Thermal Denaturation Research Articles

Assessing the compatibility of choline-based deep eutectic solvents for the structural stability and activity of cellulase: Enzyme sustain at high temperature

As a new generation of ‘green solvents’ deep eutectic solvents (DESs) represents a promising alternative to the conventional solvents. Their environmental-benign nature and designer properties promote their utility in biocatalysis. Enzymes are marginally stable when exposed to physical/chemical disturbances. One such enzyme is cellulase which is a propitious catalyst for the depolymerization of cellulose under mild conditions. Therefore, their stability is a prerequisite condition to match demands of biorefineries. To address this issue of low stability, activity and thermal denaturation of cellulase, there is a need to find a sustainable and suitable co-solvent that is biocompatible with enzymes ultimately to facilitate their application in bio-industries. In this regard, we synthesized three choline-based DESs, choline chloride (ChCl)-glycerol, ChCl-ethylene glycol and ChCl-lactic acid and employed them to analyze their suitability for cellulase. The present study systematically evaluates the influence of the mentioned DESs on stability, activity and thermal stability of cellulase with the help of various spectroscopic techniques. The spectroscopic analysis revealed that the structural stability and activity of the enzyme were improved in presence of ChCl-glycerol and ChCl-ethylene glycol. The thermal stability was also very well maintained in both the DESs. Interestingly, the relative activity of cellulase was >80 % even after incubation at 50 °C after 48 h for both the DESs. This activity preservation behaviour was more pronounced for ChCl-ethylene glycol than ChCl-glycerol. Moreover, temperature variations studies also reveal promising results by maintain conformational intactness. On the other side, ChCl-lactic acid showed a deleterious effect on the enzyme both structurally as well as thermally. The dynamic light scattering (DLS) analysis provides more specific information about the negative influence of ChCl-lactic acid towards cellulase native structure. This DES induces unavoidable alterations in the enzyme structure which leads to the unfolding of enzyme, ultimately, destabilizing it. Overall, our results present a physical insight into how the enzyme stability and activity depend on the nature of DES. Also, the findings will help to facilitate the development and application of DESs as biocatalytic process.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Binding and stabilizating effect of RNA triplex poly(U)⋅poly(A)*poly(U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(dppx)]2.

Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.

Spectroscopic and electrochemical investigation of ternary Cu (II) complex interaction with calf thymus DNA

Aiming to design unique metallic drugs with significant therapeutic activity, in this work, mixed ligand Cu (II) complex involving 2,2′‐bipyridine and phenylalanine [Cu (bpy) (phenala.)Cl].2H2O has been fabricated and characterized using IR spectroscopy. The interaction of Cu (II) complex with CT‐DNA molecules was examined through UV–vis absorption titration plots, thermal denaturation experiments, viscosity measurements, and electrochemical studies. Cyclic voltammetry, electrochemical impedance spectroscopy, and chronoamperometry measurements have been exploited to elucidate their binding style. The negative potential shift in ΔE and E1/2 values as well as the increased current density of Cu (II)/Cu(I) redox couple suggested the promoted rate of electron transfer process in presence of CT‐DNA molecules in the examined solution. Some kinetic parameters were also estimated such as the diffusion coefficient, the exchange current density, and Tafel slope values. Based on the results of these binding experiments, a groove and/or electrostatic interaction mode was expected. The antimicrobial activity of the studied Cu (II) complex was screened against a series of bacteria and fungi. An outstanding performance was shown when treating different Gram‐positive and Gram‐negative bacteria types. Moreover, an enhanced cytotoxicity behavior toward some human tumor cell lines, including MCF7, HEPG2, and HFB4, was detected with respective IC50 values of 43.02, 51.21, and >200 μM when incubated with Cu (II) complex for 72 h. These results suggest the application of [Cu (bpy) (phenala.)Cl].2H2O complex as a chemotherapeutic agent with a promising output in the drug discovery field.

Physicochemical Properties of Chicken Breast and Thigh as Affected by Sous-Vide Cooking Conditions

Sous-vide is a cooking method used to improve the tenderness and juiciness of chicken breast. However, the comparative changes in meat quality attributes of sous-vide cooked chicken breast and thigh muscles are not fully understood. The objective of this study was to investigate the effects of sous-vide cooking conditions, based on collagen denaturation temperature of intramuscular connective tissue, on the physicochemical properties of chicken breasts and thighs. Chicken breast and thigh were cooked at four sous-vide cooking conditions (55 °C for 3/6 h and 65 °C for 3/6 h) and conventional cooking at 75 °C (core temperature of 71 °C) as control. No significant differences in pH and lightness were found between the sous-vide cooking conditions. Moisture content, cooking loss, protein solubility, shear force, myofibrillar fragmentation index, and lipid oxidation were affected by sous-vide cooking conditions (p < 0.05). The decreased shear force and total collagen content of 65 °C sous-vide cooking treatment might be associated with collagen denaturation (p < 0.05). Sous-vide cooking at 55 °C could decrease cooking loss, with higher moisture than sous-vide cooking at 65 °C (p < 0.05). These tendencies on water-holding capacity and shear force at the two different temperatures were similarly observed for both chicken breast and thigh. Therefore, this study indicates that chicken breast and thigh are similarly affected by the sous-vide cooking conditions and suggests that a novel strategy to apply together two temperature ranges based on the thermal denaturation of intramuscular connective tissue would be required.

High-throughput thermal denaturation of tryptophanyl-tRNA synthetase combinatorial mutants reveals high-order energetic coupling determinants of conformational stability.

Landscape descriptions provide a framework for identifying functionally significant dynamic linkages in proteins but cannot supply details. Rate measurements of combinatorial mutations can implicate dynamic linkages in catalysis. A major difficulty is filtering dynamic linkages from the vastly more numerous static interactions that stabilize domain folding. The Geobacillus stearothermophilus (TrpRS) D1 switch is such a dynamic packing motif; it links domain movement to catalysis and specificity. We describe Thermofluor and far UV circular dichroism melting curves for all 16 D1 switch variants to determine their higher-order impact on unliganded TrpRS stability. A prominent transition at intermediate temperatures in TrpRS thermal denaturation is molten globule formation. Combinatorial analysis of thermal melting transcends the protein landscape in four significant respects: (i) bioinformatic methods identify dynamic linkages from coordinates of multiple conformational states. (ii) Relative mutant melting temperatures, δTM, are proportional to free energy changes. (iii) Structural analysis of thermal melting implicates unexpected coupling between the D1 switch packing and regions of high local frustration. Those segments develop molten globular characteristics at the point of greatest complementarity to the chemical transition state and are the first TrpRS structures to melt. (iv) Residue F37 stabilizes both native and molten globular states; its higher-order interactions modify the relative intrinsic impacts of mutations to other D1 switch residues from those estimated for single point mutants. The D1 switch is a central component of an escapement mechanism essential to free energy transduction. These conclusions begin to relate the escapement mechanism to differential TrpRS conformational stabilities.

Microcalorimetry reveals multi-state thermal denaturation of G. stearothermophilus tryptophanyl-tRNA synthetase.

Mechanistic studies of Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford an unusually detailed description-the escapement mechanism-for the distinct steps coupling catalysis to domain motion, efficiently converting the free energy of ATP hydrolysis into biologically useful alternative forms of information and work. Further elucidation of the escapement mechanism requires understanding thermodynamic linkages between domain configuration and conformational stability. To that end, we compare experimental thermal melting of fully liganded and apo TrpRS with a computational simulation of the melting of its fully liganded form. The simulation also provides important structural cameos at successively higher temperatures, enabling more confident interpretation. Experimental and simulated melting both proceed through a succession of three transitions at successively higher temperature. The low-temperature transition occurs at approximately the growth temperature of the organism and so may be functionally relevant but remains too subtle to characterize structurally. Structural metrics from the simulation imply that the two higher-temperature transitions entail forming a molten globular state followed by unfolding of secondary structures. Ligands that stabilize the enzyme in a pre-transition (PreTS) state compress the temperature range over which these transitions occur and sharpen the transitions to the molten globule and fully denatured states, while broadening the low-temperature transition. The experimental enthalpy changes provide a key parameter necessary to convert changes in melting temperature of combinatorial mutants into mutationally induced conformational free energy changes. The TrpRS urzyme, an excerpted model representing an early ancestral form, containing virtually the entire catalytic apparatus, remains largely intact at the highest simulated temperatures.

Toward a Molecular Mechanism of Complementary RNA Duplexes Denaturation.

RNA duplexes are relatively rare but play very important biological roles. As an end-product of template-based RNA replication, they also have key implications for hypothetical primitive forms of life. Unless they are specifically separated by enzymes, these duplexes denature upon a temperature increase. However, mechanistic and kinetic aspects of RNA (and DNA) duplex thermal denaturation remain unclear at the microscopic level. We propose an in silico strategy that probes the thermal denaturation of RNA duplexes and allows for an extensive conformational space exploration along a wide temperature range with atomistic precision. We show that this approach first accounts for the strong sequence and length dependence of the duplexes melting temperature, reproducing the trends seen in the experiments and predicted by nearest-neighbor models. The simulations are then instrumental at providing a molecular picture of the temperature-induced strand separation. The textbook canonical "all-or-nothing" two-state model, very much inspired by the protein folding mechanism, can be nuanced. We demonstrate that a temperature increase leads to significantly distorted but stable structures with extensive base-fraying at the extremities, and that the fully formed duplexes typically do not form around melting. The duplex separation therefore appears as much more gradual than commonly thought.

The interconnecting hairpin extension "arm": An essential allosteric element of phytochrome activity

In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals thatthe arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes.

Synthesis, physicochemical characterisation and DNA binding study of a novel azo Schiff base Ni(II) complex

The azo Schiff base ligand was synthesised, along with its Ni(II) complex, by diazotisation of salicylaldehyde with 4-nitroaniline in accordance with the accepted literature approach. Using a variety of spectroscopic techniques, the resulting complex is analysed both quantitatively and qualitatively (Elemental analysis, FT-IR spectroscopy, UV-VIS spectroscopy, 1H NMR, etc.). Spectral measurements of the complex revealed a mole ratio of 1:1. The non-electrolytic nature of the complex is confirmed by molar conductance investigation. The unique azo compound had a tetrahedral shape as a result of the tetra coordination of two phenolic oxygen and two imine nitrogen. The ability of the metal complexes to bind DNA was examined using absorption spectroscopy, fluorescence spectroscopy, viscosity tests, and thermal denaturation methods. Experimental research suggests that complexes bind to DNA through intercalation.

Thermal Behavior of Pea and Egg White Protein Mixtures.

The partial substitution of animal protein by plant protein is a new opportunity to produce sustainable food. Hence, to control the heat treatment of a composite protein ingredient, this work investigated the thermal behavior of mixtures of raw egg white (EW) and a laboratory-prepared pea protein isolate (PPI). Ten-percentage-by-weight protein suspensions prepared with different PPI/EW weight ratios (100/0, 75/25, 50/50, 25/75, 0/100) at pH 7.5 and 9.0 were analyzed by differential scanning calorimetry (DSC) and dynamic rheology in temperature sweep mode (T < 100 °C). The DSC data revealed changes in the thermal denaturation temperatures (Td) of ovotransferrin, lysozyme, and pea legumin, supposing interactions between proteins. Denaturation enthalpy (∆H) showed a high pH dependence related to pea protein unfolding in alkaline conditions and solubility loss of some proteins in admixture. Upon temperature sweeps (25-95 °C), the elastic modulus (G') of the mixtures increased significantly with the EW content, indicating that the gel formation was governed by the EW protein. Two thermal sol-gel transitions were found in EW-containing systems. In particular, the first sol-gel transition shifted by approximately +2-3 °C at pH 9.0, probably by a steric hindering effect due to the presence of denatured and non-associated pea globulins at this pH.

Insight on the mechanism of hexameric Pseudin-4 against bacterial membrane-mimetic environment.

As an alternative to antibiotics, Antimicrobial Peptides (AMPs) possess unique properties including cationic, amphipathic and their abundance in nature, but the exact characteristics of AMPs against bacterial membranes are still undetermined. To estimate the structural stability and functional activity of AMPs, the Pseudin AMPs (Pse-1, Pse-2, Pse-3, and Pse-4) from Hylid frog species, Pseudis paradoxa, an abundantly discovered source for AMPs were examined. We studied the intra-peptide interactions and thermal denaturation stability of peptides, as well as the geometrical parameters and secondary structure profiles of their conformational trajectories. On this basis, the peptides were screened out and the highly stable peptide, Pse-4 was subjected to membrane simulation in order to observe the changes in membrane curvature formed by Pse-4 insertion. Monomeric Pse-4 was found to initiate the membrane disruption; however, a stable multimeric form of Pse-4 might be competent to counterbalance the helix-coil transition and to resist the hydrophobic membrane environment. Eventually, hexameric Pse-4 on membrane simulation exhibited the hydrogen bond formation with E. coli bacterial membrane and thereby, leading to the formation of membrane spanning pore that allowed the entry of excess water molecules into the membrane shell, thus causing membrane deformation. Our report points out the mechanism of Pse-4 peptide against the bacterial membrane for the first time. Relatively, Pse-4 works on the barrel stave model against E. coli bacterial membrane; hence it might act as a good therapeutic scaffold in the treatment of multi-drug resistant bacterial strains.

Toward the analysis of functional proteoforms using mass spectrometry-based stability proteomics.

Functional proteomics aims to elucidate biological functions, mechanisms, and pathways of proteins and proteoforms at the molecular level to examine complex cellular systems and disease states. A series of stability proteomics methods have been developed to examine protein functionality by measuring the resistance of a protein to chemical or thermal denaturation or proteolysis. These methods can be applied to measure the thermal stability of thousands of proteins in complex biological samples such as cell lysate, intact cells, tissues, and other biological fluids to measure proteome stability. Stability proteomics methods have been popularly applied to observe stability shifts upon ligand binding for drug target identification. More recently, these methods have been applied to characterize the effect of structural changes in proteins such as those caused by post-translational modifications (PTMs) and mutations, which can affect protein structures or interactions and diversify protein functions. Here, we discussed the current application of a suite of stability proteomics methods, including thermal proteome profiling (TPP), stability of proteomics from rates of oxidation (SPROX), and limited proteolysis (LiP) methods, to observe PTM-induced structural changes on protein stability. We also discuss future perspectives highlighting the integration of top-down mass spectrometry and stability proteomics methods to characterize intact proteoform stability and understand the function of variable protein modifications.

Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion system (TPSS, type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) transporter that is integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal TPS domain followed by an extensive C-terminal domain with divergent sequence repeats. Two soluble, N-terminal constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447) and 1 to 606 (EtpA1-606). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical AlphaFold model of full-length EtpA largely concurs with the crystal structure adding an extended β-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal β-helix into the C-terminal domains of TpsA proteins.

The organic osmolyte betaine effectively counteracts the deleterious effect of diclofenac on protein structure and stability

Understanding the physicochemical properties of pharmacy and the concept of pharmacophore is essential to devise guidelines for the synthesis of target-oriented drugs. In the present study, thermal denaturation, circular dichroism, intrinsic fluorescence, and docking were employed for studying the effects of the non-steroidal anti-inflammatory drug, diclofenac sodium, on the stability and structure of bovine pancreatic ribonuclease (RNase A) taken as a model of an enzyme. Since drug-protein interaction is an important pharmacokinetic parameter of a drug, it was deemed significant to study the effects of diclofenac on protein structure and stability. When the thermodynamic parameters were assessed, it was shown that RNase-A was destabilized in the presence of diclofenac sodium in terms of Tm and ΔGD°, while the addition of osmolytes stabilized the protein. In the presence of diclofenac sodium and osmolyte, there was a change in a tertiary structure of the protein but not in its secondary structure. Spectral results revealed a decrease in fluorescence intensity confirming fluorescence quenching of RNase-A by diclofenac sodium and osmolytes. Molecular docking analysis suggested that hydrogen bonds and Van der Waals are involved in the binding of RNase-A to diclofenac sodium. The RNAse-A activity is decreased in the presence of diclofenac sodium while osmolyte can restore the RNase-A activity and the indices kcat increase and Km values decrease. This study presents new combination of drug therapy comprising diclofenac sodium and osmolytes to reduce the risk of adverse effects of diclofenac sodium.

Investigating structure, biological activity, peptide composition and emulsifying properties of pea protein hydrolysates obtained by cell envelope proteinase from Lactobacillus delbrueckii subsp. bulgaricus

We present the structure, biological activity, peptide composition, and emulsifying properties of pea protein isolate (PPI) after hydrolysis by cell envelope proteinase (CEP) from Lactobacillus delbrueckii subsp. bulgaricus. Hydrolysis resulted in the unfolding of the PPI structure, characterized by an increase in fluorescence and UV absorption, which was related to thermal stability as demonstrated by a significant increase in ΔH and the thermal denaturation temperature (from 77.25 ± 0.05 to 84.45 ± 0.04 °C). The hydrophobic amino acid of PPI significantly increased from 218.26 ± 0.04 to 620.77 ± 0.04 followed by 557.18 ± 0.05 mg/100 g, which was related to their emulsifying properties, with the maximum emulsifying activity index (88.62 ± 0.83 m2/g, after 6 h hydrolysis) and emulsifying stability index (130.77 ± 1.12 min, after 2 h hydrolysis). Further, the results of LC-MS/MS analysis demonstrated that the CEP tended to hydrolyze peptides with an N-terminus dominated by Ser and a C-terminus dominated by Leu, which enhanced the biological activity of pea protein hydrolysates, as supported by their relatively high antioxidant (ABTS+ and DPPH radical scavenging rates were 82.31 ± 0.32% and 88.95 ± 0.31%) and ACE inhibitory (83.56 ± 1.70%) activities after 6 h of hydrolysis. 15 peptide sequences (score > 0.5) possessed both antioxidant and ACE inhibitory activity potential according to the BIOPEP database. This study provides theoretical guidance for the development of CEP-hydrolyzed peptides with antioxidant and ACE inhibitory activity that can be used as emulsifiers in functional foods.

Cooking mediated wheat gluten aggregation behavior: Physicochemical properties and component changes

Changes in the physicochemical properties of wheat gluten (WG) during cooking are mainly dependent on its intramolecular and intermolecular interactions when subjected to high-temperature treatment. In this study, the effects and mechanisms of cooking on the aggregation behavior of WG were investigated. Results showed that an increase in cooking time led to an initial increase and subsequent decrease in WG viscoelasticity, with optimal viscoelastic properties observed at 5 min. As cooking proceeded, covalent cross-linking of WG deepened and the contribution of hydrogen bonds to maintaining WG structure weakened, while the contribution of hydrophobic interactions strengthened. Cooking increased the proportion of gauche-gauche-gauche in the disulfide (SS) bridge conformation and decreased the homogeneity of the WG network. The unfolding of gluten molecules caused by hydrogen bonds breaking and intermolecular cross-linking through hydrophobic interactions and SS formation jointly contributing to the rheological properties of WG. Notably, glutenin (Glu) exhibited more similar physicochemical changes to WG during cooking than gliadin (Gli). Glu was more sensitive to cooking than Gli and formed dense, continuous aggregates prior to Gli. Cross-links formed by Glu occupied the majority of three-dimensional network of WG, while Gli was involved only in the form of branched chains. This may explain why Glu dominates the physicochemical changes of WG during cooking. This study contributes to a deeper understanding of the thermal denaturation mechanism of WG and provides new insights into the processing of flour-based foods.

Detoxification of the post-harvest antifungal pesticide thiabendazole by cold atmospheric plasma

Cold atmospheric plasma (CAP) irradiation has a sterilizing effect without thermal denaturation or the production of residual substances. Hence, it is considered to be a safe sterilization technology with minimal damage for fresh foods. In addition, its decomposition effect on chemical substances has also been confirmed, and the application of CAP in the food and agricultural domains is increasing. In this study, we examined the potential of CAP to detoxify pesticide residues. Post-harvest chemical treatments using pesticides, such as fungicides, are frequently employed in imported agricultural products and are often disapproved by consumers. Therefore, we assessed the detoxification of thiabendazole (TBZ), a widely used post-harvest pesticide, using low-cost air plasma irradiation. We found that CAP irradiation conditions that detoxified TBZ caused little damage to the edible parts of mandarin oranges. The results of the present study suggest that CAP irradiation is useful for detoxifying and degrading pesticide residues without damaging agricultural products and that CAP irradiation is an effective means of maintaining food safety.

Quantitative Lipidome Analysis of Boiled Chicken Egg Yolk under Different Heating Intensities.

The effects of the four heating intensities (hot-spring egg yolk, HEY; soft-boiled egg yolk, SEY; normal-boiled egg yolk, NEY; and over-boiled egg yolk, OEY) on lipidomes of boiled egg yolks were investigated. The results indicated that four heating intensities had no significant effect on the total abundance of lipids and lipid categories except for bile acids, lysophosphatidylinositol, and lysophosphatidylcholine. However, of all the 767 lipids quantified, the differential abundance of 190 lipids was screened among the egg yolk samples at four heating intensities. Soft-boiling and over-boiling altered the assembly structure of the lipoproteins through thermal denaturation and affected the binding of lipids and apoproteins, resulting in an increase in low-to-medium-abundance triglycerides. The decreased phospholipid and increased lysophospholipid and free fatty acid in HEY and SEY suggests potential hydrolysis of phospholipids under relatively low-intensity heating. Results provide new insights into the effect of heating on the lipid profiles of egg yolk and would support the public's choice of cooking method for egg yolks.

Dual-frequency boiling histotripsy in an ex vivo bovine tendinopathy model.

Histotripsy fractionates most soft tissues; however, healthy tendons have shown resistance to histotripsy fractionation. Prior work has shown that pre-heating tendons increases susceptibility to histotripsy fractionation; combining multiple driving frequencies may also allow successful fractionation of tendons. Here, we evaluate single- and dual-frequency histotripsy in four healthy and eight tendinopathic ex vivo bovine tendons. First, we evaluated single-frequency (1.07, 1.5, and 3.68 MHz) and dual-frequency (1.07 and 1.5 MHz or 1.5 and 3.68 MHz) bubble dynamics with high-speed photography in a tissue-mimicking phantom. Then, tendons were treated with histotripsy. Cavitation activity was monitored with a passive cavitation detector (PCD) and targeted areas were evaluated grossly and histologically. Results in tendinopathic tendons showed 1.5 MHz or 3.68 MHz single-frequency exposure caused focal disruption, whereas 1.5 and 3.68 MHz dual-frequency exposures caused fractionated holes; all treatments caused some thermal denaturation. Exposure to 1.07 MHz alone or combined with 1.5 MHz did not show fractionation in tendinopathic tendons. In healthy tendons, only thermal necrosis was observed for all tested exposures. PCD showed some differences in cavitation activity in tendinopathic tendons but did not predict successful fractionation. These results suggest that full histotripsy fractionation is possible using dual-frequency exposures in tendinopathic tendons.