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ABSTRACT: Cow milk allergy is a common phenomenon experienced in early childhood (

Cow milk allergy is a common phenomenon experienced in early childhood (<5 yr of age) with an average occurrence rate of roughly 2.5%. The most prevalent allergen in cow milk is believed to be β-LG. The objective of this study was to evaluate the use of hydrophobic supercritical CO 2 (ScCO 2) to modify the chemical structure β-LG, thus impairing its recognition by antibodies. Whole milk powder (WMP) was selected because of its closest compositional resemblance to bovine fluid milk and its applications in reconstitution and in the beverage (infant, toddler, and adult), confectionary, bakery, and meat industries. For this study, WMP was treated with food-grade CO 2 at temperatures of 50, 63, and 75 ° C under operating pressures of 100, 150, 200, 250, and 300 bar. Proteins in WMP were examined using SDS-PAGE, western blot, and ELISA. Orbitrap Fusion liquid chromatography-tandem MS (LC-MS/MS) and periodic staining was performed to confirm post-translational modifications in β-LG. Functional properties of WMP before and after treatment were assessed by its solubility index, oil holding capacity, emulsion capacity and stability, zeta potential, particle size, and color analysis. SDS-PAGE of treated samples yielded fuzzy bands (variable mobility of molecules due to different molecular weights results in ill-defined bands) indicative of an increase in molecular weight, presumably due to chemical change in the protein, and demonstrated a maximum of 71.13 ± 0.29% decrease in the band intensity of β-LG under treatment conditions of 75 ° C/300 bar for 30 min. These changes were small with samples treated with heat only. Lighter, diffused bands were observed using western blot analysis. The ELISA tests proved that ScCO 2 treatment specifically and significantly affected the antigenicity of β-LG with a reduction of 42.9 ± 2.83% and 54.75 ± 2.43% at 63 ° C/200 bar and 75 ° C/300 bar, respectively. Orbitrap fusion detected the presence of fatty acids and sugar moieties bound to β-LG and the latter was confirmed by periodic staining. Functional properties of ScCO 2 -treated milk powder yielded a decrease in solubility index and an increase in emulsion capacity of WMP was observed under ScCO 2 treatment at 75 ° C/300 bar, with small and insignificant changes at other treatments producing a decrease in antigenicity. Color changes were small for most samples, except at 63 ° C/200 bar, where a significant increase in yellowness was observed. Zeta potential and particle size measurements indicated that most changes were temperature driven. This study demonstrates 2 approaches to mitigate β-LG antigenicity via fatty acid binding and lactosylation using hydrophobic ScCO 2. [ABSTRACT FROM AUTHOR]

The aim was to determine the genetic variation in the CSN1S1, CSN2, CSN1S2, CSN3 and LGB genes in Blanco Orejinegro cattle. 419 animals from 15 herds were genotyped with GGP Bovine 150 K (n= 70) and 50 K (n= 349) chips. Information was obtained from 43 SNPs in the mentioned genes and protein variants *B, *C and *D of aS1-CN; *A1, *A2, *B, *H2 and *F of ß-CN; *A and *D of αS2-CN, *A, *A1, *B, *I and *H of κ-CN and *A, *B, *C, *D, *E, *F and *H of ß-LG were reconstructed. Allele and genotypic frequencies were estimated for SNPs and for protein variants; Hardy-Weinberg equilibrium and FST values were evaluated for each of the SNPs under different structuring criteria. LD values and haplotypic frequencies were estimated for caseins. The most frequent variants were CSN1S1*B (0.804), CSN2*A2 (0.509), CSN1S2*A (0.997), CSN3*A (0.679) and ß-LG*B (0.657). None of the variants showed deviations from HWE, but the CSN2*A2 allele showed a slight increasing trend over time. The FST values were low (0.035) regardless of the structuring criteria. Twenty-eight CSN1S1-CSN2-CSN1S2-CSN3 haplotypes were found, 22 of them with frequencies <5%; the three most frequent were BB-A1A2-AA-AA-AA-AA (16.6%), BB-A1A2-AA-AA-AAAB (14.1%) and BB-A2A2-AA-AA-AA (10.1%). A good potential of BON cattle to produce high quality milk with functional value was reported. [ABSTRACT FROM AUTHOR]

The aim was to determine the genetic variation in the CSN1S1, CSN2, CSN1S2, CSN3 and LGB genes in Blanco Orejinegro cattle. 419 animals from 15 herds were genotyped with GGP Bovine 150 K (n= 70) and 50 K (n= 349) chips. Information was obtained from 43 SNPs in the mentioned genes and protein variants *B, *C and *D of αS1-CN; *A1, *A2, *B, *H2 and *F of β-CN; *A and *D of αS2-CN, *A, *A1, *B, *I and *H of κ-CN and *A, *B, *C, *D, *E, *F and *H of β-LG were reconstructed. Allele and genotypic frequencies were estimated for SNPs and for protein variants; Hardy-Weinberg equilibrium and FST values were evaluated for each of the SNPs under different structuring criteria. LD values and haplotypic frequencies were estimated for caseins. The most frequent variants were CSN1S1*B (0.804), CSN2*A2 (0.509), CSN1S2*A (0.997), CSN3*A (0.679) and β-LG*B (0.657). None of the variants showed deviations from HWE, but the CSN2*A2 allele showed a slight increasing trend over time. The FST values were low (0.035) regardless of the structuring criteria. Twenty-eight CSN1S1-CSN2-CSN1S2-CSN3 haplotypes were found, 22 of them with frequencies

Milk protein genes are associated with milk yield and composition in dairy animals. The present study aimed to identify milk protein genes (CSN1S1, CSN2, CSN3, and BLG) genetic variants and their association with milk yield in Sahiwal cattle and Nili-Ravi buffaloes. One hundred animals from each species were selected to collect blood samples and milk production records. Primers were designed for these milk protein genes for PCR amplification. Sequencing of resultant PCR products revealed a higher number of SNPs (13 vs. 7, 5 vs. 1, and 6 vs. 2) in Sahiwal as compared to Nili-Ravi animals in CSN1S1, CSN2, and CSN3 genes, respectively. However, a single SNP was observed in BLG gene of both species. Association analysis revealed that one SNP in BLG gene of Nili-Ravi was associated (p < 0.05) with 305-day milk yield. Two SNPs at CSN1S1 gene in Sahiwal were associated with dry-period. Similarly, one SNP at CSN1S1 and two SNPs at CSN3 gene showed significant association (p < 0.05) with average calving-interval in Sahiwal while two SNPs in CSN1S1 gene were associated (p < 0.05) with this trait in Nili-Ravi. These SNPs could be helpful as candidate variants for marker-assisted selection in cattle and buffaloes for improvement of lactation performance. [ABSTRACT FROM AUTHOR]

Background: In Spain, IgE-mediated cow's milk protein allergy (CMPA) affects approximately 0.69% of infants. Molecular diagnosis may be useful for monitoring natural spontaneous tolerance development in CMPA. The aim of this study was to retrospectively analyse a cohort of paediatric patients with IgE-mediated CMPA who were avoiding milk products awaiting natural tolerance and determine the relationship between disease persistence and major cow's milk allergens. Methods: A retrospective chart review of 200 patients diagnosed with IgE-mediated CMPA between 2011 and 2020 was conducted. Patients strictly avoided milk products until an oral food challenge was performed. The main outcome was the introduction of liquid milk following a negative oral food challenge and its correlation with IgE and SPT measurements of milk components at diagnosis. Secondary outcomes included the rate of allergic reactions and anaphylaxis during the treatment period and its correlation with IgE and SPT measurements. Results: Of the 200 charts analysed, 122 patients had a negative oral food challenge to milk (61.0%) (95% confidence interval (CI): 54.1–67.5) following a period of strict avoidance of milk. Higher levels of component-specific IgE, especially casein, were associated with failure in the oral food challenge (p = 0.02). Allergic reactions were experienced by 106 children (53%), of which 34 (17%; 95% CI: 12.4–22.8) had anaphylactic reactions. The risk of anaphylaxis was not predicted by raised IgE levels. Conclusions: While a large proportion of children acquired natural tolerance to cow's milk following a period of strict avoidance, IgE-mediated CMPA persisted in many children. Casein IgE levels at diagnosis were raised in those who failed to achieve natural tolerance. Allergic reactions to milk, including anaphylaxis, occurred commonly, but this was not predicted by raised IgE levels or SPT measurements. [ABSTRACT FROM AUTHOR]

We studied the genetic polymorphism of beta-lactoglobulin (β-Lg) whey protein in Gangatiri zebu cows for this Research Communication. The polymorphic nature of milk protein fractions and their association with milk production traits, composition and quality has attracted several efforts in evaluating the allelic distribution of protein locus as a potential dairy trait marker. Genetic variants of β-Lg have highly significant effects on casein number (B > A) and protein recovery (B > A) and also determine the yield of cheese dry matter (B > A). Molecular techniques of polyacrylamide gel electrophoresis and high-resolution accurate mass-spectroscopy were applied to characterize the β-Lg protein obtained from the Gangatiri breed milk. Sequence analysis of β-Lg showed the presence of variant B having UniProt database accession number P02754, coded on the PAEP gene. Our study can provide reference and guidance for the selection of superior milk (having β-LgB) from this indigenous breed that could potentially give a good yield of β-Lg for industrial applications. [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]

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat‐induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti‐fibrillogenic activity. Thus, the anti‐fibrillogenic effect of a series of Zr phthalocyanines with different out‐of‐plane coordinated ligands, namely positively charged (PcZrLys2), negatively charged (PcZrCitr2), and group able to form disulfide bridges (PcZrS2), on the heat‐induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2) to be 10.6 ± 3.6 and 7.3 ± 1.2 μM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2, thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti‐amyloidogenic agent suppressing heat‐induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry. [ABSTRACT FROM AUTHOR]

The food industry is constantly in search of innovative new products to meet consumer needs and expectations. As a result of consumer awareness and interest in nutritious and healthy foods, there is an increasing trend towards the functional foods including whey proteins. The aim of this review is to cover the properties and health benefits of whey proteins, which are accepted as value-added components and whose importance in their valorization has been recently recognized. Whey proteins have a high protein efficiency ratio, net protein utilization and biological value compared to all other protein sources. Whey proteins are used in a variety of products such as infant formulas, food supplements, sports bars and beverages, due to the various factors and compounds they contain that can improve health and prevent disease. Whey protein is a high-quality protein that contains all the essential amino acids needed daily by the body. The components of whey such as beta-lactoglobulin (β-LG), alpha-lactalbumin (α-LA), immunoglobulins, bovine serum albumin, lactoferrin, lactoperoxidase, and glycomacropeptide can be used in promotion of health. Most of these components have broad spectrum antimicrobial and immune-enhancing properties. Whey proteins have various functionalities on human health. They improve antioxidative activity, help protect against cancer and cardiovascular disease and enhance bone health and performance of physically active individuals. Whey proteins are potent, promise and active components, which have many various nutritional and therapeutic benefits. Consequently, whey proteins have been the subject of increasing commercial interest in the production and development of health-promoting functional foods. Thus, it is important to know the properties and health benefits of whey proteins well. In this context, this review offers a perspective to guide the reader about the importance and health benefits of whey proteins. [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]

Bovine β-lactoglobulin was crystallized from 3 M NaCl buffered at pH 3.8 with sodium citrate as thick hexagonal prisms of greater than 1 mm in edge length. Analyses of the X-ray diffraction intensities using three different current algorithms were unanimous in specifying the space group to be P6322, with unit-cell dimensions a = b = 75.47, c = 140.79 Å. No progress could be made, however, towards an acceptable solution by molecular replacement using this symmetry. In the end, it was found that the true space group was C2221, a subgroup of P6322, with a = 65.89, b = 114.12, c = 140.51 Å, with the apparent 622 symmetry arising from an unusual threefold or tritohedral twinning. An assembly based on a model of the protein in another crystal form (PDB entry 1beb) containing three molecules in the asymmetric unit was refined to 2.3 Å resolution with a final R factor of 0.23 and Rfree of 0.26. NCS restraints were maintained throughout. For the most part, the molecules found in this crystal form are virtually the same as in PDB entry 1beb, although there are numerous local variations, particularly in loop elements, rotamer conformation differences and some alterations, including additions, at the termini.

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]

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]

• Hydroxychalcone (HC) is naturally occurring molecule having clinical use. • HC derivatives differing in polarity of the substituents have been synthesized. • Aggregation of β-lg was studied by spectroscopic, imaging, and theoretical tools. • Synthesized HC promote and modulates morphology of β-lg aggregates. • HC with polar substituents are less efficient than unsubstituted HC. Hydroxychalcones are naturally occurring compounds having biological relevance. This work demonstrates the synthesis of some chalcones having hydrophobic and polar groups and the investigation of their effect on the stability of a model protein beta-lactoglobulin (β-lg). An easy and simple method (Simon-Smith) was utilized to synthesize the four chalcone compounds. The binding of the chalcone molecules to the protein destabilized the protein structure and subsequently exposed the hydrophobic cores resulting in the aggregation of β-lg under thermal conditions. Using multiple spectroscopic methods (UV-Vis, fluorescence, FT-IR), imaging techniques like TEM, and theoretical tools were used to monitor the formation of protein aggregation and to understand the underlying mechanism. Exposures of hydrophobic amino acid residues in the aqueous medium by binding of the chalcones are responsible for protein aggregation through protein-protein interactions. It is sensitive to the polarity of the hydroxychalcones. In this process, polar group-containing hydroxychalcones are less effective, and unsubstituted hydroxychalcones are most efficient in promoting the amyloid fibrillation of β-lg. [Display omitted] [ABSTRACT FROM AUTHOR]

Soft food gels are of high interest for food innovation that can cater to the specific needs of various populations. This study explores the innovative development of soft beta-lactoglobulin (β-LG) gels through a green process that combines heat treatment and transglutaminase (TGase)-catalyzed crosslinking at a moderate pH. The optimal procedure involves heating β-LG solutions, pH 7.5 at 80 °C for 30 min to facilitate protein unfolding and aggregation, followed by TGase catalysis at 50 U TGase/g β-LG. This process produced soft and moist β-LG gels at a low concentration of 5% (w/v). The softness of β-LG gels was characterized through rheological measurements, suggesting a low storage modulus and high frequency dependence. Morphological observations of β-LG gels using TEM showed a loosely arranged network. SDS-PAGE and FTIR analyses indicated the formation of covalent disulfide bonds and isopeptide bonds in β-LG gels through the heat and TGase treatments. The potential of the novel β-LG gels as traditional agar gel substitutes was comparatively analyzed on their textural properties. Both gels showed distinctive jelly-like characteristics, whereas β-LG gels exhibited superior softness and reduced brittleness, positioning them as viable protein-based alternatives to agar gels for various applications in both food and nonfood sectors. [Display omitted] • Novel soft food gels are formed using beta-lactoglobulin (β-LG). • β-LG gels are covalently crosslinked via disulfide and isopeptide bonds. • Transglutaminase catalysis enhances β-LG gel properties. • Desirable β-LG gel texture outperforms commercial agar gels. [ABSTRACT FROM AUTHOR]

Background: In Spain, IgE-mediated cow’s milk protein allergy (CMPA) affects approximately 0.69% of infants. Molecular diagnosis may be useful for monitoring natural spontaneous tolerance development in CMPA. The aim of this study was to retrospectively analyse a cohort of paediatric patients with IgE-mediated CMPA who were avoiding milk products awaiting natural tolerance and determine the relationship between disease persistence and major cow’s milk allergens. Methods: A retrospective chart review of 200 patients diagnosed with IgE-mediated CMPA between 2011 and 2020 was conducted. Patients strictly avoided milk products until an oral food challenge was performed. The main outcome was the introduction of liquid milk following a negative oral food challenge and its correlation with IgE and SPT measurements of milk components at diagnosis. Secondary outcomes included the rate of allergic reactions and anaphylaxis during the treatment period and its correlation with IgE and SPT measurements. Results: Of the 200 charts analysed, 122 patients had a negative oral food challenge to milk (61.0%) (95% confidence interval (CI): 54.1–67.5) following a period of strict avoidance of milk. Higher levels of component-specific IgE, especially casein, were associated with failure in the oral food challenge (p = 0.02). Allergic reactions were experienced by 106 children (53%), of which 34 (17%; 95% CI: 12.4–22.8) had anaphylactic reactions. The risk of anaphylaxis was not predicted by raised IgE levels. Conclusions: While a large proportion of children acquired natural tolerance to cow’s milk following a period of strict avoidance, IgE-mediated CMPA persisted in many children. Casein IgE levels at diagnosis were raised in those who failed to achieve natural tolerance. Allergic reactions to milk, including anaphylaxis, occurred commonly, but this was not predicted by raised IgE levels or SPT measurements.

The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. The use of milk protein polymorphisms as detectable molecular markers has been studied intensively because of their effect on the yield and processing properties of milk and its products. Thus, molecular markers are promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. Kappa-casein (CSN3) and beta-lactoglobulin (BLG) are two of the most important proteins in the milk of mammals that play a crucial role in the milk quality and coagulation, an essential process for cheese and butter. The A and B variant of k-casein and β-lactoglobulin were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis in 108 Romanian Simmental and 60 Holstein Friesian cattle.

Jin Chen,1,&ast; Min Gong,1,&ast; Zhuo Huang,1 Fang Wang,2 Yajing Wang,3 Zuquan Hu,1 Zhu Zeng,1 Yun Wang2 1Key Laboratory of Biology and Medical Engineering/Immune Cells and Antibody Engineering Research Center of Guizhou Province, School of Biology and Engineering, Guizhou Medical University, Guiyang, 550025, People’s Republic of China; 2Key Laboratory of Infectious Immune and Antibody Engineering of Guizhou Province, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, 550025, People’s Republic of China; 3The Affiliated Stomatological Hospital of Guizhou Medical University, Guizhou Medical University, Guiyang, 550025, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Zhu Zeng, Key Laboratory of Biology and Medical Engineering/Immune Cells and Antibody Engineering Research Center of Guizhou Province, School of Biology and Engineering, Guizhou Medical University, Guiyang, 550025, People’s Republic of China, Email zengzhu@gmc.edu.cn Yun Wang, Key Laboratory of Infectious Immune and Antibody Engineering of Guizhou Province, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, 550025, People’s Republic of China, Tel/Fax +86851-88174044, Email wangyun@gmc.edu.cnPurpose: Gastric injury is a major issue for long-term administration of aspirin. In this work, we tried to explore the possibility of using BLG to alleviate aspirin-induced gastric injury, because of excellent abilities of BLG in loading drug molecules.Methods: Various spectroscopic techniques and molecular docking methods were applied to investigate the interaction mechanism between BLG and aspirin. Animal experiments were performed to figure out the effects of taking aspirin-BLG on the stomach.Results: Our results demonstrate that aspirin could bind with BLG to form stable aspirin-BLG complex (the binding constant Kb= 2.051 × 103 M− 1). The formation process is endothermic (∆H> 0) and the main acting force is hydrophobic force. Our data also show that the aspirin-BLG complex is formed with a higher affinity in simulated gastric fluid and could remain stable for several hours, which might arise from its special binding mode under acidic condition and the resistance of BLG to gastric digestion. Furthermore, animal models (rats with aspirin-induced gastric damage) were built. The results of animal experiments reveal that the oral administration of aspirin-BLG could cause less damage to gastric tissue, and it also hardly triggers obvious inflammatory responses.Conclusion: This study would contribute to an in-depth understanding of the interaction mechanism between BLG and aspirin. It is reasonable to believe that using BLG to bind with aspirin would be a potential way to alleviate the aspirin-induced gastric injury.Graphical Abstract: Keywords: beta-lactoglobulin, aspirin, gastric injury, static quenching, molecular docking

ABSTRACT: β-Lactoglobulin (β-LG) is a major milk protein, making up more than 53% of the total whey proteins, and is seen as a valuable ingredient in food processing because of its high essential amino acid content and diverse functional applications. The Maillard reaction can occur during the storage and processing of food and generate various beneficial effects, including anti-allergenicity, antioxidant, and immunomodulatory effects. The addition of an β-LG-lactose conjugate (LGL) produced by the Maillard reaction was shown to have a strong immune-enhancing effect, increasing both nitric oxide generation and cytokine expression through activation of RAW 264.7 cells, even after in vitro digestion. Furthermore, daily LGL administration resulted in the upregulation of several immune markers in a cyclophosphamide-induced immunosuppressive mouse model, indicating that this treatment stimulates multiple immune cells, including macrophages, natural killer cells, and lymphocytes, enhancing the proliferation and activation of both the innate and adaptive immune responses. Taken together, these findings indicate that consuming LGL on a regular basis can improve immunity by increasing the natural production of various immune cells.

Milk allergy is a global health issue, which affects the quality of life and can even be life threatening. According to epidemiological investigation, 2%–6% of infants are sensitive to beta-lactoglobulin (β-LG) in milk. Avoiding allergens is still the first-rank option for vulnerable group because immunization therapy is hardly available. Therefore, exploring the allergic mechanism of β-LG and applying appropriate antiallergic treatments are the key to solving the problem of milk allergy. This review describes the occurrence of β-LG, including the type, structure, epitopes of β-LG, the performance of gastrointestinal digestion, the mechanism and allergy consequence of β-LG allergic responses. Then, various methods to reduce the sensitization of β-LG were summarized, including covalent and non-covalent interactions, non-thermal processing technologies (fermentation, irradiation, pulsed electric field, ultrasound), thermal processing technology (microwave), and combination therapies. Key findings and conclusions : β-LG allergy is an immediate type I hypersensitivity reaction involving immunoglobulin (IgE) dependent antibody responses that cause a variety of clinical symptoms, such as asthma, eczema, gastrointestinal disorders, and anaphylactic shock in severe cases. To reduce the allergenicity of the β-LG, it is necessary to adopt novel processing strategies with higher efficiency. This review covers the covalent or non-covalent binding of polyphenols, polysaccharides, metal ions and nanoparticles to β-LG, as well as emerging technologies, and finds that structural modifications and conformational epitope changes of β-LG are associated with immunomodulatory properties. The combination of various approaches is a potential new way to reduce β-LG hypersensitivity. • Specificity between IgE antibodies and epitopes is indicator of β-LG allergy. • Any technique that alters the β-LG epitopes can reduce the antigenicity of β-LG. • Dietary molecules, metal ions and nanoparticles bind to β-LG by covalent interaction. • Preparation of β-LG conjugates regulate the antigenicity and digestibility of β-LG. • Novel processing techniques for β-LG to alleviate allergic symptoms are reviewed. [ABSTRACT FROM AUTHOR]

The goal of the paper was to identify b - lactoglobulin gene polymorphism in bulls. The b - lactoglobulin (LGB) is expressed in milk and is important in the evaluation of milk production potential and butterfat and protein content. LGB is localized on bovine chromosome 11. The AA genotype of LGB is associated with higher milk yield, the BB genotype with higher fat and casein content and is more desirable for cheese making. The material involved 46 bulls (Slovak spotted breed – 41 bulls, Pinzgau breed – 3 bulls and Holstein breed – 2 bulls). Bovine genomic DNA was isolated from sperm using commercial kit NucleoSpin Tissue and used in order to estimate LGB genotypes by means of PCR RFLP method and high resolution melting analysis (HRMA). In the population of Slovak spotted breed we detected all genotypes AA, AB and BB with frequency 0.3415, 0.4390 and 0.2195, subsequently. In Pinzgau breed was detected homozygote genotypes AA and BB with frequency 0.3333 and 0.6667. In Holstein breed was observed only heterozygote genotype AB with frequency 1.

Introduction: Hypoallergenic formulas prepared from hydrolyzed cow's milk proteins are often used for the management of cow's milk allergy (CMA) in infants. In this study, both in vitro assays and an in vivo mouse model for CMA were used to assess the sensitizing and allergenic potential of a newly developed, extensive whey hydrolysate (eWH). Methods: Gel permeation chromatography was used to characterize the molecular weight distribution of the peptides. Residual antigenicity was measured using a beta-lactoglobulin ELISA as well as with immunoblotting using anti-beta-lactoglobulin (BLG) and anti-alpha-lactalbumin antibodies. In vitro residual allergenicity was assessed using huFcεRIα-RBL-2H3 cells sensitized with anti-bovine BLG human IgE. In vivo sensitizing and allergenic potential was assessed in a CMA mouse model by measuring the acute allergic skin response, anaphylactic shock score, body temperature, serum mMCP-1, whey-specific IgE, and cytokines. Results: There was no in vitro residual antigenicity and allergenicity observed of the eWH. Mice sensitized with eWH showed no acute allergic skin reaction after challenge with whey, confirmed by an absence of whey-specific IgE and anaphylactic symptoms and decrease in body temperature and mMCP-1 levels. Conclusions: Results from our in vitro and in vivo translational approach to assess sensitization capacity and residual allergenicity indicate that the newly developed eWH is safe for use in CMA infants. This was subsequently confirmed in a clinical study in which this eWH was tolerated by more than 90% (with 95% confidence) of infants or children with confirmed CMA. [ABSTRACT FROM AUTHOR]

Background: The immunopathogenesis of cow's milk protein allergy (CMPA) is based on different mechanisms related to immune recognition of protein epitopes, which are affected by industrial processing. Purpose: The purpose of this WAO DRACMA paper is to: (i) give a comprehensive overview of milk protein allergens, (ii) to review their immunogenicity and allergenicity in the context of industrial processing, and (iii) to review the milk-related immune mechanisms triggering IgE-mediated immediate type hypersensitivity reactions, mixed reactions and non-IgE mediated hypersensitivities. Results: The main cow’s milk allergens – α-lactalbumin, β-lactoglobulin, serum albumin, caseins, bovine serum albumins, and others – may determine allergic reactions through a range of mechanisms. All marketed milk and milk products have undergone industrial processing that involves heating, filtration, and defatting. Milk processing results in structural changes of immunomodulatory proteins, leads to a loss of lipophilic compounds in the matrix, and hence to a higher allergenicity of industrially processed milk products. Thereby, the tolerogenic capacity of raw farm milk, associated with the whey proteins α-lactalbumin and β-lactoglobulin and their lipophilic ligands, is lost. Conclusion: The spectrum of immunopathogenic mechanisms underlying cow's milk allergy (CMA) is wide. Unprocessed, fresh cow's milk, like human breast milk, contains various tolerogenic factors that are impaired by industrial processing. Further studies focusing on the immunological consequences of milk processing are warranted to understand on a molecular basis to what extent processing procedures make single milk compounds into allergens.

Mouse models of food allergy have contributed to our understanding of various aspects of the disease, including susceptibilities, symptom spectra, cellular mechanisms, and therapeutic approaches. Previously, we used a mouse model of non-anaphylactic cow’s milk allergy (CMA) and investigated sex- and strain-dependent differences in immunological, neurological, and behavioral sequelae. We showed that male C57BL/6J mice sensitized to a bovine whey protein, β-lactoglobulin (BLG; Bos d 5), exhibited anxiety- and depression-like behavior upon acute allergen challenge. Systemic levels of BLG-specific immunoglobulins, cytokines and chemokines were also elevated in the sensitized mice. Furthermore, neuroinflammation and intestinal dysbiosis were evident as the possible causes of the altered behavior. To assess whether frequent allergen exposure influences CMA-associated pathologies over an extended period in this subclinical model, we placed BLG-sensitized mice on a whey protein (WP)-containing or whey-free control (CTL) diet for 3 months. As expected, allergen-specific IgE was significantly elevated in the plasma after completing the 5-week sensitization phase. However, the IgE levels declined in both diet groups after 3 months. In contrast, allergen-specific IgG1 stayed elevated in sensitized mice with the CTL diet, and the WP diet to a lesser extent. Interestingly, BLG-sensitized mice on the WP diet exhibited anxiety-like behavior and a trend toward spatial memory decline compared to the sham or the sensitized mice on the CTL diet. Moreover, increased immunoreactivities for GFAP and Iba1 and elevated levels of CXCL13 and CCL12, the chemokines involved in central leukocyte recruitment and other neurological diseases, were also observed in the brain. We demonstrated that sensitization to the whey protein, particularly with continuous allergen exposure, resulted in persistent neuroinflammation and associated behavioral changes despite lowered allergen-specific immunoglobulin levels. These results suggested that continuous consumption of the offending allergen may lead to adverse consequences in the brain even after desensitization.

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.

The aim of the present paper was to summarize the gene polymorphisms of beta-lactoglobulin (BLG) gene and its effects on milk yield in 1840 genotyped Indian dairy cows reported in 17 published studies. The meta-analysis was undertaken using gene frequencies of individual studies under random effects model, whereas for association analysis of genotypes with milk yield, standardized mean differences (SMDs) along with 95% confidence interval (CI) were obtained under four genetic models such as additive (AA vs. BB), dominant (AA+AB vs. BB), completely over dominant (AA+BB vs. AB) and recessive (AA vs. AB+BB). The heterogeneity index (I2) was used to determine heterogeneity between studies. The results of meta-analysis suggested that the pooled allelic frequency of allele A was subsidiary as 0.29 (95% CI 0.24, 0.33, I2 = 88.54%) in targeted population, and also, it was non-significantly (P > 0.05) different between Bos indicus (0.28) and Bos taurus/cross cows (0.30). Egger's test indicated no risk of publication bias (P > 0.05). The results also revealed that BLG gene variants have non-significant (P > 0.05) association with milk yield under all genetic models. Although positive effects of SMDs under some models were observed, however, they failed to meet statistical significance (P > 0.05) due to high heterogeneity between studies which lead to conclusion of only uncertain influences of SNP genotypes with milk yield. It was concluded that BLG markers may not be beneficial for improving milk yield in Indian dairy cows. However, it is suggested that the revalidation of the present results should be done by using more number of studies. [ABSTRACT FROM AUTHOR]

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on beta‐lactoglobulin (BLG) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF (≥ 90% w/w dry matter protein) consists of BLG as primary component (≥ 90% of total protein), which is equivalent to BLG present in bovine milk and whey protein isolate (WPI). The NF is produced from bovine whey by crystallisation under acidic or neutral conditions. The NF is proposed to be used as a food ingredient in isotonic and sport drinks, whey powder and milk‐based drinks and similar products, and in food for special medical purposes as defined in Regulation (EU) No 609/2013. The target population is the general population. The highest daily intake of the NF was estimated for children of 3 to < 10 years of age as 667 mg/kg body weight (bw) per day. The NF presents proximate composition and content of essential amino acids similar to those in WPI. The Panel notes that the highest mean and highest 95th percentile daily protein intakes from the NF are below the protein population reference intakes for all population groups. Although a tolerable upper intake level has not been derived for protein, the protein intake from the NF may nevertheless further contribute to an already high dietary protein intake in Europe. The exposure to the reported minerals does not raise concerns. The Panel considers that the consumption of the NF is not nutritionally disadvantageous. No genotoxic concerns were identified from the standard in vitro test battery. No adverse effects were observed in the subchronic toxicity study, up to the highest dose tested, i.e. 1,000 mg NF/kg bw per day. The Panel concludes that the NF is safe under the proposed conditions of use. [ABSTRACT FROM AUTHOR]

The safety of Lacprodan® BLG, a whey-based protein, was evaluated with respect to genotoxicity and sub-chronic toxicity according to regulatory requirements. Lacprodan® BLG did not show any mutagenic potential in a bacterial reverse mutation assay or any clastogenic or aneugenic potential in an in vitro micronucleus assay performed in human lymphocytes. In a sub-chronic toxicity study, groups of 10 male and 10 female Wistar rats received the test item orally by gavage for 90 days at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group, also including 10 male and 10 female rats, received sterile water, as vehicle. No treatment-related clinical observations or toxicological effects on body or organ weights, food consumption, ophthalmic effects, hematology, clinical chemistry, fertility, urinalysis, or pathology were identified. Therefore, the no-observed-adverse-effect level (NOAEL) for Lacprodan® BLG in the 90-day toxicity study was established as 1000 mg/kg bw/day, corresponding to the highest dose level administered.

The symptoms of food allergies vary significantly between individuals, likely due to genetic determinants. In humans, allergy development is initiated by antigen-presenting cells via class II human leukocyte antigen (HLA-II). The HLA-II gene is highly polymorphic, and its allelic variance is thought to influence the susceptibility of individuals to a particular allergen. However, whether antigen presentation by different HLA-II variants contributes to symptom variation is not clear. We hypothesized that HLA-II allelic variance affects symptom phenotypes, including immediate physical reactions and delayed behavioral changes, in individuals with food hypersensitivity. To test our hypothesis, male and female mice of three transgenic strains expressing an HLA-II variant, DR3, DR15, or DQ8, were used to establish a cow's milk allergy model. Mice were sensitized to a bovine whey allergen, β-lactoglobulin (BLG; Bos d 5), weekly for 5 weeks, followed by an acute oral allergen challenge. At 30 min post-challenge, BLG-sensitized DR3 mice showed moderate to severe anaphylaxis resulting in perioral redness, swelling, and death. In contrast, DQ8 and DR15 mice were generally asymptomatic. The production of allergen-specific immunoglobulins was also HLA- and sex-dependent. Both male and female DR3 and female DR15 mice significantly increased BLG-specific IgE production, while robust elevation in BLG-specific IgG1 was observed in sensitized DQ8 mice of both sexes and, to a lesser extent, in DR15 males. Furthermore, BLG-sensitized DR15 mice showed sex-specific behavior changes, with males exhibiting mobility changes and anxiety-like behavior and females showing spatial memory impairment. When splenocytes from transgenic mice were stimulated in vitro with BLG, phenotypes of immune cells were HLA- and sex-specific, further underscoring the influence of HLA-II on immune responses. Our results support that HLA-II alleles influence behavioral responses in addition to immune and physical reactions of food allergy, suggesting that certain HLA-II variants may predispose individuals to food-allergy-associated behavioral changes.

Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on beta‐lactoglobulin (BLG) as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF (≥ 90% w/w dry matter protein) consists of BLG as primary component (≥ 90% of total protein), which is equivalent to BLG present in bovine milk and whey protein isolate (WPI). The NF is produced from bovine whey by crystallisation under acidic or neutral conditions. The NF is proposed to be used as a food ingredient in isotonic and sport drinks, whey powder and milk‐based drinks and similar products, and in food for special medical purposes as defined in Regulation (EU) No 609/2013. The target population is the general population. The highest daily intake of the NF was estimated for children of 3 to

Background: Previously, the protective farm effect was imitated using the whey protein beta‐lactoglobulin (BLG) that is spiked with iron‐flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin‐iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. Methods: Binding of iron‐catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo‐) versus empty (apo‐) BLG and for human mast cell degranulation. BLG and Bet v 1 double‐sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin‐iron complexes as ligands. Results: One major IgE and T cell epitope were masked by catechin‐iron complexes, which impaired IgE binding of milk‐allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. Conclusion: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen‐non‐specific manner, thereby conferring immune resilience against allergic symptoms. The whey protein beta‐lactoglobulin BLG binding to micronutrients such as iron‐catechin complexes (holoBLG) was recognized less by IgE antibodies of children allergic to milk, leading to reduced mast cell degranulation. BLG also facilitated catechin‐dependent activation of the anti‐inflammatory arylhydrocarbon receptor. In mice, oral application of a holoBLG‐based lozenge which also contained zinc and vitamin A, improved the allergic symptoms of double‐sensitized mice independent of the allergens they were challenged with. Double‐sensitized mice supplemented with the holoBLG lozenge also had reduced levels of allergen‐specific antibodies and a lower frequency of activated antigen presenting cells. [ABSTRACT FROM AUTHOR]

β-Lactoglobulin (β-LG) is a major milk protein, making up more than 53% of the total whey proteins, and is seen as a valuable ingredient in food processing because of its high essential amino acid content and diverse functional applications. The Maillard reaction can occur during the storage and processing of food and generate various beneficial effects, including anti-allergenicity, antioxidant, and immunomodulatory effects. The addition of an β-LG-lactose conjugate (LGL) produced by the Maillard reaction was shown to have a strong immune-enhancing effect, increasing both nitric oxide generation and cytokine expression through activation of RAW 264.7 cells, even after in vitro digestion. Furthermore, daily LGL administration resulted in the upregulation of several immune markers in a cyclophosphamide-induced immunosuppressive mouse model, indicating that this treatment stimulates multiple immune cells, including macrophages, natural killer cells, and lymphocytes, enhancing the proliferation and activation of both the innate and adaptive immune responses. Taken together, these findings indicate that consuming LGL on a regular basis can improve immunity by increasing the natural production of various immune cells. [Display omitted] [ABSTRACT FROM AUTHOR]

Summary: Optimum beta‐lactoglobulin/sodium alginate dry films (β‐LG/SA(S)) formed from casting solutions containing β‐LG 1.25% (w/v) and SA 1% (w/v) showed appropriate characteristics to be easily handled. SEM micrographs showed a less homogeneous microstructure of this films respect to sodium alginate dry films used as control (SA(F)). Tensile strength of 18 MPa and an elongation value of 5% were obtained for β‐LG/SA(S). DSC thermograms did not demonstrate changes in thermal properties of SA in presence of β‐LG. Studies based on fluorescence quenching by acrylamide did not show variations in the tertiary structure of β‐LG when β‐LG/SA(S) were dissolved in an aqueous environment. In addition, binding properties of the protein were similar to the native one when an alquilsulfonate compound was used as ligand. The maintenance of native binding properties of β‐LG after the films' dissolution would allow the development of new carriers for food bioactive compounds based on β‐LG/SA(S) dry films. [ABSTRACT FROM AUTHOR]

The genotyping of red-and-white cattle for the genes of kappa-casein and beta-lactoglobulin in the farm has revealed the presence of three genotypes, namely AA, AB and BB. In first-calf heifers, the frequency of the AA genotype of the kappa-casein gene was 33.10%, AB was 56.69%, BB was 10.21%. A allele frequency was 0.61%, B - 0.39%. In first-calf heifers, the frequency of the AA genotype of the beta-lactoglobulin gene was 16.20%, AB was 53.52%, BB was 30.28%. A allele frequency was 0.43%, B - 0.57%. The AB AB genotype prevailed in the population of red-and-white cows (29.23%) according to the frequency of occurrence of complex genotypes of kappa-casein and beta-lactoglobulin. In terms of milk yield, cows with genotype BB of the kappa-casein gene surpassed cows of the same age with genotypes AB and AA by 153-644 kg. In terms of the content of fat and protein mass fraction, the superiority was 0.06-0.19% (P=0.95; 0.99), and protein by 0.13-0.22% (P=0.99; 0.999). In terms of milk yield, the superiority of cows with the AA genotype of the beta-lactoglobulin gene over cows of the same age with the AB and BB genotypes was 208-413 kg (P=0.95). According to the mass fraction of fat and protein in milk, preference is given to cows with the BB genotype, whose milk contains 0.06-0.09% more fat and 0.08-0.17% protein (P=0.95; 0.99) compared with cows with AB and AA genotypes. The yield of milk fat in first-calf heifers with genotype BB of kappa-casein was also higher by 14.3 kg and 43.5 kg (P <0.05) than that in animals of the same age with genotypes AB and BB. Heifers with the AA genotype of the beta-lactoglobulin gene had the highest milk fat yield of 328.36 kg. This is 13.87 kg (P <0.05) more compared to the BB genotype. In terms of milk protein yield, the difference in kappa-casein genotypes was: BB to AA 36.9 kg (P <0.01), BB to AB 14.3 kg. [ABSTRACT FROM AUTHOR]

Trp fluorescent spectra appear as a log-normal function but are usually analyzed with λmax, full width at half maximum, and the first moment of incomplete spectra. Log-normal analyses have successfully separated fluorescence contributions from some multi-Trp proteins but deviations were observed in single Trp proteins. The possibility that disparate rotamer environments might account for these deviations was explored by moment spectral analysis of single Trp mutants spanning the sequence of tear lipocalin as a model. The analysis required full width Trp spectra. Composite spectra were constructed using log-normal analysis to derive the inaccessible blue edge, and the experimentally obtained spectra for the remainder. First moments of the composite spectra reflected the site-resolved secondary structure. Second moments were most sensitive for spectral deviations. A novel parameter, derived from the difference of the second moments of composite and simulated log-normal spectra correlated with known multiple heterogeneous rotamer conformations. Buried and restricted side chains showed the most heterogeneity. Analyses applied to other proteins further validated the method. The rotamer heterogeneity values could be rationalized by known conformational properties of Trp residues and the distribution of nearby charged groups according to the internal Stark effect. Spectral heterogeneity fits the rotamer model but does not preclude other contributing factors. Spectral moment analysis of full width Trp emission spectra is accessible to most laboratories. The calculations are informative of protein structure and can be adapted to study dynamic processes.

Beta-lactoglobulin (β-LG) is a lipocalin family member whose general function appears to be solubilizing and transport of hydrophobic molecules. Some properties such as avalability, ease of purification, and peculiar resistance to acidic environments can make β-LG as a carrier for hydrophobic and acid labile drugs for oral administration. In this protein vehicle, drug could be protected in acidic environment of stomach and then released within the basic small intestine. In this study, the potential of β-LG as a nanocarrier for oral delivery of a potent agent in colorectal cancer treatment, irinotecan, was evaluated. The nanoparticle was prepared by the physical inclusion complex method. Size, drug loading, encapsulation efficiency, and in vitro drug release at various pH values were investigated. The optimum formulation showed a narrow size distribution with an average diameter of 139.86 ± 13.75 nm and drug loading about 84.33 ± 5.03%. Based on the results obtained from docking simulation of irinotecan-complex, there are two distinct binding sites in this nanocarrier. Cytotoxicity of this nanocarrier on the HT-29 cancer cell line and AGS was measured by MTT assay. The cytotoxicity experiment showed that the drug-loaded nanocarrier was more effective than free drug. The higher release percent of drug from the β-LG complex at pH 7.4 compared to pH 1.2 indicated that the proposed nanocarrier could be introduced as a suitable nanovehicle for labile drugs in acidic medium targeted for colorectal segment.

Introduction: Hypoallergenic formulas prepared from hydrolyzed cow’s milk proteins are often used for the management of cow’s milk allergy (CMA) in infants. In this study, both in vitro assays and an in vivo mouse model for CMA were used to assess the sensitizing and allergenic potential of a newly developed, extensive whey hydrolysate (eWH). Methods: Gel permeation chromatography was used to characterize the molecular weight distribution of the peptides. Residual antigenicity was measured using a beta-lactoglobulin ELISA as well as with immunoblotting using anti-beta-lactoglobulin (BLG) and anti-alpha-lactalbumin antibodies. In vitro residual allergenicity was assessed using huFcεRIα-RBL-2H3 cells sensitized with anti-bovine BLG human IgE. In vivo sensitizing and allergenic potential was assessed in a CMA mouse model by measuring the acute allergic skin response, anaphylactic shock score, body temperature, serum mMCP-1, whey-specific IgE, and cytokines. Results: There was no in vitro residual antigenicity and allergenicity observed of the eWH. Mice sensitized with eWH showed no acute allergic skin reaction after challenge with whey, confirmed by an absence of whey-specific IgE and anaphylactic symptoms and decrease in body temperature and mMCP-1 levels. Conclusions: Results from our in vitro and in vivo translational approach to assess sensitization capacity and residual allergenicity indicate that the newly developed eWH is safe for use in CMA infants. This was subsequently confirmed in a clinical study in which this eWH was tolerated by more than 90% (with 95% confidence) of infants or children with confirmed CMA.

Site specific oral delivery of many biopharmaceutical classification system (BCS) class II and IV drugs is challenging due to their poor solubility, low permeability and degradation in the gastrointestinal tract. Whilst colloidal carriers have been used to improve the bioavailability of such drugs, most nanocarriers based drug delivery systems suffer from multiple disadvantages, including low encapsulation efficiency (liposomes, polymeric nanoparticles), complex synthesis methods (silica, silicon-based materials) and poorly understood biodegradability (inorganic nanoparticles). Herein, a novel pH responsive nanocolloids were self-assembled using natural compounds such as bovine β-lactoglobulin (BLG) and succinylated β-lactoglobulin (succ. BLG) cross-linked with epsilon poly l -lysine (BCEP and BCP), and found to possess high loading capacity, high aqueous solubility and site-specific oral delivery of a poorly soluble nutraceutical (curcumin), improving its physicochemical properties and biological activity in-vitro and ex-vivo. Our optimized synthesis formed colloids of around 200 nm which were capable of encapsulating curcumin with ~100% encapsulation efficiency and ~10% w/w drug loading. By forming nanocomplexes of curcumin with BLG and succ. BLG, the aqueous solubility of curcumin was markedly increased by ~160-fold and ~86-fold, respectively. Encapsulation with BLG increased the solubility, whereas succ. BLG prevent release of encapsulated curcumin when subjected to gastric fluids as it is resistant to breakdown on exposure to pepsin at acidic pH. In conditions mimicking the small intestine, Succ. BLG was more soluble resulting in sustained release of the encapsulated drug at pH 7.4. Additionally, crosslinking succ. BLG with E-PLL significantly enhanced curcumin's permeability in an in-vitro Caco-2 cell monolayer model compared to curcumin solution (dissolved in 1% DMSO), or non-crosslinked BLG/succ. and BLG. In a mouse-derived intestinal epithelial 3D organoid culture stimulated with IL-1β, BLG-CUR and crosslinked BCEP nanoparticles reduced the production of inflammatory cytokines and chemokines such as Tnfα and Cxcl10 more than curcumin solution or suspension while these nanoparticles were non-toxic to organoids. Overall this work demonstrates the promise of nutraceutical-based hybrid self-assembled colloidal system to protect hydrophobic drugs from harsh gastrointestinal conditions and improve their solubility, dissolution, permeability and biological activity. [ABSTRACT FROM AUTHOR]

The lipocalin beta-lactoglobulin (BLG) is a major protein compound in cow’s milk, and we detected it in cattle stable dust. BLG may be a novel player in the farm protective effect against atopic sensitization and hayfever. In previous studies, we demonstrated that only the ligand-filled holo-form of BLG prevented sensitization to itself. Here, we investigated whether holo-BLG could, in an innate manner, also protect against allergic sensitization to unrelated birch pollen allergens using a murine model. BALB/c mice were nasally pretreated four times in biweekly intervals with holo-BLG containing quercetin–iron complexes as ligands, with empty apo-BLG, or were sham-treated. Subsequently, mice were intraperitoneally sensitized two times with apo-BLG or with the unrelated birch pollen allergen apo-Bet v 1, adjuvanted with aluminum hydroxide. After subsequent systemic challenge with BLG or Bet v 1, body temperature drop was monitored by anaphylaxis imaging. Specific antibodies in serum and cytokines of BLG- and Bet v 1-stimulated splenocytes were analyzed by ELISA. Additionally, human peripheral blood mononuclear cells of pollen allergic subjects were stimulated with apo- versus holo-BLG before assessment by FACS. Prophylactic treatment with the holo-BLG resulted in protection against allergic sensitization and clinical reactivity also to Bet v 1 in an unspecific manner. Pretreatment with holo-BLG resulted in significantly lower BLG-as well as Bet v 1-specific antibodies and impaired antigen-presentation with significantly lower numbers of CD11c+MHCII+ cells expressing CD86. Pretreatment with holo-BLG also reduced the release of Th2-associated cytokines from Splenocytes in BLG-sensitized mice. Similarly, in vitro stimulation of PBMCs from birch pollen allergic subjects with holo-BLG resulted in a relative decrease of CD3+CD4+ and CD4+CRTh2 cells, but not of CD4+CD25+CD127− Treg cells, compared to apo-BLG stimulation. In conclusion, prophylactic treatment with holo-BLG protected against allergy in an antigen-specific and -unspecific manner by decreasing antigen presentation, specific antibody production and abrogating a Th2-response. Holo-BLG therefore promotes immune resilience against pollen allergens in an innate manner and may thereby contribute to the farm protective effect against atopic sensitization.

The separation of α-lactalbumin (α-LAC) and β-lactoglobulin (β-LG) by ultrafiltration (UF) is a technological challenge due to their similar molecular size. As high hydrostatic pressure (HHP) treatment generates β-LG complexes of a larger molecular size than native β-LG, it was suggested that pressurization of skim milk could improve the separation of α-LAC by UF. The strategy was effective, but the pressurization affected UF performance, α-LAC recovery and purity. Hence, the combination of HHP and UF might not be the most efficient strategy for producing an enriched α-LAC fraction, but this study generated significant scientific knowledge about HHP in separation technology. [ABSTRACT FROM AUTHOR]

The lipocalin beta-lactoglobulin (BLG) is a major protein compound in cow's milk, and we detected it in cattle stable dust. BLG may be a novel player in the farm protective effect against atopic sensitization and hayfever. In previous studies, we demonstrated that only the ligand-filled holo-form of BLG prevented sensitization to itself. Here, we investigated whether holo-BLG could, in an innate manner, also protect against allergic sensitization to unrelated birch pollen allergens using a murine model. BALB/c mice were nasally pretreated four times in biweekly intervals with holo-BLG containing quercetin–iron complexes as ligands, with empty apo-BLG, or were sham-treated. Subsequently, mice were intraperitoneally sensitized two times with apo-BLG or with the unrelated birch pollen allergen apo-Bet v 1, adjuvanted with aluminum hydroxide. After subsequent systemic challenge with BLG or Bet v 1, body temperature drop was monitored by anaphylaxis imaging. Specific antibodies in serum and cytokines of BLG- and Bet v 1-stimulated splenocytes were analyzed by ELISA. Additionally, human peripheral blood mononuclear cells of pollen allergic subjects were stimulated with apo- versus holo-BLG before assessment by FACS. Prophylactic treatment with the holo-BLG resulted in protection against allergic sensitization and clinical reactivity also to Bet v 1 in an unspecific manner. Pretreatment with holo-BLG resulted in significantly lower BLG-as well as Bet v 1-specific antibodies and impaired antigen-presentation with significantly lower numbers of CD11c+MHCII+ cells expressing CD86. Pretreatment with holo-BLG also reduced the release of Th2-associated cytokines from Splenocytes in BLG-sensitized mice. Similarly, in vitro stimulation of PBMCs from birch pollen allergic subjects with holo-BLG resulted in a relative decrease of CD3+CD4+ and CD4+CRTh2 cells, but not of CD4+CD25+CD127− Treg cells, compared to apo-BLG stimulation. In conclusion, prophylactic treatment with holo-BLG protected against allergy in an antigen-specific and -unspecific manner by decreasing antigen presentation, specific antibody production and abrogating a Th2-response. Holo-BLG therefore promotes immune resilience against pollen allergens in an innate manner and may thereby contribute to the farm protective effect against atopic sensitization. [ABSTRACT FROM AUTHOR]

New methods of genome editing open up promising opportunities for animal husbandry, including for correcting the composition of milk to improve its consumer qualities, as well as for obtaining recombinant biologically active proteins with milk. Beta-lactoglobulin (BLG) is the main serum protein in almost all mammals, except rodents and primates. Cow's milk is a complete food product containing proteins, fats, carbohydrates and minerals. But it often causes allergic reactions that seriously affect the absorption and use of nutrients in dairy products: the main allergen is β-lactoglobulin. Regulatory regions of the BLG gene in sheep, goats, and cattle are used as part of genetic constructs. The article provides a brief overview of successful experiments in the world practice on editing the beta-lactoglobulin gene in cattle and goats using genomic editing methods, in particular the CRISPR / Cas9 technology. The article reports on the creation of a ready-made set of crispr / Cas9 components in plasmid form for microinjection into the pronucleus of bovine zygotes in order to knock out the BLG gene and replace it with the gene of a biologically active target protein, as well as a well-thought-out strategy for analyzing its possible modifications. A plasmid containing 5'-and 3' - arms of homology to the bovine BLG gene was created as a replacement DNA matrix. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at the site for EagI restriction enzyme at the junction of the arms of homology. The resulting DNA matrix is designed for sitespecific integration by homologous recombination into the BLG gene using CRISPR/Cas9 technology. To select sites for double-stranded cuts (DSC), a polymorphic variant of the bull BLG gene was determined, whose sperm was used for fertilization of cow eggs in vitro. A strategy for making DSC in the BLG gene was developed and four pX330 plasmids encoding the CRISPR/Cas9 components were obtained. The pBLGcmvEGFP plasmid containing homology arms to the BLG gene and the green fluorescent protein gene under the cytomegalovirus promoter is intended for sitespecific integration by homologous recombination into the BLG gene to evaluate the effectiveness of site-specific operation of the CRISPR/Cas9 system components under in vitro conditions. [ABSTRACT FROM AUTHOR]

Bovine milk beta-lactoglobulin (BLG) is a small whey protein that is a common ingredient in many foods. Many of the properties of BLG relevant to the food industry are related to its unfolding processes induced by physical or chemical treatments. Unfolding occurs through a number of individual steps, generating transient intermediates through reversible and irreversible modifications. The rate of formation of these intermediates and of their further evolution into different structures often dictates the outcome of a given process. This report addresses the main structural features of the BLG unfolding intermediates under conditions that may facilitate or impair their formation in response to chemical or physical denaturing agents. In consideration of the short lifespan of the transient species generated upon unfolding, this review also discusses how various methodological approaches may be adapted in exploring the process-dependent structural modifications of BLG from a kinetic and/or a thermodynamic standpoint. Some of the conceptual and methodological approaches presented and discussed in this review can provide hints for improving the understanding of transient conformers formation by proteins present in other food systems, as well as when other physical or chemical denaturing agents are acting on proteins much different from BLG in complex food systems.

Mast cells (MCs) are the major effector cells of allergic responses and reside throughout the body, including in the brain and meninges. Previously, we showed in a mouse model of subclinical cow’s milk allergy that brain MC numbers were elevated in sensitized mice. However, the neurophysiological consequences of intracranial MC accumulation and activation are unclear. We hypothesized that centrally recruited MCs in sensitized mice could be activated by the allergen via the IgE/FcεRI mechanism and increase the blood–brain barrier (BBB) permeability to promote neuroinflammation. Furthermore, we suspected that repeated allergen exposure could sustain MC activation. To investigate our hypothesis, we sensitized C57BL6/J mice to a bovine whey allergen, β-lactoglobulin (BLG), and subsequently placed them on a whey-containing diet for two weeks. MC activity and associated changes in the brain were examined. BLG-sensitized mice showed mobility changes and depression-like behavior with significantly increased MC numbers and histamine levels in select brain regions. IgG extravasation and perivascular astrogliosis were also evident. Importantly, myelin staining revealed cortical demyelination in the BLG-sensitized mice, suggesting a potential neural substrate for their behavioral changes. Our findings support the ability of brain MCs to release histamine and other mediators to increase BBB permeability and facilitate neuroinflammatory responses in the brain.