Search Results (11)

This study aimed to investigate the impact of various low-temperature heat treatments, namely sous vide (SV), dynamic temperature roasting (ΔT), and slow roasting (R), on the quality of bovine semitendinosus (ST) muscle. The effects on textural and color properties, myoglobin denaturation cooking loss, and consumer acceptance were examined. The samples treated with the SV cooking methods at 55 °C (SV55) and 65 °C (SV65) showed the lowest WBSF values (p ≤ 0.05). Sous vide at lower temperatures (SV55 and SV65) preserved the highest levels of redness a* (p ≤ 0.05) and exhibited the smallest color differences ΔE (p ≤ 0.05) between the perimeter and center cross-sections. Dynamic temperature roasting with a constant temperature difference between the product core and the oven interior of 40 °C (ΔT40) and 20 °C (ΔT20) and roasting at 75 °C (R75) led to lower color differences in the cross-sections compared to the SV methods (p ≤ 0.05). Among all methods, slow cooking, particularly sous vide, resulted in the highest product quality, with higher consumer acceptance scores for juiciness, tenderness, and color. However, it was comparable to ΔT20 samples in terms of WBSF, tenderness, color acceptance, and overall quality. These results suggest that ΔT20 roasting can serve as an alternative to the SV method, achieving a similar quality of ST muscle product in a significantly shorter time. Full article

Currently, there is a growing demand for ready-to-eat, prepared, high-quality, preservative-free products. However, the shelf-life of these products is often so short that a relatively high percentage of these products cannot be sold and end up as food waste. In this study, pork loin with different marinades (paprika and herbs) was treated with different high hydrostatic pressures (0 MPa, 300 MPa, 450 MPa and 600 MPa) and a bioactive component (piperine) and then the quality of the meat was examined after 0, 4, 8, 12 and 14 days of cold storage. Changes were monitored using color, pH, texture and microbiological analyses. Both pressure, piperine enrichment, storage time and the interactions of different factors had a significant effect on the quality of the loin samples with different marinades. Due to the denaturation of myoglobin, meat slices were less red and lighter after HHP treatment. The addition of piperine reduced this lightness. The pH increased with increasing pressure and decreased with storage time. HHP treatment significantly increased meat hardness, with samples treated at 600 MPa being 19% harder than those treated at 450 MPa. Microbiological results indicated that HHP at 450 MPa and 600 MPa effectively reduced anaerobic total live plate counts, ensuring satisfactory sensory and microbiological quality throughout storage. Piperine fortification also resulted in a more favorable microbiological status during storage without any perceptible change in quality properties during storage. These findings underscore the effectiveness of HHP and piperine enrichment in enhancing the safety and quality of marinated meats. Full article

This study highlights a promising single washing method for producing dark-fleshed mackerel surimi aided by ultrasonication in conjunction with cold carbonated water containing 0.6% NaCl and mixed antioxidants (0.5% EDTA/0.2% sodium erythorbate/0.2% sodium tripolyphosphate) (CSA). Different washing periods (5, 10, and 15 min) with and without ultrasound were tested. Unwashed mince (A1) and conventional water-washed surimi (10 min/cycle, 3 cycles) (A2) were used as controls. A3, A4, and A5 were subjected to ultrasound-assisted washing for 5, 10, and 15 min, respectively, whereas A6, A7, and A8 had non-ultrasound-assisted washing for 5, 10, and 15 min. Results showed that the surimi yield decreased as the ultrasonic treatment time increased from 5 to 15 min (p < 0.05). Increased ultrasonic time resulted in greater protein denaturation, protein oxidation, myoglobin removal, and lipid oxidation in surimi (p < 0.05). Surimi produced by CSA ultrasonication for 5 min (A3), on the other hand, had a comparable overall quality to A2 surimi (p > 0.05). The correspondence gel (A3) outperformed the control gel (A2) in terms of gel strength, whiteness, and water-holding capacity (p < 0.05). The formation of regularly continuous, more organized, and smooth network structures in surimi gel was observed in A2 and A3 gels, whereas sparse and larger pore sizes were noticed in surimi gels produced by longer ultrasonic treatment. All of the surimi gels had identical FTIR spectra, indicating that the functional groups of the protein gel were consistent throughout. As a result, a single 5 min CSA-ultrasonic washing could potentially yield surimi of comparable quality to conventional washing. This could pave the way for the development of dark-fleshed fish surimi, which would require less washing time and produce less waste water. Full article

Hybrid meat products are an excellent strategy to incorporate plant proteins into traditional meat formulations considering recent market trends focusing on the partial reduction in red meat content. In this work, we evaluated the effects of different concentrated plant proteins (soy, pea, fava bean, rice, and sunflower) in partially replacing meat in meat emulsion model systems. Soy, pea, and sunflower proteins showed great compatibility with the meat matrix, giving excellent emulsion stability and a cohesive protein network with good fat distribution. Otherwise, adding rice and fava bean proteins resulted in poor emulsion stability. Color parameters were affected by the intrinsic color of plant proteins and due to the reduction in myoglobin content. Both viscoelastic moduli, G′ and G″ decreased with the incorporation of plant proteins, especially for rice and fava bean. The temperature sweep showed that myosin denaturation was the dominant effect on the G′ increase. The water mobility was affected by plant proteins and the proportion between immobilized and intermyofibrillar water was quite different among treatments, especially those with fava bean and rice proteins. In vitro protein digestibility was lower for hybrid meat emulsion elaborated with rice protein. It is concluded that soy, pea, and mainly sunflower proteins have suitable compatibility with the meat matrix in emulsified products. Full article

Many proteins are usually not stable under different stresses, such as temperature and pH variations, mechanical stresses, high concentrations, and high saline contents, and their transport is always difficult, because they need to be maintained in a cold regime, which is costly and very challenging to achieve in remote areas of the world. For this reason, it is extremely important to find stabilizing agents that are able to preserve and protect proteins against denaturation. In the present work, we investigate, by extensively using synchrotron small-angle X-ray scattering experiments, the stabilization effect of five different sugar-derived compounds developed at ExtremoChem on two model proteins: myoglobin and insulin. The data analysis, based on a novel method that combines structural and thermodynamic features, has provided details about the physical-chemical processes that regulate the stability of these proteins in the presence of stabilizing compounds. The results clearly show that some modified sugars exert a greater stabilizing effect than others, being able to maintain the active forms of proteins at temperatures higher than those in which proteins, in the absence of stabilizers, reach denatured states. Full article

Electrospray ionisation (ESI) is renowned for its ability to ionise intact proteins for sensitive detection by mass spectrometry (MS). However, the use of a conventional direct current ESI voltage can result in the formation of relatively large initial droplet sizes, which can limit efficient ion desolvation and sensitivity. Here, pulsed nanoESI (nESI) MS using nanoscale emitters with inner diameters of ~250 nm is reported. In this approach, the nESI voltage is rapidly pulsed from 0 to ~1.5 kV with sub-nanosecond rise times, duty cycles from 10 to 90%, and repetition rates of 10 to 350 kHz. Using pulsed nESI, the performance of MS for the detection of intact proteins can be improved in terms of increased ion abundances and decreased noise. The absolute ion abundances and signal-to-noise levels of protonated ubiquitin, cytochrome C, myoglobin, and carbonic anhydrase II formed from standard denaturing solutions can be increased by up to 82% and 154% using an optimal repetition rate of ~200 kHz compared to conventional nESI-MS. Applying pulsed nESI-MS to a mixture of four proteins resulted in the signal for each protein increasing by up to 184% compared to the more conventional nESI-MS. For smaller ions (≤1032 m/z), the signal can also be increased by the use of high repetition rates (200–250 kHz), which is consistent with the enhanced performance depending more on general factors associated with the ESI process (e.g., smaller initial droplet sizes and reduced Coulombic repulsion in the spray plume) rather than analyte-specific effects (e.g., electrophoretic mobility). The enhanced sensitivity of pulsed nESI is anticipated to be beneficial for many different types of tandem mass spectrometry measurements. Full article

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral: 7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each. Full article

The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase. Full article

The unique electrochemical properties of ionic liquids (ILs) have motivated their use as solvents for organic synthesis and green energy applications. More recently, their potential in pharmaceutical chemistry has prompted investigation into their effects on biomolecules. There is evidence that some ILs can destabilize proteins via a detergent-like manner; however, the mechanism still remains unknown. Our hypothesis is that if ILs are denaturing proteins via a detergent-like mechanism, detergent-mediated protein unfolding should be enhanced in the presence of ILs. The properties of myoglobin was examined in the presence of a zwitterionic (N,N-dimethyl-N-dodecylglycine betaine (Empigen BB^®, EBB)), cationic (tetradecyltrimethylammonium bromide (TTAB)), and anionic (sodium dodecyl sulfate (SDS)) detergent as well as ILs based on alkylated imidazolium chlorides. Protein structure was measured through a combination of absorbance, fluorescence, and circular dichroism (CD) spectroscopy: absorbance and CD were used to monitor heme complexation to myoglobin, and tryptophan fluorescence quenching was used as an indicator for heme dissociation. Notably, the detergents tested did not fully denature the protein but instead resulted in loss of the heme group. At low IL concentrations, heme dissociation remained a traditional, cooperative process; at high concentrations, ILs with increased detergent-like character exhibited a more complex pattern, which is most likely attributable to micellization of the ionic liquids or direct denaturation or heme dissociation induced by the ILs. These trends were consistent across all species of detergents. 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence was further used to characterize micelle formation in aqueous solutions containing detergent and ionic liquid. The dissociation thermodynamics show that EBB- and TTAB-induced dissociation of heme is not significantly impacted by room temperature ionic liquids (RTILs), whereas SDS-induced dissociation is more dramatically impacted by all RTILs examined. Together, these results indicate a complex interaction of detergents, likely based on headgroup charge, and the active component of RTILs to influence heme dissociation and potentially protein denaturation. Full article

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔG_dissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF₄) in aqueous solution on the ΔG_dissociation values. Absorbance experiments show the ILs had minimal effect on ΔG_dissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies. Full article

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V_H–V_L sequence with a (Gly₄Ser)₃ linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k_on/k_off), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. Full article