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Purpose of the study. To assess the effect of inclusion of lactoglobulin in complex preoperative preparation of colon cancer patients on their tumor and resection line tissue microbiota.Materials and methods. 40 patients with colon cancer stages II–III, in whom the operation was the first stage of treatment, during standard preoperative preparation, were injected with a preparation of antibodies against opportunistic intestinal microorganisms obtained from colostrum of immunized cows, 2 g twice a day orally before surgery for 3 days (total dose of 12 g) (main group); 40 patients received standard antibiotic prophylaxis (control group). The quantitative composition of the microbiota was determined in the samples of the removed tumor and tissue of the resection line.Results. The total microbial contamination of the tumor was 9.2 times lower in the main group relative to the control group; the frequency of E.coli and Clostridiae excretion was also statistically significantly lower (p = 0.004 and 0.03, respectively). In the tumors of patients of the main group out of twelve studied representatives of microorganisms, the number of six was statistically significantly lower than in control group, and three of those found in the control group were not detected. Since they were potentially pathogenic (Pseudomonas aeruginosa, Staphylococcus aureus, fungi of the Candida spp.), the microbial composition of the tumor of patients in the main group can be considered more favorable than the control group. Similar differences were noted in non-t umor intestinal tissue, in which the content of Enterobacter spp, Streptococcus, Clostridiae, Peptostreptococci was statistically significantly lower than in the control group.Conclusion. Thus oral administration of colostrum antibodies caused positive changes in tumor and colon tissue microbiota. We suggest the application of lactoglobulin to be useful for surgical treatment of such patients taking into account the possible impact of microbiota in patients’ response to chemo- and immunotherapy.

Photosensitization of proteins mediated by chromophores is a mechanism commonly employed by nature and mimicked in a broad array of laboratory research and applications. Nature has evolved specialized complexes of proteins and photosensitizers (PS) that assemble to form photoreceptor proteins (PRP). These are used by many organisms in diverse processes, such as energy conversion, protection against photodamage, etc. The same concept has been used in laboratory settings for many applications, such as the stimulation of neurons or the selective depletion of proteins in a signaling pathway. A key issue in laboratory settings has been the relationship between the photooxidation of proteins and conformational changes in host proteins. For several years, we have been interested in creating non-native PRP using porphyrin PS. In this study, we investigated the self-assembled complex between zinc protoporphyrin IX (ZnPPIX) and bovine β-lactoglobulin (BLG) as a model of non-native PRP. Since BLG undergoes a significant conformational transition near physiological pH, the study was carried out at acidic (pH 5) and alkaline (pH 9) conditions where the two conformations are respectively prevalent. We employed a series of steady-state and time-resolved optical spectroscopies as well as gel electrophoresis to experimentally characterize the photosensitization mechanisms and their effect on the host protein. Our results show that ZnPPIX prompts light-dependent modifications of BLG, which appear to be much more significant at alkaline pH. The modifications seem to be driven by photooxidation of amino acid residues that do not lead to the formation of cross-links or protein fragmentation.

Photosensitization of proteins mediated by chromophores is a mechanism commonly employed by nature and mimicked in a broad array of laboratory research and applications. Nature has evolved specialized complexes of proteins and photosensitizers (PS) that assemble to form photoreceptor proteins (PRP). These are used by many organisms in diverse processes, such as energy conversion, protection against photodamage, etc. The same concept has been used in laboratory settings for many applications, such as the stimulation of neurons or the selective depletion of proteins in a signaling pathway. A key issue in laboratory settings has been the relationship between the photooxidation of proteins and conformational changes in host proteins. For several years, we have been interested in creating non-native PRP using porphyrin PS. In this study, we investigated the self-assembled complex between zinc protoporphyrin IX (ZnPPIX) and bovine β-lactoglobulin (BLG) as a model of non-native PRP. Since BLG undergoes a significant conformational transition near physiological pH, the study was carried out at acidic (pH 5) and alkaline (pH 9) conditions where the two conformations are respectively prevalent. We employed a series of steady-state and time-resolved optical spectroscopies as well as gel electrophoresis to experimentally characterize the photosensitization mechanisms and their effect on the host protein. Our results show that ZnPPIX prompts light-dependent modifications of BLG, which appear to be much more significant at alkaline pH. The modifications seem to be driven by photooxidation of amino acid residues that do not lead to the formation of cross-links or protein fragmentation. [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]

ABSTRACT: Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).

Pickering emulsions stabilized by polysaccharides and protein complexes have attracted increasing attention. Exploring their interfacial activities and adsorption behaviors at the oil–water interface is of great significance for deepening our understanding of the emulsification mechanism and improving the properties of polysaccharide/protein-stabilized emulsions. Here, cellulose nanocrystals (CNCs) with rod-like structures and spherical β-lactoglobulin (β-LG) proteins were selected as representative stabilizers. We initially investigated the interaction between the 2 at pH 2.0–7.0 through a multiscale approach. Then, oil–water interfacial tension tests were used to assess the interfacial activities of CNCs and β-LG, revealing that β-LG exerted primary influence over the interfacial behavior in their complexes. Meanwhile, quartz crystal microbalance with dissipation was used to monitor the interfacial adsorption behaviors of CNCs, β-LG, and CNC/β-LG complexes, demonstrating that the interfacial layer formed by the CNC/β-LG complexes on the oil film (∼696.7 ng/cm2) was thicker and more stable compared with that formed by individual CNCs (∼48.4 ng/cm2) or β-LG (∼87.1 ng/cm2). Finally, we applied the CNC/β-LG complexes as stabilizers in Pickering emulsions and the emulsions prepared with the complexes at concentrations above 0.8 wt% exhibited storage stability for up to 28 d which confirmed their exceptional emulsifying ability and practical value. This study provides profound insights into the interfacial behavior of polysaccharide–protein complexes in Pickering emulsions and is expected to facilitate the design of novel food-grade emulsifiers. [Display omitted] • CNC/β-LG complexes fabricated at pH 3.0 exhibited the best stability. • β-LG dominated the interfacial activity in the CNC/β-LG complexes. • The interfacial layer formed by CNC/β-LG complexes was thicker than that of single particles. • CNC/β-LG complexes within the continuous phase promoted the stability of emulsions. [ABSTRACT FROM AUTHOR]

Coacervates of oppositely charged milk proteins are used in functional food development, mainly to encapsulate bioactives. To uncover the driving forces behind coacervates formation, we study the association of lactoferrin and β-lactoglobulin at amino-acid level detail, using molecular simulations. Our findings show that inter-protein electrostatic interactions dominate and are, surprisingly, equally divided between an isotropic part, due to monopole-monopole attraction of the oppositely charged proteins, and an anisotropic part due to uneven surface charge distributions. In good agreement with recent experimental association constants, the calculated protein-protein interaction free energy is strongly dependent on pH and salt concentration. In addition to thermodynamics, we also investigate amino acid contacts in microstates of trimeric and pentameric protein complexes, and identify interaction hot-spots that drive heteroprotein complex coacervation process., Competing Interests: Declaration of competing interest The authors declare there are no competing financial or personal interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Recombinant milk protein production is emerging as a pivotal innovation in the dairy industry, driven by the need for sustainable and ethically produced dairy alternatives. Traditional dairy production faces challenges such as greenhouse gas emissions, ethical concerns about animal welfare, and fluctuating productivity due to environmental factors. Recombinant technology offers a promising avenue, using genetically modified organisms to produce milk proteins that closely mimic their natural counterparts. This work encompasses a comprehensive review of articles and patents aimed at understanding the current state and advancements in the production of recombinant milk proteins. It addresses technical aspects such as raw milk composition, protein expression in various hosts, the importance of posttranslational modifications, and the challenges of scaling up for commercial production. The study also explores the implications and possibilities of these advancements for the food industry. Recombinant milk protein production holds significant promise in revolutionizing the dairy industry. Key findings from this review include the identification of efficient host organisms and vectors, advancements in genetic engineering and bioprocessing, and innovative strategies for large-scale production. The future of this technology is promising, especially in creating sustainable, efficient production methods. However, challenges remain in achieving cost-effectiveness, scalability, and a nutritional profile comparable to traditional milk. Continuous research and development are essential for optimizing the technology and enhancing its commercial viability to meet the increasing demand for sustainable dairy alternatives. [Display omitted] • Recombinant milk protein is a new alternative to traditional dairy protein. • Animal-free formulations with recombinant milk protein can mimic dairy products. • Different hosts have been used to express recombinant milk protein. • The vector design is a key factor for scaling up of recombinant milk protein. • Scale-up and cost-effective downstream ensure recombinant milk protein feasibility. [ABSTRACT FROM AUTHOR]

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Kidney disease is currently prevalent worldwide but only shows insidious symptoms in the early stages. The second near-infrared window (NIR-II) fluorescence imaging has become a widely used preclinical technology for evaluating renal dysfunction due to its high resolution and sensitivity. However, bright renal clearable NIR-II fluorescence nanoprobes with a simple synthesis process are still lacking. Herein, we develop a lactoglobulin (LG)@dye nanoprobe for NIR-II fluorescence imaging of kidney dysfunction in vivo based on a purification-free method. The nanoprobe was synthesized by simply mixing LG and IR820 in aqueous solutions at 70 °C for 2 h based on the covalent interaction between the meso-Cl in IR820 and LG. The synthesized LG@IR820 nanoprobe has bright and stable NIR-II fluorescence, ultra-small size (<5 nm), low toxicity, and renal-clearable ability. The high reaction efficiency and pure aqueous reaction media make the synthesis method purification-free. In a unilateral ureteral obstruction mouse model, incipient renal dysfunction assessment was achieved by LG@IR820 nanoprobe, which couldn't be diagnosed with conventional kidney function indicators. This study provides a bright and purification-free NIR-II LG@IR820 nanoprobe to visualize kidney dysfunction at the early stage. [Display omitted] • The LG@IR820 nanoprobe has bright and stable NIR-II fluorescence. • The LG@IR820 nanoprobe has ultra-small size (<5 nm) and renal-clearable ability. • The LG@IR820 nanoprobe has a purification-free synthesis method. • The LG@IR820 nanoprobe can assess kidney dysfunction by NIR-II fluorescence imaging. [ABSTRACT FROM AUTHOR]

The wide spread of drug-resistant forms of the pathogens, complicating prevention and treatment of acute intestinal infections, determines the feasibility of using bacteriophages and immune lactoglobulins for antibacterial therapy of these diseases. The aim of this study was to investigate the antibacterial efficacy of the joint use of immune lactoglobulin and Salmonella bacteriophage in the treatment of experimental salmonellosis in mice. The experimental Salmonella infection was reproduced by means of a single intragastric administration of 2LD50 of daily agar culture of S.typhimurium. As a result, the development of Salmonella infection lasting up to 14 days was noted. The animals were divided into four groups according to the pattern of the treatment. The following preparations were used for the treatment of animals: «Lactoglobulin against opportunistic bacteria and Salmonella» dry cow, «Lactoglobulin» liquid, bacteriophage of Salmonella ABCDE groups, liquid. The preparations were daily administered to mice orally after the infection for 7 days. In the study of the joint therapeutic effect of the lactoglobulin and bacteriophage the special attention was paid to the terms of elimination of the pathogen from animal organs. Analysis of the data showed that in all four groups a positive dynamics of excretion of the pathogen of mice was revealed. In groups of animals that were treated with both drugs, the elimination of Salmonella out of organs occurred 1.5 times faster than in the control groups of mice. At the same time in the experimental groups on the third day of treatment with preparations the statistically significant decrease in the number of S.typhimurium in the contents of the large intestine of animal was revealed. The study found that immune lactoglobulin used simultaneously with Salmonella phage for 7 days reduced the severity and duration of the disease course and blocked the further development of the infectious process, promoted the efficient and rapid cleansing of animals organism of the pathogen, increased the survival of mice. The obtained data suggest the applicability of joint use of immune lactoglobulin preparations and bacteriophages in the treatment of acute intestinal infections caused by antibiotic-resistant strains of Salmonella.

Current treatments for hair follicle (HF) disruption are based on 5‐α reductase inhibitors and prostaglandin modulators. Botanicals and nutraceutical compounds interfere with hair loss or stimulate its partial regrowth. Here, we used in vitro cocultures to investigate the activity of Serenoa repens (SR) and N‐acetyl glucosamine + milk proteins (NAG/Lac) on the paracrine interactions between human microvascular endothelial cells (HMVEC) and HF dermal papilla cells (FDPC). Both SR and NAG/Lac‐induced endothelial tubulogenesis were enhanced by FDPC. SR promoted proliferation of both the cell types, while NAG/Lac was effective on endothelium. Vascular endothelial growth factor production, enhanced by SR, was further augmented by FDPC. In FDPC 5‐α reductase‐II and β‐catenin expressions were modified by SR and less by NAG/Lac, with no additional effect by HMVEC. SR and NAG/Lac prevented lipid peroxidation, whereas NAG/Lac was effective on interleukin 1β production. Finally, SR and NAG/Lac differentially affected HMVEC permeability and tight junction proteins content. These data provide a mechanistic background for the potential use of these compounds as promoters of HF vascularization. 1)Paracrine communication between endothelial and follicle dermal papilla cells.2)Characterization of the role of natural compounds on in vutro cocultures systems.3)mechanistic background for the potential use of natural compounds as promoters of HF vascularization. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the role of milk protein fractions in the coagulation, curd firming, and syneresis of bovine milk. Analyses were performed on 1,271 individual milk samples from Brown Swiss cows reared in 85 herds classified into 4 types of farming systems, from the very traditional (tied cows, feed manually distributed, summer highland pasture) to the most modern (loose cows, use of total mixed rations with or without silage). Fractions αS1-casein (CN), αS2-CN, β-CN, κ-CN, β-lactoglobulin (LG), and α-lactalbumin (LA) and genotypes at CSN2,CSN3, and BLG were obtained by reversed-phase HPLC. The following milk coagulation properties were measured with a lactodynamograph, with the testing time extended to 60 min: rennet coagulation time (RCT, min), curd firming time (min), and curd firmness at 30 and 45 min (mm). All the curd firmness measures recorded over time (total of 240 observations/sample) were used in a 4-parameter nonlinear model to obtain parameters of coagulation, curd firming, and syneresis: RCT estimated from the equation (min), asymptotic potential curd firmness (mm), the curd firming and syneresis instant rate constants (%/min), and the maximum curd firmness value (CFmax, mm) and the time taken to reach it (min). All the aforementioned traits were analyzed with 2 linear mixed models, which tested the effects of the protein fractions expressed in different ways: in the first, quantitative model, each protein fraction was expressed as content in milk; in the second, qualitative model, each protein fraction was expressed as a percentage of total casein content. Besides proteins, additional nuisance parameters were herd (included as a random effect), daily milk production (only for the quantitative model), casein content (only for the qualitative model), dairy system, parity, days in milk, the pendulum of the lactodynamograph, and the CSN2, CSN3, and BLG genotypes. Both αS1-CN and β-CN showed a clear and favorable effect on CFmax, where the former effect was almost double the latter. Milk coagulation ability was favorably affected by κ-CN, which reduced both the RCT and RCT estimated from the equation, increased the curd firming and syneresis instant rate constants, and allowed a higher CFmax to be reached. In contrast, αS2-CN delayed gelation time and β-LG worsened curd firming, both resulting in a low CFmax. The results of this study suggest that modification of the relative contents of specific protein fractions can have an enormous effect on the technological behavior of bovine milk. [ABSTRACT FROM AUTHOR]

Abstract: Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β‐lactoglobulin to Toyopearl Butyl‐650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δhads, was observed to be dependent on temperature and salt concentration. Δhads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δhads for β‐lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δhads increased with increasing temperature. The influence of salt concentration on Δhads was also more pronounced for bovine serum albumin than for β‐lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques. [ABSTRACT FROM AUTHOR]

Abstract: Whey proteins are obtained from dairy industry waste. Studies involving the analysis of the bioactive compounds in whey show health benefits, as it is an excellent source of indispensable amino acids. Milk whey contains principally β‐lactoglobulin, α‐lactoglobulin, bovine serum albumin, and lactoferrin, proteins with innumerable functional and technological properties. One application of these proteins in food is the formation of interpolymer complexes, along with other proteins or anionic polysaccharides. The formation of complexes occurs mainly through electrostatic interactions between a negatively charged biopolymer and a positively charged biopolymer. This formation is influenced by factors such as pH, ionic strength, and biopolymer ratio. Because they do not use high temperatures and chemical reagents and have additional nutritional and functional value, these complexes have been used as encapsulating agents for bioactive ingredients. Recent studies on their training and applications are addressed in this review to boost new research and applications in the food industry, thus increasing opportunities for utilizing whey proteins. [ABSTRACT FROM AUTHOR]

This study aimed to investigate factors affecting protein fractions, namely α-casein (α-CN), β-casein (β-CN), κ-casein (κ-CN), β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) predicted from milk infrared spectra in milk of dairy and dual-purpose cattle breeds. The dataset comprised 735,328 observations from 49,049 cows in 1782 herds. Results highlighted significant differences of protein fractions in milk of the studied breeds. Significant variations of protein fractions were found also through parities and lactation, with the latter thoroughly influencing protein fractions percentage. Interesting correlations (r) were estimated between β-CN, κ-CN and β-LG, expressed as percentage of crude protein, and milk urea nitrogen (r = 0.31, −0.20 and −0.26, respectively) and between α-LA and fat percentage (r = 0.41). The present study paves the way for future studies on the associations between protein fractions and milk technological properties, and for the estimation of genetic parameters of predicted protein composition.

Bovine β-lactoglobulin (BLG) belong to the lipocalin family. This is a group of proteins involved in the binding and transporting of hydrophobic molecules. Experimental and theoretical reports have stated its complex structural behavior in solution, with coupled effects between homodimerization and ligand recognition. Nonetheless, structural evidence at the atomic level about the cause of this coupled effect has not been reported to date. To address this issue microsecond molecular dynamics (MD) simulations were combined with the molecular mechanics generalized Born surface area (MM/GBSA) approach, clustering analysis and principal component analysis (PCA), to explore the conformational complexity of BLG protein-protein self-association and palmitic acid (PLM) or dodecyl sulfate (SDS) ligand recognition in the monomeric and dimeric state. MD simulations, coupled to the MM/GBSA method, revealed that dimerization exerts contrasting effects on the ligand-binding capacity of BLG. Protein dimerization decreases PLM affinity, promoting dimer association. For SDS the dimeric state increases affinity, enhancing dimer dissociation. MD simulations based on PCA revealed that while few differences in the conformational subspace are observed between the free and bound monomer and dimer coupling for PLM, substantial changes are observed between the free and bound monomer and dimer coupling for SDS. [ABSTRACT FROM AUTHOR]

Potentiometric titration curves were obtained for aqueous solutions of native whey protein isolate (WPI) and β-lactoglobulin (β-lg) at different NaCl concentrations. The curves crossed at approximately the same pH, which is considered to be the isoionic point (IIP). Titration with NaCl confirmed that the pH was independent of the salt concentration up to 0.1 M at the IIP, which was 4.9 for WPI and 5.0 for β-lg. Fractal aggregates and microgels of different sizes were formed by heating protein solutions at different pH and different concentrations. The titration curves of the aggregates depended on the type of aggregates, but not on their size. The IIP increased by at most 0.2 pH units after aggregation. For a given pH larger than IIP, the charge density of the proteins (α) was reduced after denaturation and aggregation. The reduction was stronger for microgels than for fractal aggregates. Addition of NaCl or increasing the protein concentration mitigated the effect. Comparison between WPI and β-lg showed that the pH dependence of α was almost the same for pH > 5.0 both for native and aggregated proteins. [ABSTRACT FROM AUTHOR]

Oxidation results in protein deterioration in mammals, plants, foodstuffs and pharmaceuticals, via changes in amino acid composition, fragmentation, aggregation, solubility, hydrophobicity, conformation, function and susceptibility to digestion. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and H 2 O 2 alters the structure and composition of the major whey protein β-lactoglobulin. Thermal treatment induced reducible cross-links, with this being enhanced by low H 2 O 2 concentrations, but decreased by high concentrations, where fragmentation was detected. Cross-linking was prevented when the single free Cys121 residue was blocked with iodoacetamide. Low concentrations of H 2 O 2 added before heating depleted thiols, with H 2 O 2 alone, or H 2 O 2 added after heating, having lesser effects. A similar pattern was detected for methionine loss and methionine sulfoxide formation. Tryptophan loss was only detected with high levels of H 2 O 2 , and no other amino acid was affected, indicating that sulfur-centered amino acids are critical targets. No protection against aggregation was provided by high concentrations of the radical scavenger 5, 5-dimethyl-1-pyrroline N -oxide (DMPO), consistent with molecular oxidation, rather than radical reactions, being the major process. Sulfenic acid formation was detected by Western blotting and LC–MS/MS peptide mass-mapping of dimedone-treated protein, consistent with these species being significant intermediates in heat-induced cross-linking, especially in the presence of H 2 O 2 . Studies using circular dichroism and intrinsic fluorescence indicate that H 2 O 2 increases unfolding during heating. These mechanistic insights provide potential strategies for modulating the extent of modification of proteins exposed to thermal and oxidant treatment. [ABSTRACT FROM AUTHOR]

We investigated the interaction of a series of metal-protoporphyrins (PPIXs) with bovine β- lactoglobulin (BLG) using a combination of optical spectroscopy and computational simulations. Unlike other studies, the simulations were not merely used to rationalize the experimental data but were employed to refine the experimental data itself. The study was carried out at two pH values, 5 and 9, where BLG is known to have different conformation dictated by the so-called Tanford transition which occurs near pH 7.5. The transition is postulated to regulate access to the interior binding cavity of the protein, thus the pH variation was used as a parameter to investigate whether PPIXs access the central cavity of BLG. The results of our study show that indeed binding increases significantly at alkaline pH, however, the increased affinity is not due to the accessibility of the central cavity. Instead, binding appears to be determined by the tendency of PPIXs to form large inhomogeneous aggregates at acidic pH which hinders interactions with proteins. The binding site determined through a combination of experimental and computational methods is located at the interface between two BLG monomers where the long α-helix segment of the protein face each other. This region is rich in positively charged Lys residues that interact with the propionic acid chains of the protoporphyrins. Establishing the modality of binding between protoporphyrins and BLG would have important consequences for the use of BLG:PPIX complexes in applications such as artificial photoreceptors, artificial metallo-enzymes, delivery of photosensitizers for phototherapy and even solar energy conversion. [Display omitted] • Bovine β-lactoglobulin (BLG) was used as model carrier of protoporphyrin IX photosensitizers (PS) for drug delivery and artificial photoreceptor protein. • BLG binds monomeric metal-free and metal-protoporphyrins IX (PPIX) in significant amounts only at alkaline pH. • PPIXs bind BLG at the monomer-monomer interface of BLG dimers in the region between α-helices rich in Lys residues. • Combined of experimental and computational methods yield a model for binding and for correct FRET calculations. [ABSTRACT FROM AUTHOR]

Milk contains an array of proteins with useful bioactivities. Many milk proteins encompassing native or chemically modified casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin demonstrated antiviral activities. Casein and alpha-lactalbumin gained anti-HIV activity after modification with 3-hydroxyphthalic anhydride. Many milk proteins inhibited HIV reverse transcriptase. Bovine glycolactin, angiogenin-1, lactogenin, casein, alpha-lactalbumin, beta-lactoglobulin, bovine lactoferrampin, and human lactoferrampin inhibited HIV-1 protease and integrase. Several mammalian lactoferrins prevented hepatitis C infection. Lactoferrin, methylated alpha-lactalbumin and methylated beta-lactoglobulin inhibited human cytomegalovirus. Chemically modified alpha-lactalbumin, beta-lactoglobulin and lysozyme, lactoferrin and lactoferricin, methylated alpha-lactalbumin, methylated and ethylated beta-lactoglobulins inhibited HSV. Chemically modified bovine beta-lactoglobulin had antihuman papillomavirus activity. Beta-lactoglobulin, lactoferrin, esterified beta-lactoglobulin, and esterified lactoferrindisplayed anti-avian influenza A (H5N1) activity. Lactoferrin inhibited respiratory syncytial virus, hepatitis B virus, adenovirus, poliovirus, hantavirus, sindbis virus, semliki forest virus, echovirus, and enterovirus. Milk mucin, apolactoferrin, Fe-lactoferrin, beta-lactoglobulin, human lactadherin, bovine IgG, and bovine kappa-casein demonstrated antihuman rotavirus activity. [ABSTRACT FROM AUTHOR]

Interactions between bovine and goat β-lactoglobulin and tetracaine and pramocaine were investigated with isothermal titration calorimetry, X-ray crystallography and molecular modelling. Tetracaine and pramocaine binding to lactoglobulin is an entropy driven endothermic reaction. In this work, we found that determined association constants and thermodynamic parameters indicate that pramocaine has a higher affinity to lactoglobulin than tetracaine. Crystal structures that were determined with resolutions in the range from 1.90 to 2.30 Å revealed in each case the presence of a single drug molecule bound in the β-barrel in a mode similar to that observed for 14- and 16-carbon fatty acids. The position of the ligand in the β-barrel indicates the optimal fit of 6-carbon aromatic rings to the binding pocket and the major role of hydrophobic interactions in ligand binding. Calculations of tetracaine and pramocaine docking to lactoglobulin revealed that molecular modelling overestimated the role of polar protein–drug interactions. [ABSTRACT FROM AUTHOR]

Protein aggregates were formed by heating the globular protein β-lactoglobulin (β-lg) in aqueous solution in the presence of CaCl 2 leading to small strands at low CaCl 2 concentrations and larger spherical microgels at higher CaCl 2 concentrations. The structure of mixtures of protein aggregates and the polysaccharide κ-carrageenan (κ-car) was investigated at pH 7 using confocal laser scanning microscopy. Microphase separation in the form of dense protein domains was observed for mixtures with microgels even at very low κ-car concentrations (0.5 g/L). Gelation of κ-car was induced by cooling in the presence of 10 mM KCl. Addition of microgels strongly increased the gelation rate and the elastic modulus of the κ-car gels, while the effect of adding native proteins or strands was small. The effect could be attributed to the presence of calcium ions used to form the microgels. However, the effect was smaller than in pure κ-car gels if the same amount of calcium ions was added in the form of CaCl 2 . The effect is explained by the competition between κ-car and β-lg for calcium ions. A weaker effect was observed with native β-lg, which can be correlated with the weaker binding of Ca 2+ by native β-lg compared to microgels. Gelation of β-lg in the presence of κ-car was induced by heating mixtures of κ-car and native β-lg. The presence of κ-car facilitated gelation of β-lg at lower CaCl 2 concentrations. κ-car was gelled within the β-lg gel by cooling. It was found that the elastic modulus of the mixed gel was close to the sum of that of the κ-car gel with native β-lg and the β-lg gel with uncrosslinked κ-car coils. [ABSTRACT FROM AUTHOR]

ABSTRACT The bovine dairy protein β-lactoglobulin (βlg) is a promiscuous protein that has the ability to bind several hydrophobic ligands. In this study, based on known experimental data, the dynamic interaction mechanism between bovine βlg and four fatty acids was investigated by a protocol combining molecular dynamics (MD) simulations and molecular mechanics generalized Born surface area (MMGBSA) binding free energy calculations. Energetic analyses revealed binding free energy trends that corroborated known experimental findings; larger ligand size corresponded to greater binding affinity. Finally, binding free energy decomposition provided detailed information about the key residues stabilizing the complex. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1010-1018, 2014. [ABSTRACT FROM AUTHOR]

ABSTRACT Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, the detailed process of how a ligand reaches a protein binding site has been an intriguing question over decades. The short time interval between the encounter between a ligand and its receptor to the formation of the stable complex has prevented experimental observations. Bovine β-lactoglobulin (βlg) is a lipocalin member that carries fatty acids (FAs) and other lipids in the cellular environment. Βlg accommodates a FA molecule in its highly hydrophobic cavity and exhibits the capability of recognizing a wide variety of hydrophobic ligands. To elucidate the ligand entry process on βlg, we report molecular dynamics simulations of the encounter between palmitate (PA) or laurate (LA) and βlg. Our results show that residues localized in loops at the cavity entrance play an important role in the ligand penetration process. Analysis of the short-term interaction energies show that the forces operating on the systems lead to average conformations very close to the crystallographic holo-forms. Whereas the binding free energy analysis using the molecular mechanics Generalized Born surface area method shows that these conformations were thermodynamically favorable. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 744-757, 2014. [ABSTRACT FROM AUTHOR]

Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of Δ S and Δ H revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical Δ G for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Lactoglobulin is a globular milk protein for which physiological function has not been clarified. Due to its binding properties lactoglobulin might serve as a carrier for bioactive molecules. Binding of 12-, 14-, 16- and 18-carbon saturated fatty acids to bovine β-lactoglobulin has been characterised by isothermal titration calorimetry and X-ray crystallography as a part of systematic studies of lactoglobulin complexes with ligands of biological importance. The thermodynamic parameters have been determined for lauric, myristic and palmitic acid complexes revealing systematic decrease of enthalpic and increase of entropic component of ΔG with elongation of aliphatic chain. In all crystal structures determined with resolution 1.9–2.1Å, single fatty acid molecule was found in the β-barrel in extended conformation with individual pattern of interactions. Location of a fatty acid in the binding site depends on the length of aliphatic chain and influences polar interactions between protein and ligand. Systematic changes of entropic component indicate important role of water in binding process. [Copyright &y& Elsevier]

Abstract: The literature is reviewed on the formation and the structure of β-lactoglobulin and whey protein isolate (WPI) aggregates in aqueous solution induced by heating. The focus is on the effects of the pH and added salt. The use of β-lactoglobulin and WPI aggregates in cold-set gels, foams and emulsions, encapsulation, and films and coatings is also reviewed. [Copyright &y& Elsevier]

There is growing interest in the fractionation of whey proteins because of the specific properties of individual whey proteins. The objective of this work was to assess the efficiency of two membranes to obtain two fractions, one rich in β-lactoglobulin (β-Lg) and the other rich in α-lactalbumin (α-La) from a wheyprotein concentrate using microfiltration (MF). Two MF membranes were tested for the fractionation: a flat-sheet membrane VCWP and a spiral membrane MF-7002, both with nominal pore sizes of 0.1 lm. The VCWP retained 78% of the proteins in solution, and this membrane was shown to be permeable to both proteins, β-Lg and α-La. The retention of protein by MF-7002 was 65%, and there was a partial retention of lactose. The permeate collected in the MF-7002 in the concentration stage was over 50% α-La; although this was present in lower concentrations, it was passed preferentially to the permeate. The results indicated that some aggregation of the proteins may have occurred under the experimental conditions because there was only a partial separation of the proteins under study. [ABSTRACT FROM AUTHOR]

The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH(CH)COOH, where x = 6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants ( K). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K value measured in negative ion mode and using sampling conditions that minimize in-source dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values. [ABSTRACT FROM AUTHOR]

Abstract: For BSA and β-lactoglobulin adsorption to hydrophobic interaction chromatography (HIC) stationary phases leads to conformational changes. In order to study the enthalpy (ΔH ads), entropy (ΔS ads), free energy (ΔG ads) and heat capacity (Δc p,ads) changes associated with adsorption we evaluated chromatographic data by the non-linear van’t Hoff model. Additionally, we performed isothermal titration calorimetry (ITC) experiments. van’t Hoff analysis revealed that a temperature raise from 278 to 308K increasingly favoured adsorption seen by a decrease of ΔG ads from −12.9 to −20.5kJ/mol for BSA and from −6.6 to −13.2kJ/mol for β-lactoglobulin. Δc p,ads values were positive at 1.2m (NH4)2SO4 and negative at 0.7m (NH4)2SO4. Positive Δc p,ads values imply hydration of apolar groups and protein unfolding. These results further corroborate conformational changes upon adsorption and their dependence on mobile phase (NH4)2SO4 concentration. ITC measurements showed that ΔH ads is dependent on surface coverage already at very low loadings. Discrepancies between ΔH ads determined by van’t Hoff analysis and ITC were observed. We explain this with protein conformational changes upon adsorption which are not accounted for by van’t Hoff analysis. [Copyright &y& Elsevier]

The sequential deposition of pectin and protein – bovine serum albumin (BSA), β-lactoglobulin (BLG) and gelatin – to form multilayer structures was examined by Fourier transform infrared-attenuated total reflection spectroscopy (FTIR-ATR) and a quartz crystal microbalance with dissipation monitoring (QCMD). With each layer deposited there was a progressive increase in mass deposited, with a more substantial deposition of protein. Pectin deposition led to a relatively hydrated, open structure which permitted binding of protein within the layer when the biopolymers carried an opposite net charge. On increasing the pH, disassembly of the structures occurred within the vicinity of the isoelectric point of the globular proteins. No disassembly was observed for the pectin/gelatin multilayer. When a globular protein was substituted for a poly-l-lysine layer in a pectin/poly-l-lysine multilayer it was displaced by the subsequent deposition of a poly-l-lysine layer, the more highly charged polycation displacing the relatively low charged polyampholyte. The pectin/poly-l-lysine/protein multilayers remained intact upon titration to pH 8.0. [Copyright &y& Elsevier]

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Abstract: The interaction between the algal polysaccharide furcellaran and the globular proteins, bovine serum albumin and β-lactoglobulin was examined as a function of pH using potentiometric and turbidimetric titration and photon correlation spectroscopy. On decreasing pH, the furcellaran first formed a soluble complex with the globular proteins at a pHc, which showed a maximum in its dependence on ionic strength. On further decrease in pH, the onset of a more substantial aggregation, as indicated by a marked increase in turbidity occurred in the vicinity of the isoelectric point of the protein. Between these pH’s the protein/furcellaran complex had a characteristic average size which was larger than the isolated furcellaran chain in solution. Complexation occurred when the protein carried an average net charge of the same sign as the furcellaran. [Copyright &y& Elsevier]

Sedimentation field-flow fractionation (SdFFF) was applied in order to characterize particle sizes of β-lactoglobulin aggregates induced by Ca2+ or Zn2+. Aggregation induced by Zn2+ was faster than that induced by Ca2+. Effects of Zn2+ and β-lactoglobulin concentrations, as well as contact time, on the aggregation of β-lactoglobulin were examined. All factors exhibited a combined effect on the size of aggregates, whereby larger aggregates were obtained at increased concentrations of Zn2+ and β-lactoglobulin. At fixed concentrations of 2% (w/v) β-lactoglobulin and 10 mM Zn2+, the particle size of the aggregates increased from 0.19 μm (at 15 min) to 0.38 μm (at 2880 min). Further, a hyphenated technique of SdFFF and inductively coupled plasma–optical emission spectrometry (ICP–OES) was used to examine whether intermolecular ionic bridges take part in salt-induced β-lactoglobulin aggregation. With SdFFF–ICP–OES, protein–cation–protein cross-linkages were observed for β-lactoglobulin aggregation induced by Zn2+, but not for that induced by Ca2+. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Reindeer β-lactoglobulin (βLG) belongs to the lipocalin superfamily. Its DNA and protein sequences have been determined and showed that it had nine residue changes from bovine βLG. Reindeer βLG, the structure of which was finally determined at 2.1 Å resolution in space group P1, crystallized in a unit cell that is both P2-like and P21-like owing to the presence of an almost perfect (but noncrystallographic) body-centring vector. The non-body-centred data could only be observed using a very bright synchrotron beam and a novel refinement strategy was adopted to enable us to use the weak h + k + l = 2 n + 1 reflections. [ABSTRACT FROM AUTHOR]

Milk protein polymorphism genetic variants of bovine milk proteins Alfas1-CN-casein, Kapa-CN-casein, Beta-LG-lactoglobulin and polypeptide hormone prolactin (PRL) were studied in two native cattle breeds -- Lithuanian Light Grey and Lithuanian White Backed and in two modern cattle breeds -- Lithuanian Black and White and Lithuanian Red -- by polymerase chain reaction (PCR). The aim of this study was to investigate the distribution of cattle genetic markers and to evaluate them as potential markers of milk performance traits in Lithuanian dairy cattle breeds. Two different alleles of polypeptidic hormone PRL were detected in four Lithuanian cattle breeds. The PRL gene A allele was found with a higher frequency in Lithuanian White Backed cattle breed and it was the least frequent in Lithuanian Red cattle breed. Three genotypes were identified for polypeptidic hormone PRL (AA, AB and BB) in Lithuanian breeds. Prolactin gene AA genotype was the most frequent in all investigated breeds. A higher influence of PRL gene was evaluated for milk fat percentage (12.12%, p < 0.001). Lithuanian dairy cattle breeds were investigated for three milk protein systems and eight 8 different milk protein types. According to research results, the B types of Alphas1-casein were found as predominant in all four breeds studied. The most common A and B types of milk protein Kapa-CN were found at a high frequency in all cattle breeds. In Lithuanian Red, important B types were found at a higher frequency. The obtained results for three milk protein systems loci showed higher influence of Kapa-casein gene on milk protein percentage (5.9%, P<0.001) All Lithuanian dairy cattle breeds showed a high frequency of whey protein Beta-lactoglobulin B types, the highest being in Lithuanian Red breed and the lowest in Lithuanian Light Grey. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the interaction between PPIX and β-lactoglobulin (β-lg) as a function of the pH of the solution. β-lg is a small globular protein (MW ≈18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with β-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and β-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of β-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since β-lg has been shown to be stable within the range of pH adopted in our experiments (5.0–9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior β-barrel however an alternative (or concurrent) binding site may still be present. [Copyright &y& Elsevier]

Milk yield, composition and manufacturing properties are related to milk protein polymorphism genetic variants of bovine milk proteins AlfaS1-casein, Kappa-casein and Beta- lactoglobulin. Three hundred ninety four blood samples of Lithuanian dairy breeds of unrelated cows were investigated (109 LWB, 168 LR, 68 LLG ir 49 LBW). Milk protein genes were identified by polymerase chain reaction (PCR) and RFLP method. It was assessed that BB genotype of milk protein Kappa-casein locus affected major milk fat (4.50±0.5 %) and protein (3.47 ±0.04 %) averages; whereas Kappa- casein BE genotype could be characterized by higher milk yield average (5776±27 kg). The whey protein BB genotype of Beta-laktoglobulin locus had influenced major milk fat (4.67±0.01%) average. Alfas1- casein BB genotype affected higher milk yield average (5242±14 kg), whereas Alfas1-casein CC genotype was superior in protein average (3.64±0.09 %). In our study statistically higher influence of Kappa-casein gene was estimated for milk protein percentage (5.9%, P<0.001). AlfaS1- Kappa haplotypes were associated with the highest protein percentage (2.2%, P<0.001) and lowest fat, kg (0.4%, P<0.001) in bovine milk. Further, in studied Lithuanian dairy cattle population milk protein AlfaS1- Kappa casein haplotypes, BC haplotypes had highest effect on average milk protein percentage and AB haplotype was associated with average milk yield, kg. The identification of milk protein genes could be an economically important selection criteria for dairy herds designated for industrial milk production. Moreover, milk protein polymorphism can be used as selection criteria and informative molecular markers for yield, composition and technological properties of milk in cattle selection programs. [ABSTRACT FROM AUTHOR]