Thermal Denaturation Temperature Research Articles

Stable and biocompatible fibrillar hydrogels based on the self-crosslinking between collagen and oxidized chondroitin sulfate

Stable and biocompatible collagen-based hydrogels based on natural extracellular matrix (ECM) with a homogeneous microstructure are ideal scaffolds for tissue engineering; however, lack of structural stability and mechanical property have hindered their application. In this work, Chondroitin Sulfate (CS) was periodate-oxidized to prepare dialdehyde ECM-derived biopolymer with the weight average molecular weight of 3.7 × 104 g/moL to construct the stable collagen-based fibrillar hydrogels. The results of Fourier Transform Infrared (FTIR) analysis and hydroxylamine hydrochloride titration assay confirmed the presence of aldehyde groups in oxidized CS (CSox) with an oxidization degree of 30.67%. Gelation time test and scanning electron microscopy (SEM) observation showed that gel formation and fibrillogenesis process were accelerated by adding CSox, and all hydrogels exhibited a tight fibrillar structure. With an increase in CSox concentration from 0.125 to 2.000 mg/mL, the crosslinking degree increased from 24.95% to 35.05% due to the formation of covalent bonds between the ε-amino groups of collagen and aldehyde of CSox. The mechanical strength of collagen/CSox hydrogels reached a maximum value of 11.79 KPa, which was more than triple that of pure collagen hydrogel (3.58 KPa) at 0.125 mg/mL CSox. The swelling ratio, enzymatic resistance, and thermal stability were also improved with the increasing concentration of CSox. When the CSox concentration reached 0.500 mg/mL, the collagen-based hydrogels exhibited the highest thermal denaturation temperature, which was approximately 7.5 °C higher than that of the pure collagen hydrogel. Moreover, the results of the CCK-8 assay and LIVE/DEAD staining demonstrated that all the collagen/CSox hydrogels accelerated cell proliferation and showed acceptable biocompatibility. This work broadens the avenue for the design of natural collagen-based hydrogels for tissue engineering.

Structural, functional, rheological, and biological properties of the swim bladder collagen extracted from grass carp (Ctenopharyngodon idella)

The objective of this study was to develop an aquatic collagen with high thermal stability as a possible alternative to terrestrial sources. Swim bladder collagen (SBC) was extracted from grass carp with high yield of 39.2% (db), and characterized as type I collagen. X-ray diffraction indicated that SBC maintains a native triple helical collagen structure. Secondary structure analysis revealed that SBC consists of 41.93% α-helices, 45.31% β-sheets, 12.76% β-turns, and 0% random coils. Proteins from SBC exhibited foaming and emulsifying superiority to proteins from terrestrial sources, such as chicken feet collagen. The thermal denaturation temperature of SBC (34.3 °C) is similar to porcine skin collagen. Dynamic frequency sweep tests showed that elasticity plays a dominant role in the SBC solution system as the concentration increases. The SBC solution system shows viscous behavior when the temperature is below Td. In addition, the steady shear tests showed that all of the SBC solutions exhibited pseudoplasticity with shear-thinning behavior. SBC could provide a suitable environment for MC3T3-E1 cell growth and maintain normal cellular morphology. Overall, the results indicated that SBC might has potential for further applications in food, cosmetics and biomedical fields.

The water soluble zinc based metal-organic frameworks (Zn-MOFs) as potential inhibitors for collagen fibrillogenesis

Small molecules ranging from organic to inorganic systems have been reported as stabilizing agents for collagen. Various transition metal complexes have been utilized as tanning agent. However, as per the environmental norms issued by various regulatory agencies, the presence of certain metals such as Cr, Fe, Al, Zr and Ti in leather has been restricted to minimal amount (50 ppm), an unsurmountable task. To overcome the above issue and find an alternative tanning system, here in this study, we have reported the interaction of two water-soluble zinc-based metal-organic frameworks (MOFs), i.e., ZnPV (1) and ZnPA (2), with collagen using various spectroscopic techniques. Fibrillation kinetics studies showed that a significant delay in fibril formation with Zn-MOFs treated collagen was observed compared to the collagen untreated/ treated with individual ligands and metal salt. Circular dichroism studies show that at a low weight ratio (1:0.2 and 1:1::Collagen: MOF), no perturbation in the triple helical structure was observed, while at higher weight ratio (1:4), denaturation of collagen occurs. FT-IR studies showed that no perturbation was observed in the amide backbone in MOF-treated collagen. Differential scanning calorimetric data revealed that both Zn-MOFs increased the thermal denaturation temperature by 22 ± 2 °C compared to the collagen treated with individual entities. The viscosity of collagen rises with the increase in the concentration of Zn-MOFs. To the best of our knowledge, this is the first report on the use of the metal-organic framework as a stabilizing agent for collagen structure and might help in exploring the MOFs as potential tanning agents.

Functional properties of sharpnose stingray (Dasyatis zugei) skin collagen by ultrasonication extraction as influenced by organic and inorganic acids

The aim of this study was to extract acid-soluble collagen from the skin of the sharpnose stingray using organic (acetic) and inorganic (hydrochloric) acid via an ultrasound-assisted method. The pre-treated stingray skin was extracted by 0.5M acetic acid and hydrochloric acid under ultrasonication treatment for 30 min. The collagen's properties, including viscosity, rheology, water-holding capacity (WHC), emulsifying stability index (ESI) and solubility, were determined. The yield of collagen extracted using acetic acid (11.49 ± 0.03%) was significantly higher than the yield extracted using hydrochloric acid (3.50 ± 0.01%) (p < 0.05). The viscosities of both extracted collagens were thermally stable, with a high thermal denaturation temperature (Td). The viscoelastic properties of collagen extracted using acetic acid were higher than those of collagen extracted using hydrochloric acid. The WHC and ESI for extracted collagen using acetic acid were significantly higher (47.8%, 15.01 min) than those of extracted collagen using hydrochloric acid (20.08%, 12.91 min), respectively. Both extracted collagens had higher solubility in an acidic pH range (pH 1–5) and the lowest solubility at a basic pH range (pH 9–10). Hence, these findings showed that the collagen extracted by ultrasound using acetic acid gave a higher yield and better properties than collagen extracted using hydrochloric acid.

Development of pH-responsive emulsions stabilized by whey protein fibrils

Globular proteins can form fibrillar structures, for example, by heating them above their thermal denaturation temperature under suitable pH (usually highly acidic) and ionic strength (usually relatively low) conditions. These fibrils can then be used as functional ingredients in foods. In this study, we investigated the potential of whey protein isolate fibrils (WPIFs) to act as stabilizers to create pH-responsive emulsions. Initially, changes in the properties of the WPIFs were measured when the system was changed from pH 2.5 to 10.0 and then back to pH 2.5. WPIF-based emulsions were then prepared and subjected to the same pH regime. For comparison, WPI-emulsions stabilized by the molecular form of native whey protein were subjected to the same treatment. The pH-dependence of the properties of the WPIF-emulsions was different from that of the WPI-ones. Insights into the molecular origin of these effects were obtained by measuring the interfacial properties, circular dichroism spectra, and surface hydrophobicities of the WPIFs and WPI. These results indicated the relative importance of electrostatic, hydrogen bonding, and hydrophobic interactions at the oil-water interface and/or continuous phase in stabilizing the emulsions. WPIF-emulsions were more resistant to coalescence than WPI-emulsions, which was attributed to the formation of a more robust interfacial coating around the oil droplets and a strong viscoelastic network in the aqueous phase. This research shows that the functional performance of food proteins can be extended by creating fibril structures.

Heat-induced gel properties and gastrointestinal digestive properties of egg white produced by hens fed with selenium-enriched yeast

The aim of this study was to investigate the effect of selenium enrichment on the gel properties and gastrointestinal digestive properties of egg white. Results of texture profile analysis, scanning electron microscopy and differential scanning calorimetry showed that the heat-induced gel of selenium-enriched egg white (EW-2) exhibited higher gel strength, smoother microstructure, and higher thermal denaturation temperature than ordinary egg white (EW-1), which might be due to the change of ovomucin and ovotransferrin content. The gastrointestinal digestive products of the EW-2 protein contained a higher proportion of small peptides and more free amino acids than those of EW-1. Results of oxygen radical absorbance capacity and cellular antioxidant activity assays indicated that digestive products of the EW-2 protein exhibited stronger antioxidant activity than those of the EW-1 protein. In summary, Se enrichment improved heat-induced gel properties of egg white, and promoted the gastrointestinal digestion of egg white protein.

Preparation and characterization of the dialdehyde hydroxypropyl methylcellulose/collagen (DHPMC/COL) solutions

Dialdehyde hydroxypropyl methylcellulose (DHPMC)/Collagen (DHPMC/COL) solutions were prepared with different DHPMC contents (0–70 wt%). According to the viscosimetric method, all samples were compatible. The compatibility was related to Schiff’s base, which formed between the amino groups of collagen and the dialdehyde groups of DHPMC. When DHPMC content increased, the cross-linking degree increased, and the synchronous negative cross-peaks between the C-O(H) of DHPMC at 1031 cm−1 and amide bands of collagen at 1623 cm−1, 1523 cm−1, and 1238 cm−1 indicated that hydrogen bonds mainly formed between DHPMC and collagen. As a result, the thermal stability improved. The morphology showed more sheet-like structures than collagen. When the DHPMC content was 50 wt%, the thermal denaturation temperature (Td) and residual weight% were 8.5 °C and 10.22%, respectively, higher than other samples. However, the cross-linking degree decreased as the DHPMC content increased further. The intensity of negative cross-peak was decreased in synchronous correlation spectra, while the intensity of cross-peak at 1031 cm−1 was enhanced, indicating that hydrogen bonds formed between the C–O and C–O(H) of the DHPMC were enhanced. Consequently, the thermal stability decreased and more porous structures were observed compared to those when DHPMC content was 50 wt%.

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis.

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.

Physico-Chemical Characteristics and In Vitro Gastro-Small Intestinal Digestion of New Zealand Ryegrass Proteins

Being widely abundant, grass proteins could be a novel source of plant proteins for human foods. In this study, ryegrass proteins extracted using two different approaches-chemical and enzymatic extraction, were characterised for their physico-chemical and in vitro digestion properties. A New Zealand perennial ryegrass cultivar Trojan was chosen based on its higher protein and lower dry matter contents. Grass protein concentrate (GPC) with protein contents of approximately 55 and 44% were prepared using the chemical and enzymatic approach, respectively. The thermal denaturation temperature of the GPC extracted via acid precipitation and enzymatic treatment was found to be 68.0 ± 0.05 °C and 66.15 ± 0.03 °C, respectively, showing significant differences in protein’s thermal profile according to the method of extraction. The solubility of the GPC was highly variable, depending on the temperature, pH and salt concentration of the dispersion. The solubility of the GPC extracted via enzymatic extraction was significantly lower than the proteins extracted via the chemical method. Digestion of raw GPC was also studied via a gastro-small intestinal in vitro digestion model and was found to be significantly lower, in terms of free amino N release, for the GPC prepared through acid precipitation. These results suggest that the physico-chemical and digestion characteristics of grass proteins are affected by the extraction method employed to extract the proteins. This implies that selection of an appropriate extraction method is of utmost importance for achieving optimum protein functionality during its use for food applications.

DNA-binding function of c-Myb R2R3 around thermal denaturation temperature.

The minimum DNA-binding domain of the transcrip­tional factor c-Myb R2R3 remarkably fluctuates in the solution. In the present study, we evaluated the protein fluctuation of R2R3 C130I mutant, R2R3*, on its DNA-binding and folding thermodynamics. DNA-binding analysis using isothermal titration calorimetry revealed that the heat capacity change determined from the correlation between temperature and binding enthalpy change is highly negative above 35°C, indicating that the fluctuation increases with increasing temperature and elevates the conformational change on DNA binding. The results were in accordance with those of differential scanning calorimetry, which revealed that the heat capacity corresponding to thermal denatu­ration gradually increased above 35°C, followed by the broad transition peak. In contrast, the transition peak of R2R3* in the DNA-bound state was sharper and larger than that in the DNA-unbound state. The fluctuating form could transform into lesser fluctuating form upon DNA binding, resulting in a larger enthalpy change for denaturation of R2R3* in the DNA-bound state. It should also be noted that R2R3* could specifi­cally bind to DNA around thermal denaturation temperature. This would be due to proteins with numerous fluctuations. Moreover, we discuss specific and non-specific DNA binding accompanied by the conformational change between well-ordered and disordered forms of R2R3* observed around the denaturation temperature.

Improvement of thermal stability of oyster (Crassostrea gigas) ferritin by point mutation

Ferritin can be widely used as functional nanomaterial. But the physiological activity of ferritin can be damaged under excessive temperatures, which affect the self-assembly property. In this study, point mutation was produced in Asp120 to Gly120 of ferritin amino acid sequence and the heat resistance was improved significantly. The thermal denaturation temperature of mutated ferritin is 89.17 °C and has increased by 13 °C more than the wild-type oyster ferritin. The effect of thermal treatment on the denaturation, aggregation state, particle size and the structure of ferritin was not changed before 90 °C. The computational modeling and analysis indicated that mutated ferritin promotes the overall structural stability assembly via decreasing the interaction energies of 62 percent energies in 3-fold interface. Improving the thermal stability of oyster ferritin by point mutation enhances its applications as a food ingredient.

Characteristics and Functional Properties of collagen extracted from Nile tilapia (Oreochromis niloticus) skin

: Tilapia skin might be used as a new source of collagen, therefore collagen extracted from Nile tilapia skin can be utilized as food products because of its good functional properties. Two extraction methods were used to obtain acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from Nile tilapia skin. In this study, collagen was characterized in terms of FT-IR Spectrum, amino acid analysis, SDS-PAGE and Thermal denaturation temperatures (Td). Also, some major functional properties including solubility, oil and water absorption, emulsifying activity and stability, foaming ability, foam stability, and gelation properties were determined. The results showed that the yields of ASC and PSC were 4.30% and 1.84 % (on a wet weight basis), respectively. Amino acid composition analysis indicated that the imino acid content of ASC and PSC were found to be 172.10 and 164.18 residues per 1000 residues, respectively. SDS-PAGE results indicated that ASC and PSC were type I collagen. The FTIR Spectrum confirmed that the presence of a triple-helical structure characteristic of ASC and PSC. The denaturation temperature of ASC and PSC were 26⁰C and 25⁰C respectively. The solubility of ASC and PSC were highly solubilized in the pH range of 2–3. ASC and PSC had good water and oil absorption capacity. Gelation studies revealed that ASC and PSC have good gelation properties. Consequently, Nile tilapia skin could be used as a good source to extract ASC and PSC which can be used to add high value in the food industries.

Relationship between meat quality and intramuscular collagen characteristics of muscles from calf-fed, yearling-fed and mature crossbred beef cattle

Intramuscular Ehrlich Chromogen (EC) and pyridinoline (Pyr) concentrations in the gluteus medius (GM) and semitendinosus (ST) from crossbred Angus calf- (n = 14) and yearling-fed (n = 14) steer and mature cow (MC, n = 12) carcasses were related to collagen and intramuscular connective tissue (IMCT) thermal stability and peak Warner-Bratzler shear force (WBSF). In both muscles, Pyr density was greater in MC, while EC concentrations were comparable in calf- and yearling-fed steer muscles and lowest in MC muscles. Thermal denaturation temperature and enthalpy of IMCT were highest in both muscles when from MC, although only total collagen was correlated with WBSF in calf fed-yearling fed steer data. Results confirmed that EC concentration contributed to collagen thermal stability in steer muscles, but decreased it in MC muscles, while Pyr was consistently associated with collagen thermal stability.

Raw and cooked meat emulsion stability as affected by starches determined by principal component analysis

This work aimed to study the relationship between meat emulsion in both raw and cooked states employing two different types of starch, potato, and wheat, in meat batters formulated with different fat percent. Emulsion stability as related to water retention capacity, thermal denaturation temperature, and enthalpy plus thermal diffusivity were determined in the raw and cooked state. There was an inherent difference in potato starch functionality, with higher emulsion stability and yield, but the Pearson correlation analysis and PCA demonstrated that water holding capacity and the denaturation enthalpy resulted in higher cook yield as a consequence of lower expressible moisture. This results in establishing the importance to maintain meat extensors as starches functionality before and during mixing and thermal processing to obtain better yield and less water release in fat-reduced meat products, both important quality parameters, related to sensory (juiciness) and texture (hardness) properties of comminuted meat products.

Shockwave processing of beef brisket in conjunction with sous vide cooking: Effects on protein structural characteristics and muscle microstructure

We studied the effect of shockwave processing and subsequent sous vide cooking on meat proteins (molecular size and thermal stability) and muscle structures (molecular, micro- and ultrastructure). Beef briskets were subjected to shockwave (11 kJ/pulse) and were sous vide-cooked at 60 °C for 12 h. Shockwave processing alone decreased the enthalpy and thermal denaturation temperature of the connective tissue proteins (second peak in the DSC thermogram, p < 0.05) compared to the control raw samples, while the protein gel electrophoresis profile remained unaffected. It led to disorganisation of the sarcomere structure and also modified the protein secondary structure. More severe muscle fibre coagulation and denaturation were observed in the shockwave-treated cooked meat compared to the cooked control. The results show that shockwave processing, with and without sous vide cooking, promotes structural changes in meat, and thus may have the potential to improve the organoleptic quality of the tough meat cuts.

Preparation and application of dialdehyde nanocellulose reinforced jatropha oil based polymer emulsions as leather fatliquors

Bio-based acrylated epoxidized jatropha oil (AEJO) was developed using a solvent-free synthetic method, and the structure of the resulting product was confirmed by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance (1H NMR) spectra. Subsequently, an amphiphilic polymer emulsion AEJO-AA was prepared by emulsion polymerization using acrylic acid (AA) and AEJO. Moreover, 2, 3-dialdehyde nanocellulose (DNC) was prepared and combined with AEJO-AA emulsion as a fatliquoring agent for goatskin upper leather. The fatliquored leather shows improved mechanical strength and thermal stability. When the DNC loading is 1.5 wt%, the fatliquored leather exhibits the highest tensile strength of 24.3 MPa, tear strength of 62.2 N/mm and thermal denaturation temperature (Td) of 95.4 °C. The fogging values of the fatliquored leather satisfy the requirements of automobile seat leather. The biochemical oxygen demand (BOD5) and chemical oxygen demand results indicate that the DNC/AEJO-AA emulsion is easily biodegradable. Overall, the developed DNC/AEJO-AA emulsion provides a new strategy in the production of bio-based and ecological polymer fatliquor of leather.

Physicochemical properties and gelling behaviour of Bambara groundnut protein isolates and protein-enriched fractions

Plant proteins, and specifically those from legume crops, are increasingly recognised as sustainable and functional food ingredients. In this study, we expand on the knowledge of Bambara groundnut (Vigna subterranea (L.) Verdc.) [BGN] proteins, by characterising the composition, microstructure and rheological properties of BGN protein isolates obtained via wet extraction and protein-enriched fractions obtained via dry fractionation. The BGN protein isolates were compared in the context of the major storage protein, vicilin, as previously identified. Molecular weight analysis performed with gel electrophoresis and size-exclusion chromatography coupled to light-scattering, revealed some major bands (190 kDa) and elution patterns with molecular weights (205.6–274.1 kDa) corresponding to that of BGN vicilin, whilst the thermal denaturation temperature (Tp 91.1 °C, pH 7) of BGN protein isolates also coincided to that of the vicilin fraction. Furthermore, the concentration dependence of the elastic modulus G’ of the BGN protein isolates, closely resembled that of BGN vicilin (both upon NaCl addition); suggesting that vicilin is the main component responsible for gelation. Confocal laser scanning and scanning electron micrographs revealed inhomogeneous aggregate structures, which implies that fractal scaling were better suited for description of the BGN protein isolate gel networks. Concerning the BGN protein-enriched fractions, both rotor and impact milling with air jet sieving and air classification, respectively, were successfully applied to separate these fractions from those high in starch; as evident from compositional analysis, particle size distributions and microscopic imaging. When considering sustainability aspects, dry fractionation could thus be a viable alternative for producing BGN protein-enriched fractions.

Effects of synergistic modification with enzymatic hydrolysis and phosphorylation on functional and structural properties of ovalbumin

The functional and structural properties of enzymatic hydrolysis and phosphorylation synergistic modified ovalbumin (OVA) were investigated. The results showed the solubility, gel strength, and emulsifying properties of the hydrolyzed and phosphorylation OVA (HP-OVA) were higher than that of the hydrolyzed OVA (H-OVA) and unmodified OVA, whereas the foaming ability and foam stability were reduced. Moreover, the H-OVA peptide chain caused more amino acid residues to be exposed, therefore, the α-helix and β-turns increased accordingly. The HP-OVA thermal denaturation temperature increased by 7.78°C and 17.67°C, respectively, compared with the H-OVA and OVA. In addition, due to the embedding of phosphate groups, the molecular structure was changed from a spherical body to a laminated sheet structure. Practical applications As an important food ingredient, OVA can improve the taste and texture of foods, improve product stability and extend shelf life. The functional properties of OVA can be improved by modification and its application can be further expanded in food. In this study, we used compound protease and phosphate to modify with OVA in order to further explore the functional and structural changes of OVA. The effect of the modified OVA on the solubility, gelation, emulsifying, and foaming properties were investigated, which will be helpful for the further understanding of the relationship between the structure and function properties of OVA. The present investigation was to provide the basic framework for deeply processed protein products, and compare with our previous research, which provide a system for its functional research and development, and a theoretical basis for the application of OVA in the food industry to adapt to the needs of different products.

The effects of L-arginine on protein stability and DNA binding ability of SaeR, a transcription factor in Staphylococcus aureus

The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production and stability of the recombinant protein SaeR, l-arginine (L-Arg) was added to the purification buffers. L-Arg enhanced the solubility and stability of the recombinant protein SaeR. The thermal denaturation temperature of SaeR in 10 mM L-Arg buffer was significantly increased compared to the buffer without L-Arg. Microscale Thermophoresis (MST) analysis results showed that the SaeR protein could bind to the P1 promoter under both phosphorylated and non-phosphorylated status in buffer containing 10 mM L-Arg. These results illustrate an effective method to purify SaeR and other proteins.

Heterologous Expression of Recombinant Transglutaminase in Bacillus subtilis SCK6 with Optimized Signal Peptide and Codon, and Its Impact on Gelatin Properties.

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50°C and 8.0, respectively. MTG activity increased 1.42- fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8°C. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.