-8H (P ≤ 0.05) reduced the WBSF in both muscles; however, the ST appeared more tender than BF.

Effect of neurodegenerative mutations in NEFL gene on thermal denaturation of the neurofilament light chain protein

The effects of amino acid substitutions E90K, N98S and A149V in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule was investigated. By using the circular dichroism spectroscopy, it was shown that these substitutions do not lead to a changes in the α-helical structure of NFL, but they caused a noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using the differential scanning calorimetry. It was shown that the E90K replacement lead to the disappearance of the low-temperature thermal transition (domain 1). The mutations lead to changes in the enthalpy of melting of NFL domains, as well as lead to significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these amino acid substitutions are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coiled-coil domain 1A, they differently effects on the structure and stability of the NFL molecule.

Effect of Neurodegenerative Mutations in the NEFL Gene on Thermal Denaturation of the Neurofilament Light Chain Protein.

Effects of E90K, N98S, and A149V mutations in the light chain of neurofilaments (NFL) on the structure and thermal denaturation of the NFL molecule were investigated. By using circular dichroism spectroscopy, it was shown that these mutations did not lead to the changes in α-helical structure of NFL, but they caused noticeable effects on the stability of the molecule. We also identified calorimetric domains in the NFL structure by using differential scanning calorimetry. It was shown that the E90K replacement leads to the disappearance of the low-temperature thermal transition (domain 1). The mutations cause changes in the enthalpy of NFL domains melting, as well as lead to the significant changes in the melting temperatures (Tm) of some calorimetric domains. Thus, despite the fact that all these mutations are associated with the development of Charcot-Marie-Tooth neuropathy, and two of them are even located very close to each other in the coil 1A, they affect differently structure and stability of the NFL molecule.

Assembly of plant-based meat analogs using soft matter physics: A coacervation-shearing-gelation approach

High-energy processing equipment, like extruders or shear cells, are normally required to create fibrous plant-based meat analogs with textures and mouthfeels designed to mimic those of real meat. Here, we propose a simple low-energy method of creating fibrous structures using a soft matter physics approach, which involves complex coacervation, shearing, and gelation of protein/polysaccharide mixtures. A mixture of potato protein and gellan gum was used to create the chicken analogs. Initially, potato protein (10, 15, or 20 wt%) and gellan gum (3 or 4 wt%) were mixed to produce aqueous solutions under conditions where coacervates should form due to electrostatic attraction between the proteins and polysaccharides. This solution was then sheared to elongate and align the coacervates into fiber-like structures. Finally, the mixtures were heated (90 °C, 30 min) to induce thermal denaturation of the proteins, which stabilized the fibrous structures by gelling the proteins. The textural, rheological, and thermal properties of the chicken analogs were characterized and compared to those of real chicken. The texture and rheology of the chicken analogs became increasingly close to real chicken as the protein content of the plant-based chicken was increased from 10 to 20 wt%. Differential scanning calorimetry showed that the thermal denaturation of the potato proteins in the chicken analogs occurred over a similar temperature range as the myosin proteins in real chicken. Thermogravimetric analysis showed that the chicken analogs containing 20 wt% protein behaved the most similarly to real chicken during heating. Analysis of the appearance and microstructure of the chicken analogs showed they had aligned fibrous structures like those found in real chicken. This study suggests that soft matter physics approaches can be used for the creation of fibrous structures and the development of plant-based meat analogs.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Effect of Potato Dietary Fiber on the Quality, Microstructure, and Thermal Stability of Chicken Patty.

A total of 150 chicken patties containing different concentrations of potato dietary fiber (PDF) (0.0-4.0%) (30 for every treatment) with three replicates were used to access the influence of PDF on their quality, microstructure, and thermal stability. PDF improved the quality of chicken patty, including significantly inhibiting dimensional change and improving water- and fat-binding properties and textural properties (p < 0.05). Moreover, PDF promoted a more homogeneous and dense meat-protein network structure to be formed. The results of thermal stability showed that PDF did not affect the thermal denaturation of proteins (p > 0.05). The samples with PDF (<3.0%) did not have a significant negative effect on sensory properties of chicken patty; meanwhile, there were more abundant nutrients and a lower energy value in samples with PDF compared with the control. Therefore, PDF could be a promising ingredient to improve the properties of chicken patties, which was related to the amount of PDF added and performed best at 3.0% level.

Effect of calcium on the thermal denaturation of whey proteins and subsequent fouling in a benchtop fouling device: An experimental and numerical approach

The key role of calcium in whey protein fouling behavior is well known in plate heat exchangers (PHEs), as it affects both the thermal denaturation of proteins and deposition reaction on the surface. However, the complex flow pattern and the closed configuration of PHEs make it hard to investigate this phenomenon in situ. In this paper, a microchannel benchtop fouling device was designed, making it possible to achieve a similar temperature profile to that performed in PHEs but in a laminar regime. A 3D simulation was developed to predict the thermal denaturation of β-lactoglobulin (BLG) as well as its deposition in the microchannel using computational fluid dynamics (CFD). The thermodynamics and heat transfer of the numerical model was validated by experimentally measuring the bulk fluid temperature profile in the microchannel using fluorescence microscopy, where Pyrromethene 556 was used as a temperature indicator. Results revealed a quasi-linear relationship between the pre-exponential factor of deposition reaction and the calcium concentration, implying that the fouling was built in such a pattern that only one calcium ion per one BLG molecule is involved. The imaging of the fouling deposit in situ showed spherical structures of deposits at low calcium levels, while denser and more rod-like ones were found at higher calcium concentrations. The fouling behavior was found to follow a crystallization-like pattern with preference upon the previously fouled layer instead of clean stainless steel surface. These findings confirm the essential role of ionic calcium on the formation of fouling deposits by anchoring the denatured BLG protein upon the surface as well as strengthening the protein-protein interactions for fouling build-up.

Cosolvent Exclusion Drives Protein Stability in Trimethylamine N-Oxide and Betaine Solutions.

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.

Thermal Denaturation of Milk Whey Proteins: A Comprehensive Review on Rapid Quantification Methods Being Studied, Developed and Implemented

Heat treatment of milk signifies a certain degree of protein denaturation, which modifies the functional properties of dairy products. Traditional methods for detecting and quantifying the denaturation of whey proteins are slow, complex and require sample preparation and qualified staff. The world’s current trend is to develop rapid, real-time analytical methods that do not destroy the sample and can be applied on/in-line during processing. This review presents the rapid methods that are being studied, developed and/or applied to determine and quantify the thermal denaturation of whey proteins, including spectroscopic, electrochemical and miniaturized methods. The selected methods save a significant amount of time and money compared to the traditional ones. In addition, the review emphasizes the methods being applied directly to milk and/or that have potential for on/in/at-line application. There are interesting options to quantify thermal denaturation of whey proteins such as biosensors, nanosensors and microchips, which have fast responses and could be automated. In addition, electrochemical sensors are simple to use and portable, while spectroscopy alternatives are suitable for on/in/at-line process.

CalFitter 2.0: Leveraging the power of singular value decomposition to analyse protein thermostability.

The importance of the quantitative description of protein unfolding and aggregation for the rational design of stability or understanding the molecular basis of protein misfolding diseases is well established. Protein thermostability is typically assessed by calorimetric or spectroscopic techniques that monitor different complementary signals during unfolding. The CalFitter webserver has already proved integral to deriving invaluable energy parameters by global data analysis. Here, we introduce CalFitter 2.0, which newly incorporates singular value decomposition (SVD) of multi-wavelength spectral datasets into the global fitting pipeline. Processed time- or temperature-evolved SVD components can now be fitted together with other experimental data types. Moreover, deconvoluted basis spectra provide spectral fingerprints of relevant macrostates populated during unfolding, which greatly enriches the information gains of the CalFitter output. The SVD analysis is fully automated in a highly interactive module, providing access to the results to users without any prior knowledge of the underlying mathematics. Additionally, a novel data uploading wizard has been implemented to facilitate rapid and easy uploading of multiple datasets. Together, the newly introduced changes significantly improve the user experience, making this software a unique, robust, and interactive platform for the analysis of protein thermal denaturation data. The webserver is freely accessible at https://loschmidt.chemi.muni.cz/calfitter.

Enfuvirtide biosynthesis in thermostable chaperone-based fusion

Synthetic peptides are in high demand as biologically active substances. Solid phase synthesis is the primary method of peptide production. However, it has drawbacks: large amount of chemical waste and rapid increase in price with peptide length. Biosynthesis is intended as method to bypass these flaws. Direct biosynthesis is usually not effective and among other approaches for improving quality and quantity of target product fusion partners are widely used. In this study we used a thermostable chaperon-based fusion partner developed by us to produce enfuvirtide in Escherichia coli expression system. Fusion partner's thermal stability provided additional purification mode by thermal denaturation of host proteins in lysate. Fusion protein was purified by ion exchange chromatography after lysate heating step and was then hydrolyzed with cyanogen bromide to release enfuvirtide. Enfuvirtide was isolated by RP-HPLC up to 94% purity with total yield of 2.86–3.31 mg per 1 L of low-density culture. The data demonstrate the posibility of thermostable chaperone-based fusion partner GroEL use for effective peptide biosynthesis.

Effect of Protein Thermal Denaturation on the Texture Profile Evolution of Beijing Roast Duck.

To investigate the mechanism of the texture formed by protein thermal denaturation, the profile and formation of texture and thermal denaturation of protein were evaluated using texture profile analysis (TPA) and transmission electron microscopy (TEM) combined with differential scanning calorimeter (DSC). Results indicated that the surface temperature of Beijing roast duck increased from 23.9 to 174.4 °C, while the center temperature rose from 20.6 to 99.3 °C during roasting. Shear force decreased significantly during the first 20 min, and the texture profile largely changed at 20 and 40 min. Firstly, Band I was broken and twisted, Band A was overstruck, and Z-line was diffused and finally disappeared, resulting in a blurred myofibril structure. The sarcomere considerably contracted within 30 min. Secondly, the main myofibrillar proteins were denatured at 20 and 40 min, respectively. The formation of hydrophobic interactions and the reduction of ionic bonds were observed. Thirdly, roasting induced protein thermal denaturation, which was correlated with interprotein forces, texture profile, and the shear force. Muscle fibers were damaged and shrunken, accompanied by the formation of hydrophobic interactions and the reduction of ionic bonds. The turning points were at 20 and 40 min, and the main proteins were denatured, leading to the formation of tenderness of Beijing roast duck.

Effects of immersion freezing on the conformational changes of myofibrillar proteins in pork under ultrasonic power densities of 0, 15, 30 and 45 W L−1

SummaryThe objective of this study was to assess the effects of ultrasonic‐assisted immersion freezing (UIF) of 0, 15, 30 and 45 W L−1 on the conformation changes of pork myofibrillar protein (MP). The structure and conformation of MP were evaluated, and the solubility, thermal stability and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) were also discussed. The sample treated with 30 W L−1 (UIF‐30) had the highest total protein solubility (116.6 mg g−1) and thermal denaturation temperature (57.07℃) compared with other treatments. UIF‐30 has higher α‐helix content, and fluorescence intensity together with surface hydrophobicity had insignificant changes compared with the control. In SDS–PAGE, only the sample treated with 45 W L−1 (UIF‐45) showed slight band degradation. In general, UIF‐30 effectively minimised the changes of protein conformation, which may be related to the reduction of mechanical damage to muscle cells caused by rapid freezing to form regular and uniform ice crystals. This is more conducive to reducing pork quality degradation caused by protein freeze denaturation.

Effects of the thermal denaturation degree of a whey protein isolate on the strength of acid milk gels and the dissociation of κ-casein.

In this study, the effects of the degree of thermal denaturation of whey protein (WP) added to milk on the dissociation of κ-casein from casein micelles were investigated, since they are related to the strength of acid milk gel and its factors. Acid milk gels were prepared by heating thermally denatured WP isolate (WPI) and undenatured milk mixtures and treating them with glucono-δ-lactone as a coagulant. The strength of these gels was negatively correlated with the WPI denaturation degree and strongly positively correlated with the extent of κ-casein dissociation from casein micelles. This behavior was ascribed to the fact that α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) contained in WPI denatured after heating and engaged in disulfide bond formation with each other. With an increase in the degree of denaturation and disulfide bond formation, the bonding between β-lactoglobulin and κ-casein was suppressed to decrease the amount of κ-casein-WPI complexes. When β-Lg forms SS bonds with α-La, the number of highly reactive, free SH groups decreases, which complicates the formation of SS bridges between β-Lg and κ-casein. Thus, the denaturation degree of WPI largely determined the degree of κ-casein dissociation from casein micelles and, consequently, the strength of acid milk gels. Adding WP to milk increases the strength of acid milk gel, and it can be controlled by changing the degree of thermal denaturation of the WP. Furthermore, it was clarified for the first time that the dissociation of κ-casein from casein micelles influences this effect. Further studies are needed to elucidate the structural features of κ-casein-dissociated micelles.

Impact of germination time on protein solubility and anti-inflammatory properties of Pisum sativum L grains

During germination processes take place that modify the major components of the grain, such is the case of proteins that are hydrolyzed to generate peptides that can lead to the generation of bioactivity. The objective of the present work was to germinate grains of Pisum sativum to evaluate the effect on the soluble protein content and the anti-inflammatory activity. The grains were subjected to 10 days of germination at 24 °C and relative humidity of 75%. Sprouts were lyophilized, milled, and phenolic compounds were extracted to avoid interferences. Soluble protein content varied significantly during the 10 days of germination. In vitro assays indicate that sprouts protein inhibits thermal denaturation of proteins, protease activity, and stabilize cell membranes. The IC50 values indicate that after germination the bioactivity increased between 1.4 and 3.5 times, with respect to the ungerminated grains. Results indicated that Pisum sativum sprouts may constitute promising health-promoting foods.

Effects of three treatments on protein structure and gel properties of Perccottus glenii myofibrillar protein

Abstract In order to improve Perccottus glenii myofibrillar protein (MP) gel properties, three treatments were evaluated: ultrasonic, transglutaminase (TGase) and combined ultrasonic-transglutaminase treatments. Combined ultrasonic-transglutaminase treatment altered protein structure and gel properties most dramatically. As compared with untreated control group protein, treated protein gels possessed decreased sulfhydryl group content and increases in water holding capacity, whiteness value and hydrophobic interactions that increased gel strength value by up to 3.79 times that of untreated protein gel. Protein structural and Differential scanning calorimetry (DSC) analyses revealed that combined ultrasonic-TGase treatment increased both protein thermal denaturation temperature and UV absorbance (as compared to control and other treatment groups) that supported formation of MP gels with desirable characteristics. These results provide a theoretical basis for development of superior MP gels to promote greater utilization of this fish protein resource by the food industry.

Characteristics of rising temperatures through laser irradiation using a temperature sensor integrated optical fiber probe for soft tissue ablation

A new structure composed of silica optical fibers for soft tissue ablation is proposed and fabricated in this study. Laser irradiation and temperature monitoring are simultaneously performed using a wavelength-division multiplexing technique with a single optical fiber probe. A 1.55-μm wavelength range light is used for temperature measurement. The fabricated Fabry–Perot interference temperature sensor comprising a glass capillary and a graded-index multimode fiber exhibits good temperature sensor characteristics. The probe end is terminated with a glass capillary having a declined glass cap. This portion suppresses the reflected light backward to the incident fibers. Because the reflected light at this portion does not affect the interference of internal reflected light, regardless of the outer medium, stable temperature measurement is confirmed. A 1.48-μm laser irradiation method is investigated as a thermal source for soft tissue ablation. The characteristics of rising temperatures through laser irradiation of chicken-breast meat are investigated and indicate sufficient temperature increases for thermal ablation of soft tissue. A good estimation of the thermal protein denaturation region is achieved through a simple analytical equation using the measured value at the fiber tip temperature sensor.

Effect of temperature on thermal denaturation of skimmed coconut milk to produce a new product, coconut water

This study investigated the effect of temperature on the thermal denaturation of skimmed coconut milk which is the residue of the virgin coconut oil extraction industry to produce a new product, coconut meat water. Skimmed coconut milk is a waste product that is expensive to dispose. The thermal denaturation of protein was applied to skimmed coconut milk to obtain coconut meat water and protein sludge. The optimum conditions were at 100°C for 10 min which had a significant effect on the amount of protein sediment. The separated coconut meat water was sterilized and stored which were analyzed the properties. The results indicated that the color (L* and a* values) decreased over time. The pH and total soluble solids content were 6.19–6.29 and 5.0–5.1°Brix, respectively. No microorganisms were detected. The nutrients identified included potassium, magnesium, and phosphorus. Consumer sensory testing using a 9-point hedonic scale indicated the product was in the range of 6.45–6.92 which was acceptable. Practical applications The study focused on the utilization of skimmed coconut milk (a residue of the virgin coconut oil extraction industry) to produce a new product. The developed sterilized coconut meat water has high nutritional value, good sensory quality, and low raw material cost. The technology to produce this product is simple and accessible to small-scale manufacturers. The sterilized coconut meat water could be used as a valuable new beverage product with a taste similar to commercial coconut water.

Role of a high centrality residue in protein dynamics and thermal stability

Centralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 °C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 °C. 1H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the distribution of thermal energy through HisF structure.