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Food Chemistry Oct 2024This study explored the effect of constant-current pulsed electric field thawing (CC-T) on the proteins and water-holding capacity of pork. Fresh meat (FM), and frozen...
This study explored the effect of constant-current pulsed electric field thawing (CC-T) on the proteins and water-holding capacity of pork. Fresh meat (FM), and frozen meat after constant-voltage thawing (CV-T), air thawing (AT) and water immersion thawing (WT) were considered as controls. The results indicated that CC-T had a higher thawing rate than conventional thawing during ice-crystal melting stage (-5 to -1 °C). It also showed a lower water migration and thawing loss, maintaining pH and shear force closer to FM. Meanwhile, CC-T decreased myoglobin oxidation, resulting in a favorable surface color. The results of protein solubility, differential scanning calorimetry, total sulfhydryl, carbonyl and surface hydrophobicity demonstrated that CC-T reduced myofibrillar protein oxidative denaturation by suppressing the formation of disulfide and carbonyl bonds, thus enhancing solubility and thermal stability. Additionally, microstructural observation found that CC-T maintained a relatively intact muscle fiber structure by reducing muscle damage and myosin filament denaturation.
Topics: Animals; Swine; Water; Freezing; Muscle, Skeletal; Solubility; Muscle Proteins; Electricity; Meat; Oxidation-Reduction; Food Handling
PubMed: 38815321
DOI: 10.1016/j.foodchem.2024.139784 -
The Analyst Jun 2024This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the...
This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.
Topics: Lactoferrin; Humans; Recombinant Proteins; Saccharomycetales; Scattering, Small Angle; Amino Acid Sequence; Glycosylation; X-Ray Diffraction
PubMed: 38814097
DOI: 10.1039/d4an00333k -
Heliyon May 2024Physico-chemical properties of fish flesh are reliable predictors of fillet quality and nutritional value. In our study, the age-related variations of the chemical...
Physico-chemical properties of fish flesh are reliable predictors of fillet quality and nutritional value. In our study, the age-related variations of the chemical composition, pH, water activity (aw), water holding capacity (WHC), color and texture analysis, protein thermal stability, myofibrillar fragmentation index (MFI), glycogen content, protein oxidation and protein profiles were investigated in (rainbow trout) fillet. The results revealed that protein denaturation temperatures (T1 and T2) decreased by 2 % and 11.6 % depending on fish age. T1 and T2 values in the same groups were raised 71 % at 11 months' fish and this increase was 58 % at 23 months' fish. An age-related reduction by 66.6 % and 31.25 % was noticed for protein oxidation markers sulfhydryl groups and disulfide bonds. MFI value increased by 86.6 % connected with age. The characteristics of fish meat quality are complex and are influenced by various factors that affect the degree of freshness of the product and its acceptance in the market. Taking into account the different demands of the consumer, this study has shown that age at slaughter has an impact on final product quality and that the recommended age at slaughter, taking into account market weight, positively affects meat quality.
PubMed: 38813226
DOI: 10.1016/j.heliyon.2024.e31146 -
International Journal of Biological... Jun 2024Our previous experiments found that rapeseed protein (RP) has applicability in low-moisture textured proteins. The amount of RP added is limited to <20 %, but the...
Understanding the mechanism for sodium tripolyphosphate in improving the physicochemical properties of low-moisture extrusion textured protein from rapeseed protein and soybean protein blends.
Our previous experiments found that rapeseed protein (RP) has applicability in low-moisture textured proteins. The amount of RP added is limited to <20 %, but the addition of 20 % RP still brings some negative effects. Therefore, in order to improve the quality of 20%RP textured protein, this experiment added different proportions of sodium tripolyphosphate (STPP) to improve the quality of the product, and studied the physical-chemical properties and molecular structure changes of the product to explore the possible modification mechanism. The STPP not only improved the expansion characteristics of extrudates, but also increased the brightness of the extrudates, the rehydration rate. In addition, STPP increased the specific mechanical energy during extrusion, decreased the material mass flow rate. Furthermore, STPP decreased the starch digestibility, increased the content of slow-digesting starch and resistant starch. STPP increased the degree of denaturation of extrudate proteins, the proportion of β-sheets in the secondary structure of proteins, as well as the intermolecular hydrogen bonding interactions. The gelatinization degradation degree of starch molecules also decreased with the addition of STPP. STPP also increased the protein-starch interactions and enhanced the thermal stability of the extrudate. All these indicate that STPP can improve the physical-chemical properties of extrudate.
Topics: Soybean Proteins; Plant Proteins; Polyphosphates; Brassica rapa; Chemical Phenomena; Starch; Water; Hydrogen Bonding
PubMed: 38810848
DOI: 10.1016/j.ijbiomac.2024.132656 -
MBio May 2024Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted...
UNLABELLED
Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen (). Critical to virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection.
IMPORTANCE
is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.
PubMed: 38809022
DOI: 10.1128/mbio.00743-24 -
Journal of Pharmaceutical Sciences May 2024Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the...
Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.
PubMed: 38801973
DOI: 10.1016/j.xphs.2024.05.024 -
The Journal of Nutrition May 2024Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein...
BACKGROUND
Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein digestion and subsequent amino acid (AA) absorption.
OBJECTIVES
The objective was to understand the impact of the protein ingredient quality within IFs on postprandial plasma AA profiles.
METHODS
Four isonitrogenous and isocaloric IFs were produced at a semi-industrial scale using whey proteins from different origins (cheese compared with ideal whey) and denaturation levels (IF-A, -B, -C), and caseins with different supramolecular organizations (IF-C, -D). Ten Yucatan minipiglets (12- to 27-d-old) were used as a human infant model and received each IF for 3 d according to a Williams Latin square followed by a 2-d wash-out period. Jugular plasma was regularly sampled from 10 min preprandial to 4 h postprandial on the third day to measure free AAs, urea, insulin, and glucose concentrations. Data were statistically analyzed using a mixed linear model with diet (IFs), time, and sex as fixed factors and piglet as random factor.
RESULTS
IFs made with cheese whey (IF-A and -B) elicited significantly higher plasma total and essential AA concentrations than IFs made with ideal whey (IF-C and -D), regardless of the pre- and postprandial times. Most of the differences observed postprandially were explained by AA homeostasis modifications. IFs based on cheese whey induced an increased plasma concentration of Thr due to both a higher Thr content in these IFs and a Thr-limiting degrading capability in piglets. The use of a nonmicellar casein ingredient led to reduced plasma content of AA catabolism markers (IF-D compared with IF-C).
CONCLUSIONS
Overall, our results highlight the importance of the protein ingredient quality (composition and structure) within IFs on neonatal plasma AA profiles, which may further impact infant protein metabolism.
PubMed: 38801861
DOI: 10.1016/j.tjnut.2024.05.009 -
Protein Science : a Publication of the... Jun 2024Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning...
Thermal stability of proteins is a primary metric for evaluating their physical properties. Although researchers attempted to predict it using machine learning frameworks, their performance has been dependent on the quality and quantity of published data. This is due to the technical limitation that thermodynamic characterization of protein denaturation by fluorescence or calorimetry in a high-throughput manner has been challenging. Obtaining a melting curve that derives solely from the target protein requires laborious purification, making it far from practical to prepare a hundred or more samples in a single workflow. Here, we aimed to overcome this throughput limitation by leveraging the high protein secretion efficacy of Brevibacillus and consecutive treatment with plate-scale purification methodologies. By handling the entire process of expression, purification, and analysis on a per-plate basis, we enabled the direct observation of protein denaturation in 384 samples within 4 days. To demonstrate a practical application of the system, we conducted a comprehensive analysis of 186 single mutants of a single-chain variable fragment of nivolumab, harvesting the melting temperature (T) ranging from -9.3 up to +10.8°C compared to the wild-type sequence. Our findings will allow for data-driven stabilization in protein design and streamlining the rational approaches.
Topics: Protein Stability; Thermodynamics; Protein Denaturation; High-Throughput Screening Assays; Brevibacillus
PubMed: 38801228
DOI: 10.1002/pro.5029 -
Journal of Computational Chemistry May 2024According to the Hofmeister series, thiocyanate is the strongest "salting in" anion. In fact, it has a strong denaturant activity against the native state of globular...
According to the Hofmeister series, thiocyanate is the strongest "salting in" anion. In fact, it has a strong denaturant activity against the native state of globular proteins. A molecular level rationalization of the Hofmeister series is still missing, and therefore the denaturant activity of thiocyanate also awaits a robust explanation. In the last years, different types of experimental studies have shown that thiocyanate is capable to directly interact with both polar and nonpolar groups of polypeptide chains. This finding has been scrutinized via a careful computational procedure based on density functional theory approaches. The results indicate that thiocyanate is able to make H-bonds via both the nitrogen and sulfur atom, and to make strong van der Waals interactions with almost all the groups of polypeptide chains, regardless of their polarity.
PubMed: 38795315
DOI: 10.1002/jcc.27440 -
International Journal of Molecular... May 2024CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density...
Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities.
CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL and HDL remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.
Topics: Apolipoprotein A-I; Animals; Zebrafish; Antioxidants; Anti-Inflammatory Agents; Lipoproteins, HDL; Lipoproteins, LDL; Oxidation-Reduction; Drug Synergism
PubMed: 38791598
DOI: 10.3390/ijms25105560