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Organophosphorus are typically hazardous chemicals used in the pharmaceutical, agricultural, and other industries. They pose a serious risk to human life and can be fatal upon direct exposure. Hence, studying the interaction between such compounds with proteins is crucial for environmental, health, and food safety. In this study, we investigated the interaction mechanism between azinphos‐methyl (AZM) and β‐lactoglobulin (BLG) at pH 7.4 using a combination of biophysical techniques. Intrinsic fluorescence investigations revealed that BLG fluorescence was quenched in the presence of increasing AZM concentrations. The quenching mechanism was identified as static, as evidenced by a decrease in the fluorescence quenching constant (1.25 × 104, 1.18 × 104, and 0.86 × 104 M−1) with an increase in temperatures. Thermodynamic calculations (ΔH > 0; ΔS > 0) affirmed the formation of a complex between AZM and BLG through hydrophobic interactions. The BLG's secondary structure was found to be increased due to AZM interaction. Ultraviolet –visible spectroscopy data showed alterations in BLG conformation in the presence of AZM. Molecular docking highlighted the significant role of hydrophobic interactions involving residues such as Val43, Ile56, Ile71, Val92, Phe105, and Met107 in the binding between BLG and AZM. A docking energy of −6.9 kcal mol−1, and binding affinity of 1.15 × 105 M−1 suggest spontaneous interaction between AZM and BLG with moderate to high affinity. These findings underscore the potential health risks associated with the entry of AZM into the food chain, emphasizing the need for further consideration of its impact on human health. [ABSTRACT FROM AUTHOR]

The aim of this study was to understand the effect of electromagnetic field (EMF) and ionic strength on the electrostatic interactions between β-lactoglobulin(β-lg) and gum Arabic (GA). Therefore, different EMF intensities (5.2, 6.4, 7.6, 8.8, and 10 mT) and NaCl concentrations (30, 35, and 40 mM) were used. The turbidimetric results showed that an increase in the EMF intensity shifted the curves to more acidic regions in comparison to the control sample. In other words, more interactions occurred in the EMF-treated samples while addition of salt distinctly decreased the turbidity and inhibited the interaction. The particle size observations were evaluated by static light scattering machine (SLS). The results showed that the particle size significantly increased (up to 0.753 ± 0.01 μm) by raising the EMF intensities to 10 mT. The addition of NaCl at a concentration of 30 mM showed a slight increase in the particle size diameter. By increasing the salt concentration to 35 and 40 mM, a decrease in the particle diameter was observed which could be due to the reduction in protein/polysaccharide reaction as a result of the prohibiting effect of salts. SEM micrographs revealed almost the same structures in the EMF-treated and untreated samples. However, more aggregation was observed in EMF-treated samples. The presence of salt also established small aggregations. This study showed that EMF can have interesting effects on protein/polysaccharide interactions and formation of particles used as delivery systems. [ABSTRACT FROM AUTHOR]

α-zeins are amphiphilic maize seed storage proteins with material properties suitable for a multitude of applications e.g., in renewable plastics, foods, therapeutics and additive manufacturing (3D-printing). To exploit their full potential, molecular-level insights are essential. The difficulties in experimental atomic-resolution characterization of α-zeins have resulted in a diversity of published molecular models. However, deep-learning α-zein models are largely unexplored. Therefore, this work studies an AlphaFold2 (AF2) model of a highly expressed α-zein using molecular dynamics (MD) simulations. The sequence of the α-zein cZ19C2 gave a loosely packed AF2 model with 7 α-helical segments connected by turns/loops. Compact tertiary structure was limited to a C-terminal bundle of three α-helices, each showing notable agreement with a published consensus sequence. Aiming to chart possible α-zein conformations in practically relevant solvents, rather than the native solid-state, the AF2 model was subjected to MD simulations in water/ethanol mixtures with varying ethanol concentrations. Despite giving structurally diverse endpoints, the simulations showed several patterns: In water and low ethanol concentrations, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At ≥ 50 mol% ethanol, extended conformations prevailed, consistent with previous SAXS studies. Tertiary structure was partially stabilized in water and low ethanol concentrations, but was disrupted in ≥ 50 mol% ethanol. Aggregated results indicated minor increases in helicity with ethanol concentration. β-sheet content was consistently low (∼1%) across all conditions. Beyond structural dynamics, the rapid formation of branched α-zein aggregates in aqueous environments was highlighted. Furthermore, aqueous simulations revealed favorable interactions between the protein and the crosslinking agent glycidyl methacrylate (GMA). The proximity of GMA epoxide carbons and side chain hydroxyl oxygens simultaneously suggested accessible reactive sites in compact α-zein conformations and pre-reaction geometries for methacrylation. The findings may assist in expanding the applications of these technologically significant proteins, e.g., by guiding chemical modifications. [ABSTRACT FROM AUTHOR]

l -Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff–Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP- d -mannose 3',5'-epimerase (GME), l -galactose dehydrogenase (l -GalDH), and l -galactono-1,4-lactone dehydrogenase (l -GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The Md GME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Md l -GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Md l -GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications. [ABSTRACT FROM AUTHOR]

β-lactoglobulin (BLG) forms amyloid-like aggregates at high temperatures, low pH, and low ionic strengths. At a pH below 2, BLG undergoes hydrolysis into peptides, with N-terminal peptides 1–33 and 1–52 being prone to fibrillization, forming amyloid-like fibrils. Due to their good mechanical properties, BLG amyloids demonstrate great potential for diverse applications, including biosensors, nanocomposites, and catalysts. Consequently, further studies are essential to comprehensively understand the factors governing the formation of BLG amyloid-like morphologies. In this study, all-atom molecular dynamics simulations were employed to explore the aggregation of N-terminal 1–33 and 1–52 BLG peptides under conditions of pH 2 and at 10 mM NaCl concentration. The simulations revealed that the peptides spontaneously assembled into aggregates of varying sizes. The aggregation process was enabled by the low charge of peptides and the presence of hydrophobic residues within them. As the peptides associated into aggregates, there was a concurrent increase in β-sheet structures and the establishment of hydrogen bonds, enhancing the stability of the aggregates. Notably, on average, 1–33 peptides formed larger aggregates compared to their 1–52 counterparts, while the latter exhibited a slightly higher content of β-sheets and higher cluster orderliness. The applied approach facilitated insights into the early stages of amyloid-like aggregation and molecular-level insight into the formation of β-sheets, which serve as nucleation points for further fibril growth. [ABSTRACT FROM AUTHOR]

[Display omitted] Nonionic surfactants can counter the deleterious effect that anionic surfactants have on proteins, where the folded states are retrieved from a previously unfolded state. However, further studies are required to refine our understanding of the underlying mechanism of the refolding process. While interactions between nonionic surfactants and tightly folded proteins are not anticipated, we hypothesized that intermediate stages of surfactant-induced unfolding could define new interaction mechanisms by which nonionic surfactants can further alter protein conformation. In this work, the behavior of three model proteins (human growth hormone, bovine serum albumin, and β-lactoglobulin) was investigated in the presence of the anionic surfactant sodium dodecylsulfate, the nonionic surfactant β-dodecylmaltoside, and mixtures of both surfactants. The transitions occurring to the proteins were determined using intrinsic fluorescence spectroscopy and far-UV circular dichroism. Based on these results, we developed a detailed interaction model for human growth hormone. Using nuclear magnetic resonance and contrast-variation small-angle neutron scattering, we studied the amino acid environment and the conformational state of the protein. The results demonstrate the key role of surfactant cooperation in defining the conformational state of the proteins, which can shift away or toward the folded state depending on the nonionic-to-ionic surfactant ratio. Dodecylmaltoside, initially a non-interacting surfactant, can unexpectedly associate with sodium dodecylsulfate-unfolded proteins to further impact their conformation at low nonionic-to-ionic surfactant ratio. When this ratio increases, the protein begins to retrieve the folded state. However, the native conformation cannot be fully recovered due to remnant surfactant molecules still adsorbed to the protein. This study demonstrates that the conformational landscape of the protein depends on a delicate interplay between the surfactants, ultimately controlled by the ratio between them, resulting in unpredictable changes in the protein conformation. [ABSTRACT FROM AUTHOR]

Whey, a component of milk and a useful by-product of the dairy industry's casein and cheese-making, has been used for generations to augment animal feed. It contains a range of proteins, including α-lactalbumin, β-lactoglobulin, bovine serum albumin, heavy and light chain immunoglobulins, lactoferrin, glycomacropeptide, and lactoperoxidase. Whey proteins exhibit great potential as biopolymers for creating bioactive delivery systems owing to their distinct health-enhancing characteristics and the presence of numerous amino acid groups within their structures. Whey has considerable factors such as antitumor, anti-inflammatory, antihypertensive, hypolipidemic, antiviral, and antibacterial properties in addition to chelating. The global market of whey protein stood at USD 5.33 billion in 2021, with a projected compound annual growth rate of 10.48% spanning the interval from 2022 to 2030. The escalating demand for whey protein is intrinsically linked to the amplifying consciousness surrounding healthy lifestyles. Notably, protein supplements are recurrently endorsed by fitness and sports establishments, thereby accentuating the focal point of customers toward whey protein. This review focuses on nutritional composition, whey bioactives, and their bioavailability with potential health benefits. [ABSTRACT FROM AUTHOR]

Xanthine oxidase (XO), a rate-limiting enzyme in uric acid production, is the pivotal therapeutic target for gout and hyperuricemia. In this study, 57 peptides from α-lactalbumin and β-lactoglobulin were obtained via virtual enzymatic hydrolysis, and 10 XO inhibitory peptides were virtually screened using molecular docking. Then toxicity, allergenicity, solubility, and isoelectric point of the obtained 10 novel peptides were evaluated by in silico tools. The XO activity of these synthetic peptides was tested using an in vitro assay by high-performance liquid chromatography. Their inhibitory mechanism was further explored by molecular docking. The results showed that 4 peptides GL, PM, AL, and AM exhibited higher inhibitory activity, and their half maximal inhibitory concentration in vitro was 10.20 ± 0.89, 23.82 ± 0.94, 34.49 ± 0.89, and 40.45 ± 0.92 m M , respectively. The peptides fitted well with XO through hydrogen bond, hydrophobic interaction, and van der Waals forces, and amino acid residues Glu802, Leu873, Arg880, and Pro1076 played an important role in this process. Overall, this study indicated 4 novel peptides GL, PM, AL, and AM from whey protein exhibited XO inhibitory activity, and they might be useful and safe XO inhibitors for hyperuricemia prevention and treatment. [ABSTRACT FROM AUTHOR]

Piceatannol (PIC) and epigallocatechin gallate (EGCG) are polyphenolic compounds with applications in the treatment of various diseases such as cancer, but their stability is poor. β-lactoglobulin (β-LG) is a natural carrier that provides a protective effect to small molecule compounds and thus improves their stability. To elucidate the mechanism of action of EGCG, PIC, and palmitate (PLM) in binding to β-LG individually and jointly, this study applied molecular docking and molecular dynamics simulations combined with in-depth analyses including noncovalent interaction (NCI) and binding free energy to investigate the binding characteristics between β-LG and compounds of PIC, EGCG, and PLM. Simulations on the binary complexes of β-LG + PIC, β-LG + EGCG, and β-LG + PLM and ternary complexes of (β-LG + PLM) + PIC, (β-LG + PLM) + EGCG, β-LG + PIC) + EGCG, and (β-LG + EGCG) + PIC were performed for comparison and characterizing the interactions between binding compounds. The results demonstrated that the co-bound PIC and EGCG showed non-beneficial effects on each other. However, the centrally located PLM was revealed to be able to adjust the binding conformation of PIC, which led to the increase in binding affinity with β-LG, thus showing a synergistic effect on the co-bound PIC. The current study of β-LG co-encapsulated PLM and PIC provides a theoretical basis and research suggestions for improving the stability of polyphenols. [ABSTRACT FROM AUTHOR]

Lipid and energy metabolism are major constituents of mammal growth and thus fattening performance of cattle. This study was designed to evaluate the effects of polymorphisms in lipid and energy metabolism-related genes including oxidized low-density lipoprotein receptor 1 (OLR1), lactoferrin (LTF), stearoyl-CoA desaturase (SCD), beta-lactoglobulin (LGB), thyroglobulin (TG), annexin A9 (ANXA9), myogenic factor 5 (MYF5), protein kinase AMP-activated non-catalytic subunit gamma 3 (PRKAG3), and pituitary-specific transcriptional factor 1 (PIT1), on fattening performance in Simmental cattle. A total of 72 purebred Simmental bulls with a similar initial age and weight were fattened on the same farm for 10 months. Association analysis was performed using linear mixed models. The OLR1 marker was significantly associated with the final weight (FW), hot carcass weight (HCW), chilled carcass weight (CCW), dressing percentage (DP), and total weight gain (TWG). SCD affected the FW, TWG, and average daily live weight gain (ADWG). The present results clearly demonstrated the significant impact of the TG marker on fattening performance. It was highly significantly associated with the FW, HCW, CCW, and TWG. The SCD × TG and the OLR1 × TG interactions had remarkable effects on the traits analyzed. The GACC and CCCC haplotypes of the SCD × TG and OLR1 × TG, respectively, were found to be powerful markers for fattening performance in Simmentals. Novel associations in this study may be useful for further genetic evaluations to improve beef cattle breeding. [ABSTRACT FROM AUTHOR]

Colloidal gas aphrons (CGAs) are highly stable, spherical, micrometer-sized bubbles encapsulated by surfactant multilayers. They have several intriguing properties, including: high stability, large interfacial area, and the ability to maintain the same charge as their parent molecules. The physical properties of CGAs make them ideal for biotechnological applications such as the recovery of a variety of: biomolecules, particularly proteins, yeast, enzymes, and microalgae. In this review, the bio-application of CGAs for the recovery of natural components is presented, as well as: experimental results, technical challenges, and critical research directions for the future. Experimental results from the literature showed that the recovery of biomolecules was mainly determined by electrostatic or hydrophobic interactions between polyphenols and proteins (lysozyme, β-casein, β-lactoglobulin, etc.), yeast, biological molecules (gallic acid and norbixin), and microalgae with CGAs. Knowledge transfer is essential for commercializing CGA-based bio-product recovery, which will be recognized as a viable technology in the future. [ABSTRACT FROM AUTHOR]

[Display omitted] Protein adsorption plays a key role in membrane fouling in liquid processing, but the specific underlying molecular mechanisms of β-lactoglobulin adsorption on ceramic silica surfaces in premix membrane emulsification have not been investigated yet. In this study, we aimed to elucidate the β-lactoglobulin adsorption and its effect on the premix membrane emulsification of β-lactoglobulin-stabilized oil-in-water emulsions. In particular, the conformation, molecular interactions, layer thickness, surface energy of the adsorbed β-lactoglobulin and resulting droplet size distribution are investigated in relation to the solvent properties (aggregation state of β-lactoglobulin) and the treatment of the silica surface (hydrophilization). The β-lactoglobulin adsorption is driven by attractive electrostatic interactions between positively charged amino acid residues, i.e. , lysin and negatively charged silanol groups, and is stabilized by hydrophobic interactions. The strong negative charges of the treated silica surfaces result in a high apparent layer thickness of β-lactoglobulin. Although the conformation of the adsorbed β-lactoglobulin layer varies with membrane treatment and the solvent properties, the β-lactoglobulin adsorption offsets the effect of hydrophilization of the membrane so that the surface energies after β-lactoglobulin adsorption are comparable. The resulting droplet size distribution of oil-in-water emulsions produced by premix membrane emulsification are similar for treated and untreated silica surfaces. [ABSTRACT FROM AUTHOR]

It is well established that deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) exhibit a reentrant condensation (RC) phase behavior in the presence of the trivalent hexamine cobalt(III) cations (Hac) which can be important for their packing and folding. A similar behavior can be observed for negatively charged globular proteins in the presence of trivalent metal cations, such as Y3+ or La3+. This phase behavior is mainly driven by charge inversion upon an increasing salt concentration for a fixed protein concentration (cp). However, as Hac exhibits structural differences compared to other multivalent metal cations, with six ammonia ligands (NH3) covalently bonded to the central cobalt atom, it is not clear that Hac can induce a similar phase behavior for proteins. In this work, we systematically investigate whether negatively charged globular proteins β-lactoglobulin (BLG), bovine serum albumin (BSA), human serum albumin (HSA) and ovalbumin (OVA) feature Hac-induced RC. Effective protein–protein interactions were investigated by small-angle X-ray scattering. The reduced second virial coefficient (B2/B2HS) was obtained as a function of salt concentration. The virial coefficient analysis performed confirms the reentrant interaction (RI) behavior for BLG without actually inducing RC, given the insufficient strengths of the interactions for the latter to occur. In contrast, the strength of attraction for BSA, HSA and OVA are too weak to show RC. Model free analysis of the inverse intensity 1 / I q → 0 also supports this finding. Looking at different q-range by employing static (SLS) and dynamic light scattering experiments, the presence of RI behavior can be confirmed. The results are further discussed in view of metal cation binding sites in nucleic acids (DNA and RNA), where Hac induced RC phase behavior. [ABSTRACT FROM AUTHOR]

Protein lactosylation is a significant modification that occurs during the heat treatment of dairy products, causing changes in proteins' physical-chemical and nutritional properties. Knowledge of the detailed lactosylation information on milk proteins under various heat treatments is important for selecting appropriate thermo-processing and identifying markers to monitor heat load in dairy products. In the present study, we used proteomics techniques to investigate lactosylated proteins under different heating temperatures. We observed a total of 123 lactosylated lysines in 65 proteins, with lactosylation even occurring in raw milk. The number of lactosylated lysines and proteins increased moderately at 75°C to 130°C, but dramatically at 140°C. We found that 6 out of 10, 9 out of 16, 6 out of 12, and 5 out of 15 lysine residues in κ-casein, β-lactoglobulin, α-lactalbumin, and α S1 -casein, respectively, were lactosylated under the applied heating treatment. Moreover, different lactosylation states of individual lysines and proteins can indicate the intensity of heating processes. Lactosylation of K14 in β-lactoglobulin could distinguish pasteurized and UHT milk, while lactosylation of lactotransferrin can reflect moderate heat treatment of products. [ABSTRACT FROM AUTHOR]

β-Lactoglobulin is one of the proteins in milk possessing antioxidant activity. The peptides derived from β-Lactoglobulin exhibit higher antioxidant activities than the most commonly used antioxidant. Furthermore, the detailed structure–activity relationship of these antioxidant peptides has not been elucidated. Therefore, in the present work, two-dimensional quantitative structure–activity relationship (2D-QSAR) and three-dimensional quantitative structure–activity relationship (3D-QSAR) models were constructed to investigate the structural factors affecting activities and gave information for the rational design of novel antioxidant peptides. After calculation and screening of molecular descriptors, linear and nonlinear models were developed by multiple linear regression (MLR), partial least squares regression (PLSR) and support vector machines (SVM) approaches. The statistical parameters are as follows: R2 = 0.643, Q2 = 0.553/MLR, R2 = 0.612, Q2 = 0.5278/PLSR, R2 = 0.7085, Q2 = 0.6887/SVM, indicating that the SVM model is superior to the MLR and PLSR models. In addition, in the 3D-QSAR models, the Dragon-CoMFA (R2cv = 0.537, R2pred = 0.5201) and Dragon-CoMSIA (R2cv = 0.665, R2pred = 0.6489) methods were conducted to predict the antioxidant activities. Comparison of statistical parameters illustrates that the suitability of Dragon-CoMSIA is superior to the Dragon-CoMFA model. The results show the robustness and excellent prediction of the proposed models, and would be applied for modifying and designing novel and potent antioxidant peptides. [ABSTRACT FROM AUTHOR]

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER–ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection. [ABSTRACT FROM AUTHOR]

Dietary protein-derived peptides are effective in improving dyslipidemia and hypercholesterolemia. We previously identified a novel cholesterol-lowering pentapeptide IIAEK from milk beta-lactoglobulin. However, it remains unclear whether IIAEK affects the micellar solubility of cholesterol and the bile acid-binding ability to lower cholesterol. Moreover, there is no direct evidence that IIAEK inhibits intestinal cholesterol absorption and affects hepatic cholesterol and fecal steroid excretion in vivo. Herein, we showed that IIAEK did not affect the micellar solubility of cholesterol and the bile acid-binding ability. However, we found that IIAEK decreased serum and liver cholesterol levels and increased fecal steroid excretion in mice. Interestingly, IIAEK markedly suppressed the intestinal absorption of [3 H]-cholesterol in mice. In conclusion, we found that IIAEK ameliorated cholesterol metabolism by suppressing intestinal cholesterol absorption without affecting in vitro micellar solubility of cholesterol and the bile acid-binding ability in mice. Pentapeptide IIAEK ameliorates cholesterol metabolism via the suppression of intestinal cholesterol absorption in mice. [ABSTRACT FROM AUTHOR]

Cow milk is an important source of food protein for children; however, it could lead to allergy, especially for infants. α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) from whey protein make up a relatively high proportion of milk proteins and have received widespread attention as major allergens in milk. However, few studies have identified the epitopes of both proteins simultaneously. In this study, ImmunoCAP and indirect ELISA were first used for detection of sIgE to screen sera from allergic patients with high binding capacity for α-LA and β-LG. Subsequently, the mimotopes was biopanned by phage display technology and bioinformatics and 17 mimic peptide sequences were obtained. Aligned with the sequences of α-LA or β-LG, we identified one linear epitope on α-LA at AA 11–26 and 5 linear epitopes on β-LG at AA 9–29, AA 45–57, AA 77–80, AA 98–101, and AA 121–135, respectively. Meanwhile, the 8 conformational epitopes and their distributions of α-LA and β-LG were located using the Pepitope Server. Finally, glutamine and lysine were determined as common AA residues for the conformational epitopes both on α-LA and β-LG. Moreover, we found the addition of mouse anti-human IgE during the biopanning process did not significantly affect the identification of the epitopes. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is β-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration. Methods: In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated. Results: Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI. Conclusions: The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future. [ABSTRACT FROM AUTHOR]

The binding interaction of milk protein β-lactoglobulin (β-LG) with plant alkaloid berberine (BER) has been thoroughly investigated using spectroscopic, calorimetric, and small-angle X-ray scattering studies aided by computational methods. The electronic UV-Vis and steady-state fluorescence spectroscopy experiments have suggested the creation of a ground-state complex between β-LG and BER. The origin of protein fluorescence is identified to be from Trp19, which is located in the hydrophobic pocket of β-LG. Time-resolved fluorescence spectroscopy confirmed the static quenching of β-LG fluorescence in the presence of BER. Binding parameters allied with the β-LG – BER complex have been estimated from the spectroscopic studies and correlated with the results directly obtained from isothermal titration calorimetry (ITC) studies. The fluorescence resonance energy transfer (FRET) from β-LG to BER has been explored. The synchronous fluorescence measurement has revealed variations in the microenvironment of the tyrosine and tryptophan residues of β-LG in the presence of BER. Circular dichroism (CD) results have revealed no or minimal conformational changes in the secondary structure of β-LG dimer due to the interaction with the BER molecule. The detailed small-angle X-ray scattering (SAXS) data further presages minimal structural alterations of β-LG in the presence of BER. Theoretical docking and molecular dynamics simulation investigations validated the experimental findings. The detailed investigation of β-LG – BER binding interactions may allow β-LG – BER or other protein–alkaloid complexes as potential drug carriers against various diseases as the protein structure is almost retained in the interaction. [ABSTRACT FROM AUTHOR]

In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER–ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection. [ABSTRACT FROM AUTHOR]

The dynamic surface properties of aqueous dispersions of α-lactalbumin (ALA) amyloid fibrils differ noticeably from the properties of the fibril dispersions of other globular proteins. As a result, the protocol of the application of ALA fibrils to form stable foams and emulsions has to be deviate from that of other protein fibrils. Unlike the fibrils of β-lactoglobulin and lysozyme, ALA fibrils can be easily purified from hydrolyzed peptides and native protein molecules. The application of the oscillating barrier method shows that the dynamic surface elasticity of ALA fibril dispersions exceeds the surface elasticity of native protein solutions at pH 2. ALA fibrils proved to be stable at this pH, but the stability breaks at higher pH levels when the fibrils start to release small peptides of high surface activity. As a result, the dynamic surface properties of ALA coincide with those of native protein solutions. The ionic strength strongly influences the adsorption kinetics of both fibril dispersions and native protein solutions but have almost no impact on the structure of the adsorption layers. [ABSTRACT FROM AUTHOR]

BACKGROUND: Due to the extrusion black box effect, polysaccharides determine the formation of meat‐like fibrous structures by modulating the flow behaviour and structural changes of plant proteins under high‐moisture extrusion conditions. However, there is limited knowledge on the mechanism of resolution. This study simulated the rheological properties of soy protein–wheat protein under 57% moisture conditions with addition of 4% sodium alginate (SA), 2% xanthan gum (XG), and 2% maltodextrin (MD). The effect of these polysaccharides on the aggregation behaviour and conformation of raw protein during high‐moisture extrusion was investigated. RESULTS: It was revealed that the three polysaccharides were effective in increasing the interaction between proteins and between proteins and water. Among them, 4% SA elicited a significantly stronger storage modulus (gelation behaviour) compared to the control. Analysis of different zones of extrudates by protein electrophoresis, particle size, and turbidity showed that SA‐4% was able to form more high molecular protein aggregates (> 245 kDa) and promoted crosslinking of low molecular subunits (< 48 kDa), resulting in moderately sized protein aggregated particles. Fluorescence and ultraviolet spectra showed the transformation of protein tertiary structures in different extrusion zones, confirming that the key extrusion zone for protein conformational transformation by polysaccharides is the die–cooling zone. Furthermore, stretching of polypeptide chains and accelerated protein rearrangement facilitated the formation of more fibrillar structures. CONCLUSION: Theoretical support for polysaccharide modulation of plant protein quality in high moisture extruded products is provided by this study. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Combined with template molecular pre-positioning, surface imprinting method, and post-modification strategy, a molecularly imprinted surface plasmon resonance (SPR) sensor was constructed on a gold substrate and used for detection of β-lactoglobulin (BLG) in camel milk powder. Firstly, the template molecule BLG was fixed by mercapto-terminated polyacrylic acid (PAA-SH) on the gold substrate which was modified by polydopamine in advance. Then imprinting was performed using dopamine as functional monomer and crosslinking agent. Finally, partially hydrolyzed poly (2-methyl-2- oxazoline) (PMOXA-EI) with anti-protein adsorption function was grafted on the non-imprinted part, and the BLG molecularly imprinted SPR sensor was prepared after eluting the template. The changes of sensor ’s surface hydrophilicity/hydrophobicity were studied by water contact angle (WCA) and the characterization of created sensor was performed by Fourier transform infrared spectrometer (FT-IR), atomic force microscopy (AFM), and variable angle [ABSTRACT FROM AUTHOR]

In recent years, the popularity of dietary nanoparticles (NPs) in the food industry as additives has raised concerns because of the lack of knowledge about potential adverse health outcomes ensuing from the interactions of NPs with components of the food matrix and gastrointestinal system. In this study, we used a transwell culture system that consisted of human colorectal adenocarcinoma (Caco‐2) cells in the apical insert and Laboratory of Allergic Diseases 2 mast cells in the basal compartment to study the effect of NPs on milk allergen delivery across the epithelial layer, mast cell responses and signaling between epithelial and mast cells in allergenic inflammation. A library of dietary particles (silicon dioxide NPs, titanium dioxide NPs and silver NPs) that varied in particle size, surface chemistry and crystal structures with or without pre‐exposure to milk was used in this investigation. Milk‐interacted particles were found to acquire surface corona and increased the bioavailability of milk allergens (casein and β‐lactoglobulin) across the intestinal epithelial layer. The signaling between epithelial cells and mast cells resulted in significant changes in the early phase and late‐phase activation of the mast cells. This study suggested that antigen challenge in mast cells with the presence of dietary NPs may cause the transition of allergic responses from an immunoglobulin E (IgE)–dependent mechanism to a mixed mechanism (both IgE‐dependent and IgE‐independent mechanisms). [ABSTRACT FROM AUTHOR]

Protein–polysaccharide Maillard conjugate has recently got much attention for improving protein functionality. In this study, soy protein isolate (SPI) was conjugated to maltodextrin (MD) at the protein-to-polysaccharide ratio of 1:2, 1:1 and 2:1 (w/w) via Maillard reaction by dry heating (60 °C, 0–9 days) or wet heating (90 °C, 0–120 min). The influences on the degree of graft (DG), functionalities and structure of conjugates were evaluated. The mass ratio of reactants and reaction time greatly impacted the DG of conjugates. The conjugates with protein-to-polysaccharide ratio of 1:2 (w/w) prepared via dry- and wet-heating methods exhibited the highest DG. The DG of dry heat-induced conjugates raised significantly as incubation for 3 days and prolonged heating has no effect of DG, whereas the DG of wet-heat conjugates increased as extended heating period. The emulsifying properties were sharply improved in the conjugates by dry heating as prolonging the incubation time. Protein solubility and foaming properties of the conjugates by dry heating incubated for a day were enhanced. Extending the reaction period negatively impacted those functionalities. On the other hand, wet heating profoundly enhanced the protein solubility and foaming properties of the conjugates. The structural characteristics by Fourier transform infrared spectrum (FTIR) and circular dichroism (CD) confirmed the conformational changes in the secondary structure of the conjugates prepared by dry- and wet-heating methods. The appropriate Maillard reaction conditions could formulate the conjugate with better functionalities that will be useful for food applications. [ABSTRACT FROM AUTHOR]

Prospects for predicting the fragmentation of polypeptide chains during their enzymatic hydrolysis using proteolysis models are considered. The opening of the protein substrate during proteolysis and the exposure of its internal peptide bonds for a successful enzymatic attack, the so-called demasking process, were taken into account. The two-step proteolysis model was used, including the parameters of demasking and the rate constants of hydrolysis of enzyme-specific peptide bonds. Herein, we have presented an algorithm for calculating the concentrations of intermediate and final peptide fragments depending on the time of hydrolysis or the degree of hydrolysis. The intermediate peptide fragments with two or one internal specific peptide bond were considered. The fragmentation of β-lactoglobulin (β-LG) by trypsin was predicted, and the calculated concentration curves for peptide fragments were compared with the experimental dependences of the concentrations on the degree of hydrolysis. Numerical parameters were proposed that characterize the concentration curves for intermediate and final peptide fragments, and they were used to compare the calculated and experimental dependences. The predicted distribution of the peptide fragments corresponded to the experimental data on the peptide release during the proteolysis of β-LG by trypsin. [ABSTRACT FROM AUTHOR]

Purpose: Whey protein isolate (WPI) has previously been shown to be a promising new excipient for the development of amorphous solid dispersions (ASD) at a high drug loading of 50% (w/w). Whilst WPI is a protein mixture, comprising mainly the three proteins β-lactoglobulin (BLG), α-lactalbumin (ALA), casein glycomacropeptides (CGMP), the individual contributions of these three proteins to the overall performance of whey protein based ASDs has still not been investigated. In addition, the limitations of the technology at even higher drug loadings (i.e., more than 50%) have not yet been explored. In this study, BLG, ALA, CGMP and WPI were each prepared as ASDs with the two poorly water-soluble drugs (Compound A and Compound B) at 50%, 60% and 70% drug loadings. Methods: Solid state characterization, dissolution rate and physical stability of the obtained samples were analyzed. Results: All the obtained samples were amorphous and showed faster dissolution rates compared to the respective pure crystalline drugs. However, the BLG based formulations—at least for Compound A—were outperforming the other ASDs in terms of stability, dissolution enhancement and solubility increase. Conclusion: Overall, the study confirmed that the investigated whey proteins showed their potential in developing ASDs even at high drug loadings of up to 70%. [ABSTRACT FROM AUTHOR]

Background: The allergists´ tool box in cat allergy management is limited. Clinical studies have shown that holo beta‐lactoglobulin (holoBLG) can restore micronutritional deficits in atopic immune cells and alleviate allergic symptoms in a completely allergen‐nonspecific manner. With this study, we aimed to provide proof of principle in cat allergy. Methods: A novel challenge protocol for cat allergy in a standardized ECARF allergen exposure chamber (AEC) was developed. In an open pilot study (NCT05455749), patients with clinically relevant cat allergy were provoked with cat allergen for 120 min in the AEC before and after a 3‐month intervention phase (holoBLG lozenge 2x daily). Nasal, conjunctival, bronchial, and pruritus symptoms were scored every 10 min– constituting the total symptom score (TSS). Peak nasal inspiratory flow (PNIF) was measured every 30 min. In addition, a titrated nasal provocation test (NPT) was performed before and after the intervention. Primary endpoint was change in TSS at the end of final exposure compared to baseline. Secondary endpoints included changes in PNIF, NPT, and occurrence of late reactions up to 24 h after exposure. Results: 35 patients (mean age: 40 years) completed the study. Compared to baseline, holoBLG supplementation resulted in significant improvement in median TSS of 50% (p < 0.001), as well as in median nasal flow by 20 L/min (p = 0.0035). 20% of patients reported late reactions after baseline exposure, but 0% after the final exposure. Conclusions: Cat allergic patients profited from targeted micronutrition with the holoBLG lozenge. As previously seen in other allergies, holoBLG supplementation also induced immune resilience in cat allergies, resulting in significant symptom amelioration. [ABSTRACT FROM AUTHOR]

Correspondence analysis (CA) is one of the multivariate statistical analysis techniques which examines the relationships between different nominal variable categories in two-way or contingency tables. The aim of this study is to investigate the relationships between Alpha-S1 casein and Beta-lactoglobulin gene polymorphisms in Norduz sheep by the CA method. In this study, genotype frequencies of CSN1S1 and BLG genes in Norduz sheep (n=102) were used as categorical variables. As a statistical method, descriptive statistics of characteristics were presented as count and percent and the chi-square (X²) test and CA in this study were performed to explore the relationships among the genotype frequencies of Beta-lactoglobulin and Alpha-S1 casein genes. The results of this study indicated that CA can be contributed to a graphical display for categories of nominal variables. Animal breeders can utilize CA as an analytical technique and graphical representation for categorical data. According to the results of this study, the first and second dimensions jointly accounted for 52.25% of the total inertia and "AA" allele of CSN1S1 has the greatest significance in the first dimension, while "BB" allele of BLG has the greatest significant in the second dimension. Therefore, it would be useful to investigate the effects of ßLactoglobulin and aS1-Casein genotypes on various yield traits in larger population of Norduz sheep. [ABSTRACT FROM AUTHOR]

Diploptera punctata, also known as the Pacific beetle cockroach, is a viviparous cockroach that gives birth to live offspring and secretes a highly concentrated mixture of glycosylated proteins as a source of nourishment for developing embryos. These proteins are lipocalins that bind to lipids and crystallize in the gut of the embryo. A structure of milk crystals harvested from the embryos showed that the milk-derived crystals were heterogeneous and made of three proteins (called Lili-Mips). We hypothesized that the isoforms of Lili-Mip would display different affinities for fatty acids due to the ability of the pocket to bind multiple acyl chain lengths. We previously reported the structures of Lili-Mip from crystals grown in vivo and recombinantly expressed Lili-Mip2. These structures are similar, and both bind to several fatty acids. This study explores the specificity and affinity of fatty acid binding to recombinantly expressed Lili-Mip 1, 2 & 3. We show that all isoforms can bind to different fatty acids with similar affinities. We also report the thermostability of Lili-Mip is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. We show that thermostability is an inherent property of the protein, and glycosylation and ligand binding do not change it significantly. Measuring the pH in the embryo's gut lumen and gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. In various crystal structures (reported here and previously by us), Phe-98 and Phe-100 occupy multiple conformations in the binding pocket. In our earlier work, we had shown that the loops at the entrance could adapt various conformations to change the size of the binding pocket. Here we show Phe-98 and Phe-100 can reorient to stabilize interactions at the bottom of the cavity–and change the volume of the cavity from 510 Å3 to 337 Å3. Together they facilitate the binding of fatty acids of different acyl chain lengths. [ABSTRACT FROM AUTHOR]

Simple Summary: Milk protein polymorphisms, especially κ-casein and β-lactoglobulin, are essential for the dairy and cheese industries. However, BB CSN3 is a more favorable polymorphism, as it is responsible for a higher casein content. Statistically significant differences (p ≤ 0.05) in interpregnancy periods values were observed between cows with the BB kappa-casein genotype and cows with the AA genotype—the former exhibited the shortest interpregnancy periods. Additionally, cows with the BB genotype showed more favorable reproductive indices, i.e., the lowest age at first calving and the lowest insemination index. However, these values were not statistically significant. There was no significant influence of any of the analyzed kappa-casein and beta-lactoglobulin genotypes on the course of cows' delivery. This research sought to determine the effect of selected milk protein polymorphisms on the reproduction parameters of 598 black and white Polish Holstein–Friesian cattle. The analyzed genes were kappa-casein (CSN3) and beta-lactoglobulin (BLG). The following reproductive indexes were selected: the age at first calving, the interpregnancy period, the calving interval, and the insemination index. The influence of the identified genotypes on the course of parturition in cows was also analyzed. Source data were collected for each cow from the beginning of the herd life and reproduction to its culling from the herd or the end of its third lactation. Data on the age at first calving, the amount of semen portions for artificial insemination (insemination index), the interpregnancy period, and the calving interval for each cow were also collected. A contingency analysis was performed through contingency tables using a Pearson's chi-squared test for each CSN3 and BLG genotype. The results show that the BB genotype of the kappa-casein gene was associated with the most favorable values of reproductive indicators. In the case of the calving interval, the values were significantly more favorable than those of other genotypes (p ≤ 0.05). No effect of beta-lactoglobulin polymorphism on the analyzed reproductive indices was observed. On the other hand, in the case of the interpregnancy period, significant statistical differences were obtained between the AA and BB genotypes. The analyzed kappa-casein and beta-lactoglobulin genotypes did not significantly influence the course of parturition in cows. To conclude, the genotype polymorphism BB CSN3 is the most favorable for the performance of the cows in the examined herd. [ABSTRACT FROM AUTHOR]

Summary: In this study, the effects of H2O2, 2,2′‐Azobis(2‐amidinopropane) dihydrochloride (AAPH) and metal ion Fe3+ on β‐lactoglobulin (BLG) glyco‐oxidation were investigated. The results demonstrated all the three inducers caused BLG morphology change and higher molecular weight BLG‐glucose (BLG‐Glu) adducts formation. H2O2 and AAPH significantly accelerated BLG oxidation by increasing carbonyl content and decreasing freesulphydryls (P < 0.05). However, Fe3+ showed little influence on carbonyl and free sulphydryls contents. All the three inducers promoted fluorescent‐advanced glycation end products (fl‐AGEs) and carboxymethyl‐lysine (CML) formation. Fe3+ and H2O2 (0.5% and 1%) could increase methylglyoxal (MGO) level and caused less time to reach its peak concentration than control group. Compared with the control group, new glycation sites were determined in H2O2, AAPH and Fe3+ treatments, indicating the inductive effect of those free radicals or metal ion on BLG glycation. The results will provide important information for the control of protein‐based products glyco‐oxidation during food processing and storage. [ABSTRACT FROM AUTHOR]

β-lactoglobulin I (β-LG I) is one of the most important whey proteins in donkey milk. However, to our knowledge, there has been no study focusing on the full nucleotide sequences of this gene (BLG I). Current investigation of donkey BLG I gene is very limited with only 2 variants (A and B) characterized so far at the protein level. Recently, a new β-LG I variant, with a significantly higher mass (+1,915 Da) than known variants has been detected. In this study, we report the whole nucleotide sequence of the BLG I gene from 2 donkeys, whose milk samples are characterized by the β-LG I SDS-PAGE band with a normal electrophoretic mobility (18,514.25 Da, β-LG I B1 form) the first, and by the presence of a unique β-LG I band with a higher electrophoretic mobility (20,428.5 Da, β-LG I D form) the latter. A high genetic variability was found all over the 2 sequenced BLG I alleles. In particular, 16 polymorphic sites were found in introns, one in the 5′ flanking region, 3 SNPs in the 5′ untranslated region and one SNP in the coding region (g.1871G > A) located at the 40th nucleotide of exon 2 and responsible for the AA substitutions p.Asp28 > Asn in the mature protein. Two SNPs (g.920–922CAC > TGT and g.1871G/A) were genotyped in 93 donkeys of 2 Italian breeds (60 Ragusana and 33 Amiatina, respectively) and the overall frequencies of g.920–922CAC and g.1871A were 0.3065 and 0.043, respectively. Only the rare allele g.1871A was observed to be associated with the slower migrating β-LG I. Considering this genetic diversity and those found in the database, it was possible to deduce at least 5 different alleles (BLG I A, B, B1, C, D) responsible for 4 potential β-LG I translations. Among these alleles, B1 and D are those characterized in the present research, with the D allele of real novel identification. Haplotype data analysis suggests an evolutionary pathway of donkey BLG I gene and a possible phylogenetic map is proposed. Analyses of mRNA secondary structure showed relevant changes in the structures, as consequence of the g.1871G > A polymorphism, that might be responsible for the recognition of an alternative initiation site providing an additional signal peptide. The extension of 19 AA sequence to the mature protein, corresponding to the canonical signal peptide with an additional alanine residue, is sufficient to provide the observed molecular weight of the slower migrating β-LG I encoded by the BLG I D allele. [ABSTRACT FROM AUTHOR]

[Display omitted] Protein nanofibrils (PNF) resulting from the self-assembly of proteins or peptides can present structural ordering triggered by numerous factors, including the shear flow. We hypothesize that i) depending on the contour length of the PNF and the magnitude of the shear rate applied to the PNF dispersion, they exhibit specific orientation, and ii) it is possible to predict the alignment of PNF by establishing a flow-alignment relationship. Understanding such a relationship is pivotal to improving the fundamental knowledge and application of fibril systems. We use β -lactoglobulin PNF aqueous dispersions with different average contour lengths but equal persistence lengths. We employ simple shear-dominated microfluidic devices with state-of-the-art imaging techniques: flow-induced birefringence (FIB) and micro-particle image velocimetry (μ -PIV), to probe the effect of shear flow on PNF alignment. We provide an empirical relationship connecting the birefringence Δ n (quantifying the extent of PNF alignment), and the Péclet number Pe (correlating the shear rate of the flow relative to the rotational diffusion of PNF) to understand the flow-alignment behavior of PNF under shear-dominated flows. Furthermore, we assess the alignment and flow profile of PNF at both high and low flow rates. The length of PNF emerges as a controlling parameter capable of modulating PNF alignment at specific shear rates. Our results shed new insights into the hydrodynamic behavior of PNF, which is highly relevant to various industrial processes involving the fibril systems. [ABSTRACT FROM AUTHOR]

We have shown that many proteins and enzymes (ovalbumin, β-lactoglobulin, lysozyme, insulin, histone, papain) undergo concentration-dependent reversible aggregation as a result of the interaction of the studied biomolecules. Moreover, irradiation of those protein or enzyme solutions under oxidative stress conditions results in the formation of stable soluble protein aggregates. We assume that protein dimers are mainly formed. A pulse radiolysis study has been made to investigate the early stages of protein oxidation by N 3 • or •OH radicals. Reactions of the N 3 • radical with the studied proteins lead to the generation of aggregates stabilized by covalent bonds between tyrosine residues. The high reactivity of the •OH with amino acids contained within proteins is responsible for the formation of various covalent bonds (including C–C or C–O–C) between adjacent protein molecules. In the analysis of the formation of protein aggregates, intramolecular electron transfer from the tyrosine moiety to Trp• radical should be taken into account. Steady-state spectroscopic measurements with a detection of emission and absorbance, together with measurements of the dynamic scattering of laser light, made it possible to characterize the obtained aggregates. The identification of protein nanostructures generated by ionizing radiation using spectroscopic methods is difficult due to the spontaneous formation of protein aggregates before irradiation. The commonly used fluorescence detection of dityrosyl cross-linking (DT) as a marker of protein modification under the influence of ionizing radiation requires modification in the case of the tested objects. A precise photochemical lifetime measurement of the excited states of radiation-generated aggregates is useful in characterizing their structure. Resonance light scattering (RLS) has proven to be an extremely sensitive and useful technique to detect protein aggregates. [ABSTRACT FROM AUTHOR]

Simple Summary: Food proteins from new sources such as vegetable origin (pulses, legumes, cereals), fungi, bacteria and insects are being introduced into the market. However, these novel foods, which had often not been consumed by humans, pose an important risk to public health. The biggest challenge is to ensure food safety by analysing in detail their compositional, nutritional, toxicological and allergenic properties. As a massive preliminary screening, proteomic methods should be employed to search for potential allergens. This review focuses on proteomic and bioinformatic tools for food researchers to identify allergens in novel foods. There is a multitude of highly valuable online tools and protein databases based on sequence alignment, motif identification or 3-D structure predictions. Thus, plant and animal food allergens, including lipid transfer proteins, profilins, seed storage proteins, lactoglobulins, caseins, tropomyosins, parvalbumins and other similar proteins, could be detected in novel food matrices. Furthermore, novel potential allergens could be found for further analysis. This would imply a major simplification. In recent years, novel food is becoming an emerging trend increasingly more demanding in developed countries. Food proteins from vegetables (pulses, legumes, cereals), fungi, bacteria and insects are being researched to introduce them in meat alternatives, beverages, baked products and others. One of the most complex challenges for introducing novel foods on the market is to ensure food safety. New alimentary scenarios drive the detection of novel allergens that need to be identified and quantified with the aim of appropriate labelling. Allergenic reactions are mostly caused by proteins of great abundance in foods, most frequently of small molecular mass, glycosylated, water-soluble and with high stability to proteolysis. The most relevant plant and animal food allergens, such as lipid transfer proteins, profilins, seed storage proteins, lactoglobulins, caseins, tropomyosins and parvalbumins from fruits, vegetables, nuts, milk, eggs, shellfish and fish, have been investigated. New methods for massive screening in search of potential allergens must be developed, particularly concerning protein databases and other online tools. Moreover, several bioinformatic tools based on sequence alignment, motif identification or 3-D structure predictions should be implemented as well. Finally, targeted proteomics will become a powerful technology for the quantification of these hazardous proteins. The ultimate objective is to build an effective and resilient surveillance network with this cutting-edge technology. [ABSTRACT FROM AUTHOR]

Milk is one of the most common and widespread allergenic foods. Milk allergy is an adverse immunological reaction to milk proteins of different mammalian species. β-Lactoglobulin (β-LG) is one of the main allergenic proteins of cow's milk. There is an urgent need to develop an accurate and traceable method to accurately quantify β-LG. Based on the known β-lactoglobulin sequence, the Skyline tool was used to simulate the β-lactoglobulin digestion process. After digestion of β-LG with trypsin, the tryptic peptides in the samples were detected selectively by MS/MS. In short, the peptides were searched with MS full scan of respective digested milk protein. Three characteristic peptides of β-lactoglobulin were screened by primary local sequence search tool (basic local alignment search tool, BLAST) and Uniprot database. Finally, one of the characteristic peptides with the highest response intensity and the best stability was selected for further verification by HPLC-MS/MS and quantitative studied by multiple reaction monitoring (MRM). In this work, the specific peptides were quantified by isotope dilution mass spectrometry (IDMS) after trypsin digestion of β-LG. At the same time, the effects of deuterium-labeled characteristic peptide IDAL*NENK(D6-Leu) and carbon-nitrogen dual-labeled characteristic peptide IDAL*NENK(13C6,15N-Leu) as internal standards on chromatographic behavior and detection results were investigated, and the methodology was verified. The method showed a good linear relationship within its own range. The limits of detection and limits of quantification were 0.001 9-0.002 2 g/L and 0.006 4-0.007 4 g/L, respectively. The recoveries ranged from 90.1% to 102.7%, and the coefficients of variation (CVs) were less than 8.0%. Analysis of 5 samples from the domestic market showed CVs were less than 6.5%. Deuterium-labeled peptides with lower synthetic cost could be used as reliable substitutes for 13C and 15N-labeled peptides in protein quantification. The established quantitative method of isotope dilution mass spectrometry had strong anti-interference ability, high sensitivity, high accuracy and good reproducibility. It is expected that the comparability of β-LG quantitative results between different laboratories will be improved. In addition, the combination of new ultrasensitive mass spectrometry with stable-isotope labelling techniques has advanced to a point that some studies with radioactive isotopes can now be readily replaced by stable-isotope techniques. This trend is expected to continue and this isotope method will be a key component of the design of new clinical studies currently under way. At the same time, with the widespread use of stable isotope markers in food allergy, metabolomics, proteomics, clinical pharmacology and other fields, the demand for stable isotope labeled compounds is expected to further increase. [ABSTRACT FROM AUTHOR]

Disulfide bond rearrangement is a common occurrence during protein analysis or treatment. A convenient and rapid method has been developed to investigate heat‐induced disulfide rearrangement of lactoglobulin using matrix‐assisted laser desorption/ionization‐in‐source decay (MALDI‐ISD) technology. By analyzing heated lactoglobulin in reflectron and linear mode, we demonstrated that cysteines C66 and C160 exist as free residues other than linked ones in some protein isomers. This method provides a straightforward and expeditious way to assess the cysteine status and structural changes of proteins under heat stress. [ABSTRACT FROM AUTHOR]

β-lactoglobulin (β-Lg) is a protein found in milk that can cause severe allergic reactions, including rash, vomiting, and diarrhea. Thus, it is crucial to develop a sensitive β-Lg detection method to protect people who are susceptible to allergies. Here, we introduce a novel and highly sensitive fluorescent aptamer biosensor for detecting β-Lg. First, a fluorescein-based dye (FAM)-labeled β-lactoglobulin aptamer (β-Lg aptamer) is adsorbed on the surface of tungsten disulfide (WS2) nanosheets via van der Waals forces, resulting in fluorescence quenching. When β-Lg is present, the β-Lg aptamer selectively binds to β-Lg, causing a conformational change in the β-Lg aptamer and releasing it from the surface of WS2 nanosheets, which restores the fluorescence signal. Simultaneously, DNase I in the system cleaves the aptamer bound to the target, producing a short oligonucleotide fragment and releasing β-Lg. The released β-Lg then binds to another β-Lg aptamer adsorbed on WS2, initiating the next round of cleavage, resulting in significant amplification of the fluorescence signal. This method has a linear detection range of 1–100 ng mL−1, and the limit of detection is 0.344 ng mL−1. Furthermore, this approach has been successfully used for detecting β-Lg in milk samples with satisfactory results, providing new opportunities for food analysis and quality control. [ABSTRACT FROM AUTHOR]

To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae . Sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) analysis indicated that the D28N/P126A was conjugated with a ~4 kDa high-mannose chain. D28N/P126A retained ~61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG. [ABSTRACT FROM AUTHOR]

The study was carried out on milk from cold-blooded Sokólski mares and warm-blooded Polish Halfbred mares. Milk samples were analysed for content of solids, protein, fat, lactose, and ash; density; energy value; percentage of α-lactalbumin (α-La), β-lactoglobulin (β-Lg), serum albumin (SA), immunoglobulin (Ig), lactoferrin (Lf) and lysozyme (Lz) in the total protein; and fatty acid profile. Milk from cold-blooded mares was found to be similar in protein and fat content to that of warm-blooded mares, but had higher content of solids, including lactose and ash. It also had significantly (P≤0.01) higher content of α-La, SA and Lz, and lower content of β-Lg, Ig and Lf in comparison to milk from warm-blooded mares. Milk from Sokólski horses had a significantly (P≤0.01) higher proportion of SFA, including C10:0, C12:0, C14:0, C16:0 (P≤0.01) and C18:0 (P≤0.05), while the milk of warm-blooded mares had a significantly (P≤0.01) higher proportion of MUFA, including C16:1n-9 and C18:n-9, and PUFA, including C18:2n-6 and C18:3n3. The share of n-6 and n-3 PUFA was significantly (P≤0.01) higher in milk from warm-blooded horses, but their ratio (n-6/n-3) was lower (more favourable) in milk from cold-blooded mares. In addition, milk from warm-blooded horses had a lower (P≤0.01) share of HSFA and a higher proportion of DFA compared to cold-blooded horses, as well as lower values for AI and TI. Despite these minor differences in comparison with warmblooded mares (Polish Halfbred), milk from cold-blooded Sokólski mares was shown to be a food product of high nutritional value, which is one argument in favour of the use of this horse breed for dairy purposes. [ABSTRACT FROM AUTHOR]

Summary: Whey protein isolate (WPI) is unstable near isoelectric point with simultaneous precipitation or phase separation, which negatively affects its application. In the current study, four kinds of edible fungal polysaccharides were selected to improve the WPI stability through non‐covalent interaction. The addition of edible fungal polysaccharides significantly improved the WPI stability. Zeta potential analysis showed that edible fungal polysaccharides reduced the zeta potential of WPI. The analysis of rheological properties showed that complex solutions exhibited higher viscosity and behaved as pseudoplastic fluids. Bio‐layer interferometry demonstrated the interactions between edible fungal polysaccharides and WPI, and the KD between Tremella fuciformis (TFP) and WPI was the lowest. LC–MS/MS results indicated that the peptide fragments "VGINYWLAHK" and "TPEVDDEALEKFDK" in α‐lactalbumin and β‐lactoglobulin were probably binding regions interacted with the four kinds of edible fungal polysaccharides. Furthermore, molecular docking results inferred GLN43 and GLU127 were the major residues in the α‐lactalbumin and β‐lactoglobulin. [ABSTRACT FROM AUTHOR]

The present study aimed at developing novel encapsulate materials of calcium-alginate and β-lactoglobulin complex for polyphenols using the jet-flow nozzle vibration method. Encapsulated microbeads were characterized using SEM, FTIR, DSC, and MSI. The encapsulation efficiency of the microbeads varied depending upon the coating material in the range of 74.17–84.87%. Calcium-alginate-β-lactoglobulin microbeads (CABM) exhibited a smooth surface and uniform shape with an average particle size of 1053.73 nm. CABM also showed better thermal and storage stabilities as compared to calcium alginate microbeads. The CABM resulted in excellent target release of polyphenols (84%) in the intestine, which was more than 3-fold the bio-accessibility offered by free polyphenol powder. Further study on individual phenolic acids after simulated in-vitro digestion (SIVD), photo-oxidative and osmotic stress revealed that CABM significantly retained a higher amount of polyphenols and exhibited improved antioxidant capacity after SIVD environment, and may have high industrial application for nutraceutical production. [ABSTRACT FROM AUTHOR]

The effectiveness of cattle selection for quantitative and qualitative traits that are of economic importance in dairy cattle breeding largely depends on the identification of the genes that control these traits, as well as their allelic polymorphisms. One such gene is the LGB gene that encodes the protein beta-lactoglobulin. This review provides brief information about the structure and biological role of this protein and its gene polymorphism. An analysis of the literature data of various studies was carried out, which made it possible to identify and evaluate the effect of the two most common alleles of the LGB gene (A and B) on the efficiency indicators of dairy production. [ABSTRACT FROM AUTHOR]

Amorphous solid dispersions (ASDs) based on proteins as co-formers have previously shown promising potential to improve the solubility and bioavailability of poorly water-soluble drugs. In particular, whey proteins have shown to be promising co-formers and amorphous stabilizers in ASD formulations, including at high drug loading. In this study, the feasibility of the whey protein β-lactoglobulin (BLG) as a co-former in ASDs was compared to the more traditional ASD co-formers based on synthetic polymers (hydroxypropyl methylcellulose acetate succinate and Eudragit® L) as well as to a nanocrystalline formulation. The poorly water-soluble drug rifaximin (RFX) was chosen as the model drug. All drug/co-former formulations were prepared as fully amorphous ASDs by spray drying at 50% (w/w) drug loading. The BLG-based ASD had the highest glass transition temperature and showed a faster dissolution rate and higher drug solubility in three release media with different pH values (1.2, 4.5, and 6.5) compared to the polymer-based ASDs and the nanocrystalline RFX. In conclusion, BLG is a promising co-former and amorphous stabilizer of RFX in ASD formulations, superior to the selected polymer-based ASD systems or the nanocrystalline formulation. [ABSTRACT FROM AUTHOR]

The use of polymorphic gene markers is a widely used alternative to traditional methods of trait selection in livestock. This study aims to determine the polymorphism of three milk gene markers: κ-casein, β-casein, and β-lactoglobulin in the Siquijor native (SN, n=124) and Holstein x Sahiwal (HS, n=86) cattle. Genotyping was done primarily by PCR-RFLP analysis while PCR-sequencing was followed only for the β-casein of the SN herd. Results showed that for the β-lactoglobulin, the genotypic frequencies of AA, AB, and BB in the HS were 0.174, 0.464, and 0.362, whereas in the SN these were 0.025, 0.099, and 0.876 respectively. The distribution of κ-casein genotypes was 0.589, 0.286, and 0.125 for the AA, AB, and BB genotypes for the HS respectively, and 0.442, 0.425, and 0.133 for the SN. β-casein genotypes showed that the A1A1, A1A2, and A2A2 were 0.143, 0.755, and 0.102 respectively in the HS and 0.600, 0.320, and 0.080 in the SN. Our results show that the three genes are polymorphic in the SN and HS cattle. It is recommended to conduct an association study between these polymorphic variants and milk quality traits to confirm their usability as gene markers for future selection. [ABSTRACT FROM AUTHOR]

The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis. [ABSTRACT FROM AUTHOR]

[Display omitted] The surface dilatational and shear moduli of surfactant and protein interfacial layers can be derived from surface pressures measured with a Wilhelmy plate parallel, Δ Π par and perpendicular Δ Π perp to the barriers in a Langmuir trough. Applying area oscillations, A 0 + Δ A e i ω t , in a rectangular Langmuir trough induces changes in surface pressure, Δ Π par and Δ Π perp for monolayers of soluble palmitoyl-lysophosphatidylcholine (LysoPC), insoluble dipalmitoylphosphatidylcholine (DPPC), and the protein β -lactoglobulin to evaluate E s ∗ + G s ∗ = A 0 Δ Π par Δ A and E s ∗ - G s ∗ = A 0 Δ Π perp Δ A . G s ∗ was independently measured with a double-wall ring apparatus (DWR) and E s ∗ by area oscillations of hemispherical bubbles in a capillary pressure microtensiometer (CPM) and the results were compared to the trough measurements. For LysoPC and DPPC, A 0 Δ Π par Δ A ≅ A 0 Δ Π perp Δ A meaning E s ∗ ≫ G s ∗ and E s ∗ ≅ A 0 Δ Π par Δ A ≅ A 0 Δ Π perp Δ A. Trough values for E s ∗ were quantitatively similar to CPM when corrected for interfacial curvature. DWR showed G ∗ was 4 orders of magnitude smaller than E s ∗ for both LysoPC and DPPC. For β -lactoglobulin films, A 0 Δ Π par Δ A > A 0 Δ Π perp Δ A and E s ∗ and G s ∗ were in qualitative agreement with independent CPM and DWR measurements. For β -lactoglobulin, both E s ∗ and G s ∗ varied with film age and history on the trough, suggesting the evolution of the protein structure. [ABSTRACT FROM AUTHOR]

Enhancing the solubility of rice glutelin in neutral aqueous solution is the prerequisite for the development of rice protein drinks and ingredients. Herein, glutelin was first dissolved in an aqueous solution of pH 12, and then heated at 121 °C for 20 min. The results showed that the solubility of glutelin increased from 2.55 mg/mL to 20.7 mg/mL at pH 7. The size of glutelin aggregates at pH 7 decreased from 900 nm to 400 nm after high temperature treatment (HTT), which was confirmed by atomic force microscopy. The results of small angle X-ray scattering showed that HTT induced the conformational unfolding of glutelin, and the protein in the aggregate was rod like shape as well as the mean square rotation radius decreased from 64.9 to 54.8 Å. Furthermore, Raman spectrum results also agree with the unfolding of glutelin conformation, which was mainly reflected in the changes of tyrosine and tryptophan residues, as well as the decreasing of α-helix content and increasing of β-sheet content. After being freeze-dried, HTT glutelin has a re-solubilization capacity of 15.48 mg/mL in pH 7 aqueous solution, which was superior to that of spray dried glutelin powder (pH 7, 9.19 mg/mL). [ABSTRACT FROM AUTHOR]