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The work aims to analyze the associations of polymorphic variants of the PRL and BLG genes with resistance and susceptibility to mastitis in Holstein cows. The experimental study consisted of the selection of biomaterial samples from 250 heads of Holstein cows aged 3 years divided into two groups (healthy and with a confirmed diagnosis of mastitis). The determination of animal genotypes was carried out using polymerase chain reaction and restriction fragment length polymorphism. The study of the nature of the association of polymorphic variants of the PRL and BLG gene with resistance/increased risk of mastitis established a significant deviation from the theoretically expected distribution of bBLG-HaeIII genotypes in the group of animals suffering from mastitis (the value of χ2 was 0.24). The bBLG-HaeIIIBB genotype can act as a marker of an increased risk of developing mastitis in Holstein cows; its frequency in the group of sick animals exceeds the frequency in the control group by more than 2 times (44.0 compared to 17.0%, respectively). The bBLG-HaeIIIAB genotype is significantly associated with mastitis resistance in Holstein cows; its frequency is 2 times lower than in the control group (28.0 compared to 54.0%).

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ß-Lactoglobulin (ß-Lg) is the main protein in whey and is known for its allergenicity and resistance to the digestion of pepsin and trypsin. The UV-C photoinduced cleavage of disulfide bonds in ß-Lactoglobulin, as promoted by excitation of tryptophan residues (Trp), is shown to induce changes in the protein's secondary structure, significantly reducing the protein's resistance to pepsin digestion. The UV-C light-induced changes in the protein secondary structure are marked by an increase in the contribution of ß-sheet and α-helix structures with a concomitantly smaller contribution of the ß-turn structural motif. The photoinduced cleavage of disulfide bonds in ß-Lg has an apparent quantum yield of ф = 0.0015 ± 0.0003 and was shown by transient absorption laser flash photolysis to arise by two different pathways: a) the reduction of the disulfide bond Cys66Cys160 occurs by direct electron transfer from the triplet-excited 3Trp to the disulfide bond due to the existence of a CysCys/Trp triad (Cys66Cys160/Trp61) and b) the reduction of the buried Cys106Cys119 disulfide bond involves a reaction with a solvated electron originated by the photoejection of electrons from the triplet-excited 3Trp decay. The in vitro gastric digestion index for UV-C-treated ß-Lg is revealed to have increased significantly by 36 ± 4 % and 9 ± 2 % under simulated elderly and young adult digestive conditions, respectively. When compared to the native protein, the peptide mass fingerprint profile of digested UV-C-treated ß-Lg shows a higher content and variety of peptides, including the production of some exclusive bioactive peptides such as PMHIRL and EKFDKALKALPMH.

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Despite some thermodynamics studies about ß-lactoglobulin (ßLG) and resveratrol (RES) interactions, there is a gap regarding kinetics data about ßLG-RES complex formation. Here, we determined the kinetic and thermodynamic parameters of ßLG-RES interactions by using surface plasmon resonance (SPR). The kinetic association parameters were dependent on the 3D water structure present on the solvation shell of both interacting molecules. At lower temperature (285.15 K), all activation energies were positive (Eacta‡= 82.86 kJ.mol-1,TΔSa‡= 32.26 kJ.mol-1, and ΔCpa‡= 4.15 kJ.mol-1K-1) due to the higher water structuration on the RES and ßLG solvation shell. All these energetic barriers become mainly from the energetic cost for the desolvation process of RES and ßLG. At higher temperature (301.15 K), the solvation water structure decreases and all the above activation energies become negative (Eacta‡=-121.58 kJ.mol-1,TΔSa‡=-173.59 kJ.mol-1, and ΔCpa‡=-29.92 kJ.mol-1K-1) because the direct interaction between desolvated RES and ßLG molecules released more energy than it is absorbed by desolvation process. However, kinetic dissociation parameters were not dependent on the hydrogen bond density of the water solvation shell as showed by the temperature independence of dissociation energetic parameters. This non-dependence of the dissociation process from the desolvation step probably is because the water molecules interacting with the ßLG-RES complex is not concentrated around/inside the protein site of interaction. The association of free molecules was 1.5 times faster than the dissociation of the thermodynamically stable complex (ΔG(a)‡â€¯â‰… 48.15 kJ.mol-1, ΔG(d)‡â€¯â‰… 73.10 kJ.mol-1). The lower free energy barrier observed for the association came from an isokinetic process where entropic and enthalpic parameters compensated for each other. The ΔG° values indicate that the thermodynamically stable complex predominates over free molecules. At low temperature (285.15 K), the hydrophobic interaction (ΔH° = 73.06 kJ.mol-1; TΔS° = 99.60 kJ.mol-1) drove the ßLG-RES complex formation while at high temperature (301.15 K), hydrophilic interactions became dominant (ΔH° = -142.50 kJ.mol-1; TΔS° = -118.18 kJ.mol-1).

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Dynamic high-pressure homogenization microfluidization (DHPM) is a versatile emerging technology that may be applied to food processing to achieve several goals. DHPM may, depending on nature of the molecules and the working parameters, induce changes in protein structure, which may improve or impair their techno-functional properties and ability to bind other molecules. In this context, DHPM (12 passes, 120 MPa), coupled or not to a cooling device, was applied to ß-lactoglobulin (ß-lg) and whey protein isolate (WPI) dispersions. Minor changes in the structure of whey proteins were induced by DHPM with sample cooling; although, when sample cooling was not applied, aggregation and increases of around 30% of protein surface hydrophobicity were noticeable for the WPI dispersion. The association constant between the proteins and lutein was in the magnitude of 104 M-1, and lutein photodegradation constant diminished about 3 times in the presence of proteins, compared to in their absence.

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κ-Cn and ß-lactoglobulin are important candidate genes associated with milk yield and milk protein content. The present investigation is carried out to determine the polymorphisms status of κ-Cn and ß-lactoglobulin genes in Anatolian Black cattle and Holstein breeds. PCR-RFLP technique was used to determine Kappa-Casein and ß-lactoglobulin polymorphisms in both cattle breeds. The allele frequency of Anatolian Black cattle in terms of κ-Cn and ß-lactoglobulin genes were 0.50 (A) 0.50 (B) and 0.20 (A) 0.80 (B) respectively, whereas in Holstein were 0.29 (A) 0.71 (B) and 0.44 (A) 0.56 (B) respectively. The chi-square test showed that each cattle breed was in Hardy-Weinberg equilibrium (P>0.05).

κ-Cn e ß-lactoglobulina são importantes genes candidatos associados à produção de leite e ao teor de proteína do leite. A presente investigação é realizada para determinar o status de polimorfismos dos genes κ-Cn e ß-lactoglobulina nas raças Anatolian Black cattle e Holstein. A técnica PCR-RFLP foi utilizada para determinar os polimorfismos Kappa-Casein e ß-lactoglobulina em ambas as raças de gado. A freqüência dos alelos do gado negro anatólio em termos de κ-Cn e ß-lactoglobulina foi de 0,50 (A) 0,50 (B) e 0,20 (A) 0,80 (B) respectivamente, enquanto que em Holstein foi de 0,29 (A) 0,71 (B) e 0,44 (A) 0,56 (B) respectivamente. O teste do qui-quadrado mostrou que cada raça bovina estava em equilíbrio de Hardy-Weinberg (P>0,05).

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X-ray analysis has provided structural data about a pH-driven conformational change in ß-lactoglobulin (BLG) known as the Tanford transition, which occurs at around pH 7 and involves the EF loop, which acts as a lid closing the internal cavity of the protein below pH 7 and opening it above pH 7. NMR studies using wild-type BLG have encountered problems trying to explain the Tanford transition, however, they have provided important insight using a dimeric BLG mutant, revealing that the opening and closure of the EF loop consists of two types of motions in the microsecond and milliseconds timescales. This provides valuable information indicating that the dimeric state is a good model to study the Tanford transition, although the understanding of this structural change is still lacking at the atomic level. We performed microsecond molecular dynamics (MD) simulations starting from different conformations of BLG in the monomeric and dimeric state, with protonated and deprotonated E89, in order to explore the Tanford transition. Our results provide structural information for the transition from the closed to the open conformation in BLG and show it occurs in the dimeric state in the microsecond timescale, in line with the fast motion observed through NMR experiments. In addition, MD simulations coupled to MMGBSA approach indicated that the most populated conformer of BLG in the open state is able to bind ligands with similar affinity to that of BLG at neutral pH obtained through crystallographic experiments.Communicated by Ramaswamy H. Sarma.

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This study investigated the use of Novo Pro-D® (NPD) and Ficin (FC) as alternative proteases for the production of bioactive peptides with reduced allergenicity from whey protein concentrate (WPC). In addition, the use of high hydrostatic pressure processing as pre-treatment of WPC and its impact on the final characteristics of hydrolysates were also evaluated. NPD treatments generated hydrolysates with a 98% reduction of soluble proteins, greater in vitro antioxidant capacity, and less immunoreactivity when compared to FC ones. However, pre-treatment was an essential tool to improve WPC hydrolysis when FC was used, resulting in hydrolysates with less soluble proteins, enhanced antioxidant capacity, and less allergenicity compared with conventional hydrolysis. As for NPD, the pre-treatment of WPC improved the in vitro antioxidant capacity and resulted in a 100% reduction in immunoreactivity to ß-lactoglobulin in a shorter processing time. Importantly, bioactive peptides generated by FC displayed an improved ability to induce in vitro arterial relaxation, compared with those obtained from NPD process. Therefore, this study provides innovative evidence regarding how the proteases used for production of whey hydrolysates can improve its biological effects, and discloses the use of high hydrostatic pressure combined with enzymatic hydrolysis as a promising alternative to produce hydrolysates with improved properties.

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Infant formulas, designed to provide similar nutritional composition and performance to human milk, are recommended when breastfeeding is not enough to provide for the nutritional needs of children under 12 months of age. In this context, the present study aimed to assess the protein quality and essential amino acid content of both starting (phase 1) and follow-up (phase 2) formulas from different manufacturers. The chemical amino acid score and protein digestibility corrected by the amino acid score were calculated. The determined protein contents in most formulas were above the maximum limit recommended by FAO and WHO guidelines and at odds with the protein contents declared in the label. All infant formulas contained lactoferrin (0.06 to 0.44 g·100 g-1) and α-lactalbumin (0.02 to 1.34 g·100 g-1) below recommended concentrations, whereas ĸ-casein (8.28 to 12.91 g·100 g-1), α-casein (0.70 to 2.28 g·100 g-1) and ß-lactoglobulin (1.32 to 4.19 g·100 g-1) were detected above recommended concentrations. Essential amino acid quantification indicated that threonine, leucine and phenylalanine were the most abundant amino acids found in the investigated infant formulas. In conclusion, infant formulas are still unconforming to nutritional breast milk quality and must be improved in order to follow current global health authority guidelines.

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Whey is a cheese co-product with high protein content used in the food industry due to its techno-functional properties and nutritive value. This study aims to optimize the production of ß-lactoglobulin (ß-lg) nanostructures, to characterize their techno-functional properties and stability, and to apply them as a carrier of bioactive molecules. Box-Behnken planning was applied to determine the best conditions to obtain the ß-lg nanostructure, which consists in treatment at 100 °C in NaCl 50 mmol·L-1 for 60 min. TEM analysis showed a fibril structure in the observed nanostructures. The nanostructured systems formed foam and emulsion with higher stability than the systems composed of the native protein. The results for encapsulation efficiency of bioactive compounds were 96.50%, 89.04%, 67.78%, and 36.39% for quercetin, rutin, naringin, and vitamin B2, respectively. Thus, ß-lg nanostructure's great capacity to encapsulate hydrophobic molecules was verified.

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Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

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