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Food Research International (Ottawa,... Aug 2024The structural and functional properties of whey-quercetin and whey hydrolysate-quercetin conjugates synthesized using alkaline and free radical-mediated methods (AM and...
The structural and functional properties of whey-quercetin and whey hydrolysate-quercetin conjugates synthesized using alkaline and free radical-mediated methods (AM and FRM) coupled with sonication were studied. FTIR showed new peaks at 3000-3500 cm (N-H stretching regions) and the 1000-1100 cm region with the conjugates. Conjugation increased the random coils and α-helix content while decreasing the β-sheets and turns. It also increased the particle size and surface hydrophobicity which was significantly (p < 0.05) higher in AM than FRM conjugates. AM conjugates had higher radical scavenging activity but lower quercetin content than FRM conjugates. Overall, the functional properties of whey-quercetin conjugates were better than whey hydrolysate-quercetin conjugates. However, hydrolysate conjugates had significantly higher denaturation temperatures irrespective of the method of production. Sonication improved the radical scavenging activity and quercetin content of FRM conjugates while it decreased both for AM conjugates. This study suggested that whey-quercetin conjugates generally had better quality than whey hydrolysate conjugates and sonication tended to further improve these properties. This study highlights the potential for using camel whey or whey hydrolysate-quercetin conjugates to enhance the functional properties of food products in the food industry.
Topics: Quercetin; Animals; Camelus; Sonication; Hydrophobic and Hydrophilic Interactions; Protein Hydrolysates; Whey; Antioxidants; Whey Proteins; Free Radical Scavengers; Spectroscopy, Fourier Transform Infrared; Free Radicals; Particle Size; Hydrogen-Ion Concentration
PubMed: 38945563
DOI: 10.1016/j.foodres.2024.114562 -
Peptide bonds cleaved by pepsin are affected by the morphology of heat-induced ovalbumin aggregates.Food Chemistry Jun 2024The study aimed to assess the extent to which protein aggregation, and even the modality of aggregation, can affect gastric digestion, down to the nature of the...
The study aimed to assess the extent to which protein aggregation, and even the modality of aggregation, can affect gastric digestion, down to the nature of the hydrolyzed peptide bonds. By controlling pH and ionic strength during heating, linear or spherical ovalbumin (OVA) aggregates were prepared, then digested with pepsin. Statistical analysis characterized the peptide bonds specifically hydrolyzed versus those not hydrolyzed for a given condition, based on a detailed description of all these bonds. Aggregation limits pepsin access to buried regions of native OVA, but some cleavage sites specific to aggregates reflect specific hydrolysis pathways due to the denaturation-aggregation process. Cleavage sites specific to linear aggregates indicate greater denaturation compared to spherical aggregates, consistent with theoretical models of heat-induced aggregation of OVA. Thus, the peptides released during the gastric phase may vary depending on the aggregation modality. Precisely tuned aggregation may therefore allow subtle control of the digestion process.
PubMed: 38944927
DOI: 10.1016/j.foodchem.2024.140260 -
European Journal of Pharmaceutics and... Jun 2024Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage,...
Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage, and administration processes, these proteins encounter various stressors such as temperature fluctuations, vibrations, and light exposure, able to induce chemico-physical modifications to their structure. Viral inactivation is a key step in downstream processes, and it is achieved by titration of the mAb at low pH, followed by neutralization. The changes of the pH pose a significant risk of unfolding and subsequent aggregation to proteins, thereby affecting their manufacturing. This study aims to investigate whether a combined exposure to light during the viral inactivation process can further affect the structural integrity of Ipilimumab, a mAb primarily used in the treatment of metastatic melanoma. The biophysical and biochemical characterization of Ipilimumab revealed that pH variation is a considerable risk for its stability with irreversible unfolding at pH 2. The threshold for Ipilimumab denaturation lies between pH 2 and 3 and is correlated with the loss of the protein structural cooperativity, which is the most critical factor determining the protein refolding. Light has demonstrated to exacerbate some local and global effects making pH-induced exposed regions more vulnerable to structural and chemical changes. Therefore, specific precautions to real-life exposure to ambient light during the sterilization process of mAbs should be considered to avoid loss of the therapeutic activity and to increase the yield of production. Our findings underscore the critical role of pH optimization in preserving the structural integrity and therapeutic efficacy of mAbs. Moreover, a detailed conformational study on the structural modifications of Ipilimumab may improve the chemico-physical knowledge of this effective drug and suggest new production strategies for more stable products under some kind of stress conditions.
PubMed: 38944210
DOI: 10.1016/j.ejpb.2024.114387 -
Journal of the Science of Food and... Jun 2024The increasing demand for sustainable alternatives to traditional protein sources, driven by population growth, underscores the importance of protein in a healthy diet....
BACKGROUND
The increasing demand for sustainable alternatives to traditional protein sources, driven by population growth, underscores the importance of protein in a healthy diet. Pecan (Carya illinoinensis (Wangenh.) K. Koch) nuts are currently underutilized as plant-based proteins but hold great potential in the food industry. However, there is insufficient information available on pecan protein, particularly its protein fractions. This study aimed to explore the physicochemical and functional properties of protein isolate and the main protein fraction glutelin extracted from pecan nuts.
RESULTS
The results revealed that glutelin (820.67 ± 69.42 g kg) had a higher crude protein content compared to the protein isolate (618.43 ± 27.35 g kg), while both proteins exhibited amino acid profiles sufficient for adult requirements. The isoelectric points of protein isolate and glutelin were determined to be pH 4.0 and pH 5.0, respectively. The denaturation temperature of the protein isolate (90.23 °C) was higher than that of glutelin (87.43 °C), indicating a more organized and stable conformation. This is further supported by the fact that the protein isolate had a more stable main secondary structure than glutelin. Both proteins demonstrated improved solubility, emulsifying, and foaming properties at pH levels deviating from their isoelectric points in U-shaped curves. Compared to the protein isolate, glutelin displayed superior water and oil absorption capacity along with enhanced gelling ability.
CONCLUSION
The protein isolate and glutelin from pecan nuts exhibited improved stability and competitive functional properties, respectively. The appropriate utilization of these two proteins will support their potential as natural ingredients in various food systems. © 2024 Society of Chemical Industry.
PubMed: 38940359
DOI: 10.1002/jsfa.13702 -
Current Research in Food Science 2024Whitespotted conger () muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations...
Whitespotted conger () muscle proteins were susceptible to oxidative denaturation during frozen storage. The objective of this study was to investigate the alterations in quality through physicochemical analysis and proteomics after whitespotted conger stored at temperatures of -18 °C and -60 °C. The microstructural observation revealed the noticeable variations such as increased interstitial space and fractured muscle fibre with extension of frozen storage time, and the muscle fibre of whitespotted conger stored at -60 °C were more intact than those stored at -18 °C. The raised TVB-N value indicated that the freshness of whitespotted conger decreased during 120-day frozen storage period. Analysis of myofibrillar protein content and SDS-PAGE demonstrated that compared to -18 °C, lower storage temperature (-60 °C) could better maintain the structure of whitespotted conger muscle by inhibiting protein degradation and oxidation. To reveal the mechanism of protein degradation, label-free quantitative proteomic analysis was performed through LC-MS/MS. The structural proteins including domain-associated proteins and actin-related proteins were up-regulated during frozen storage, but the phosphoglycerate kinase, phosphoglycerate mutase, and fructose-bisphosphate aldolase were down-regulated. Storage at -18 °C accelerated the up- or down-regulation of those differentially abundant proteins. According to KEGG analysis, up- or down-regulated pathways such as glycolysis/gluconeogenesis, carbon metabolism, biosynthesis of amino acids, and calcium signalling pathway mainly accounted for the protein degradation and quality reduction of whitespotted conger at low temperature. These results provided a theoretical basis for improving the quality stability of whitespotted conger during frozen storage.
PubMed: 38939611
DOI: 10.1016/j.crfs.2024.100779 -
Journal of Molecular Biology Jun 2024Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional...
The structural stability of membrane proteins revisited: Combined thermodynamic and spectral phasor analysis of SDS-induced denaturation of a thermophilic Cu(I)-transport ATPase.
Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.
PubMed: 38936696
DOI: 10.1016/j.jmb.2024.168689 -
International Journal of Molecular... Jun 2024A high alkaline pH was previously demonstrated to enhance the extraction yield of brewer's spent grains (BSG) proteins. The effects of extraction pH beyond the...
A high alkaline pH was previously demonstrated to enhance the extraction yield of brewer's spent grains (BSG) proteins. The effects of extraction pH beyond the extraction yield, however, has not been investigated before. The present work examined the effects of extraction pH (pH 8-12) on BSG proteins' (1) amino acid compositions, (2) secondary structures, (3) thermal stability, and (4) functionalities (i.e., water/oil holding capacity, emulsifying, and foaming properties). The ideal extraction temperature (60 °C) and BSG-to-solvent ratio (1:20 /) for maximizing the extraction yield were first determined to set the conditions for the pH effect study. The results showed that a higher extraction pH led to more balanced compositions between hydrophilic and hydrophobic amino acids and higher proportions of random coils structures indicating increased protein unfolding. This led to superior emulsifying properties of the extracted proteins with more than twofold improvement between pH 8 and a pH larger than 10. The extraction pH, nevertheless, had minimal impact on the water/oil holding capacity, foaming properties, and thermal denaturation propensity of the proteins. The present work demonstrated that a high alkaline pH at pH 11-12 was indeed ideal for both maximizing the extraction yield (37-46 wt.%) and proteins' functionalities.
Topics: Hydrogen-Ion Concentration; Amino Acids; Protein Structure, Secondary; Protein Stability; Hydrophobic and Hydrophilic Interactions; Grain Proteins; Temperature; Edible Grain
PubMed: 38928076
DOI: 10.3390/ijms25126369 -
Food Chemistry Jun 2024In response to the increasing demand for nutritionally rich foods, consumer preference for protein-enriched beverages has grown. However, heat-induced protein...
In response to the increasing demand for nutritionally rich foods, consumer preference for protein-enriched beverages has grown. However, heat-induced protein aggregation and gelation significantly hinders the production of high-protein drinks. In this study, oil-in-water (O/W) emulsions with exceptional thermal stability were formulated using modified soy protein particles (MSPs). These MSPs effectively resisted gel formation, even at a protein concentration of up to 20% (w/v). In contrast, emulsions prepared with untreated soy proteins (SPs) experienced pronounced gelation under identical conditions. The compact structure of MSPs, in comparison to SPs, imparted resistance to heat-induced denaturation and aggregation. Additionally, the emulsion displayed heightened heat processing insensitivity, due to the enhanced hydrophobicity of MSPs and their rapid adsorption at the oil-water interface, resulting in a denser, more elastic, and resilient interfacial film. These findings provide practical insights for the formulation of protein-rich milk alternatives, meeting the evolving market demands.
PubMed: 38924918
DOI: 10.1016/j.foodchem.2024.140157 -
FEBS Open Bio Jun 2024Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral,...
Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni as a physiological metal substrate, but also Co as a nonphysiological substrate with higher activity than for Ni. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.
PubMed: 38923868
DOI: 10.1002/2211-5463.13849 -
Animal Science Journal = Nihon Chikusan... 2024We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS....
We hypothesized that teats with a teat apex score (TAS) of 4 on a 4-point scale would exhibit elevated levels of denatured collagen compared with teats with lower TAS. We procured keratin layer and smooth muscle samples from Holsteins with TAS ranging from 1 to 4, as well as from crossbred heifers (Japanese Black male and Holstein female) with TAS of 1. Teats with a TAS of 4 demonstrated increased total collagen content, higher amounts of type I collagen (the harder, thicker variant), and reduced amounts of type III collagen (the softer, thinner variant) compared with teats with lower TAS. Teats with TAS of 3 and 4 exhibited evidence of damaged collagen in smooth muscle layers compared with teats with TAS of 1. Additionally, we identified 47-kDa heat shock protein-positive fibroblasts in the smooth muscles of teats with TAS of 3 and 4. Therefore, the smooth muscle of teats with a TAS of 4 exhibited increased amounts of denatured collagen in comparison to teats with lower TAS.
Topics: Animals; Cattle; Female; Muscle, Smooth; Collagen; Keratins; Mammary Glands, Animal; Protein Denaturation; Male; Collagen Type I; Fibroblasts; Collagen Type III
PubMed: 38923230
DOI: 10.1111/asj.13969