Effects of physical form of ß-lactoglobulin and calcium ingestion on GLP-1 secretion, gastric emptying and energy intake in humans: a randomised crossover trial.

The aim of this study was to assess whether adding Ca2+ to aggregate or native forms of ß-lactoglobulin alters gut hormone secretion, gastric emptying rates and energy intake in healthy men and women. Fifteen healthy adults (mean ± sd: 9M/6F, age: 24 ± 5 years) completed four trials in a randomised, double-blind, crossover design. Participants consumed test drinks consisting of 30 g of ß-lactoglobulin in a native form with (NATIVE + MINERALS) and without (NATIVE) a Ca2+-rich mineral supplement and in an aggregated form both with (AGGREG + MINERALS) and without the mineral supplement (AGGREG). Arterialised blood was sampled for 120 min postprandially to determine gut hormone concentrations. Gastric emptying was determined using 13C-acetate and 13C-octanoate, and energy intake was assessed with an ad libitum meal at 120 min. A protein × mineral interaction effect was observed for total glucagon-like peptide-1 (GLP-1TOTAL) incremental AUC (iAUC; P < 0·01), whereby MINERALS + AGGREG increased GLP-1TOTAL iAUC to a greater extent than AGGREG (1882 ± 603 v. 1550 ± 456 pmol·l-1·120 min, P < 0·01), but MINERALS + NATIVE did not meaningfully alter the GLP-1 iAUC compared with NATIVE (1669 ± 547 v. 1844 ± 550 pmol·l-1·120 min, P = 0·09). A protein × minerals interaction effect was also observed for gastric emptying half-life (P < 0·01) whereby MINERALS + NATIVE increased gastric emptying half-life compared with NATIVE (83 ± 14 v. 71 ± 8 min, P < 0·01), whereas no meaningful differences were observed between MINERALS + AGGREG v. AGGREG (P = 0·70). These did not result in any meaningful changes in energy intake (protein × minerals interaction, P = 0·06). These data suggest that the potential for Ca2+ to stimulate GLP-1 secretion at moderate protein doses may depend on protein form. This study was registered at clinicaltrials.gov (NCT04659902).

Co-encapsulation of (-)-epigallocatechin-3-gallate and piceatannol/oxyresveratrol in ß-lactoglobulin: effect of ligand-protein binding on the antioxidant activity, stability, solubility and cytotoxicity.

The co-encapsulation of multiple bioactive components in a carrier may produce synergistic effects and improve health benefits. In this study, the interactions of ß-lactoglobulin (ß-LG) with epigallocatechin-3-gallate (EGCG) and/or piceatannol (PIC)/oxyresveratrol (OXY) were investigated by multispectroscopic techniques, isothermal titration calorimetry, and molecular docking. The static quenching mechanism of ß-LG by EGCG, PIC and OXY was confirmed by fluorescence spectroscopy and UV-vis absorption difference spectroscopy. The binding sites of these three polyphenols in ß-LG were identified by site marking fluorescence experiments and molecular docking. The thermodynamic parameters of the ß-LG + EGCG/PIC/OXY binary complex and ß-LG + EGCG + PIC/OXY ternary complex were obtained from fluorescence data and used to analyze the main driving force for complex formation. The exothermic binding process was further confirmed by isothermal titration calorimetry. The α-helical content, particle size and morphology of free and ligand-bound ß-LG were determined by circular dichroism spectroscopy, dynamic light scattering and transmission electron microscopy, respectively. The effect of EGCG, PIC and OXY on the conformation of ß-LG was studied by Fourier transform infrared spectroscopy. In addition, the maximum synergistic antioxidant activity between EGCG and PIC/OXY was obtained by response surface analysis. The effects of ß-LG in the binary and ternary systems on the antioxidant activity, stability, solubility and cytotoxicity of the polyphenols were also studied. Finally, the different cytotoxicities of the complexes and nanoparticles of the binary and ternary systems were compared. The results of this study are expected to provide a theoretical basis for the development of ß-LG-based carriers co-encapsulating a variety of bioactive components.

Evaluation of colostrum bioactive protein transfer and blood metabolic traits in neonatal lambs in the first 24 hours of life.

Colostrum is a unique resource that contributes to the passive transfer of immunity and plays a central role in the health status of neonatal ruminants. However, digestion and absorption of colostral proteins in the gut remain incompletely understood. Therefore, this study aimed to investigate the effect of bovine colostrum feeding on blood metabolic traits and to quantify colostral bioactive proteins in the gastrointestinal digesta and blood to evaluate intestinal transfer in neonatal lambs in the first 24 h of life. Fifty-four newborn lambs were used in this study, including 27 lambs fed pooled bovine colostrum and slaughtered at 6 (C6h), 12 (C12h), or 24 h (C24h) after birth; 18 lambs not fed any colostrum or milk and slaughtered at birth (N0h) or 24 h (N24h) after birth; and 9 milk-fed lambs slaughtered at 24 h (M24h) after birth. Lambs receiving colostrum or milk were bottle-fed within the first 2 h to obtain intakes of 8% of body weight at birth. Samples of blood and digesta from the abomasum, jejunum, and ileum were collected after slaughter. Serum concentrations of glucose, insulin, total protein, and aspartate aminotransferase were higher in colostrum-fed lambs than in N0h lambs. Serum concentrations of insulin, total protein, insulin-like growth factor 1, and γ-glutamyl transpeptidase were higher in C24h lambs than in N24h or M24h lambs. Apparent efficiencies of IgG absorption in C6h, C12h, and C24h lambs were 14.4, 26.8, and 17.2%, respectively, whereas apparent efficiencies of lactoferrin (LF), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) absorption were very low in colostrum-fed lambs, with mean values of 0.06, 0.002, and 0.003%, respectively. Concentrations of IgG, LF, α-LA, and ß-LG in the digesta of the abomasum, jejunum, and ileum rapidly decreased from C6h to C24h lambs, and the disappearance rates of IgG, LF, α-LA, and ß-LG were higher in lambs from C6h to C12h (62.1, 75.7, 91.3, and 95.0% for IgG, LF, α-LA, and ß-LG, respectively) than from C12h to C24h (34.6, 22.5, 7.5, and 2.2% for IgG, LF, α-LA, and ß-LG, respectively). These results indicated that bovine colostrum feeding improved the metabolic and immunological status of lambs, and that ingested colostral IgG was prone to intact uptake into the blood, whereas almost all ingested LF, α-LA, and ß-LG disappeared in the lumen of the gastrointestinal tract in a time-dependent manner. The findings provide novel information for exploring selective absorption of colostral compounds in the small intestine of lambs.

Beta-lactoglobulin-nutrition allergen and nanotransporter of different nature ligands therapy with therapeutic action.

ß-lactoglobulin is one of the nutrition allergens present in the milk of many mammals, with the exception of human. This protein belongs to the family of lipocalins, consisting of nine antiparallel ß-strands (ß-A to ß-I) and one α-helix. This structure allows it to serve as a nanotransporter of various nature ligands in a pH dependent manner, which allows us to confidently consider it as a reliable carrier of drugs directly into the intestine, bypassing the destructive acidic environment of the stomach. Based on the latest data, this review describes the currently known methods of reducing the allergenicity of beta-lactoglobulin, as well as the mechanisms and methods of forming complexes of this protein with ligands, which emphasizes its importance and versatility and explains the growing interest in studying its properties in recent decades, and also opens up prospects for its practical application in medicine and pharmaceuticals.

Modulation of gastrointestinal digestion of ß-lactoglobulin and micellar casein following binding by (-)-epigallocatechin-3-gallate (EGCG) and green tea flavanols.

The effect of binding of flavonoids, (-)-epigallocatechin-3-gallate (EGCG) and green tea extract (GTE), to beta-lactoglobulin (ß-Lg) and micellar casein (micellar casein isolate, MCI) on protein digestibility was investigated. ß-Lg resisted digestion by pepsin, but in the presence of EGCG the digestion of ß-Lg was enhanced. Binding of EGCG to ß-Lg was identified by nitro blue tetrazolium (NBT) staining and found, by isothermal titration calorimetry, to be an enthalpy-driven exothermic process, with a binding constant of 19 950 L mol-1. Binding promoted a more rapid digestion of ß-Lg during simulated upper duodenal digestion. NBT staining indicated a loss of binding of EGCG to ß-Lg during combined gastric and distal small intestinal digestion and correlated with the cleavage of ß-Lg. However, increased ß-Lg heteromer formation and reduced ß-Lg monomer digestibility were observed for the ß-Lg-GTE complex. MCI was more digestible than ß-Lg during pepsin digestion, but reduced digestibility was observed for both MCI-EGCG and MCI-GTE complexes, with loss of binding during intestinal digestion. The free radical scavenging capacity (FRSC) of EGCG remained stable for the ß-Lg-EGCG complex throughout the gastric and intestinal phases of digestion, but this was significantly lowered for the MCI-EGCG complex. These results indicated that polyphenols bind to milk proteins modulating the in vitro digestibility and FRSC of ß-Lg and MCI as a result of the formation of complexes.

New insights into the binding mechanism between osthole and ß-lactoglobulin: Spectroscopic, chemometrics and docking studies.

Osthole, a type of coumarin derivative, owns many biological functions, but the poor water solubility and low bioavailability limit its usage in food and pharmaceutical fields. ß-Lactoglobulin (BLG) is a major protein in bovine milk whey which is usually used as a carrier of poorly soluble bioactive components. Researching the interaction between osthole and BLG can help us in understanding their binding mechanism and design an osthole delivery system using BLG as carrier protein to further promote the application of osthole in functional food and drug. Hence, this study was devoted to explore the binding properties of osthole with BLG and the effect on the protein structure by employing multispectroscopic approaches, chemometrics and molecular simulation studies. The results of the concentration profiles and pure spectra of the components resolved from highly overlapping spectral signals by the multivariate curve resolution-alternating least squares (MCR - ALS) algorithm verified the formation of an osthole-BLG complex. Moreover, osthole was found to strong quench the intrinsic fluorescence of BLG and the quenching mechanism was determined to be a static procedure. The binding constant of osthole with BLG at 298 K was (4.06 ±â€¯0.03) × 104 L mol-1. Hydrophobic forces and hydrogen bonds played major roles in the binding process. The analysis of synchronous fluorescence, circular dichroism and Fourier transform infrared spectra found that the presence of osthole caused BLG structure compact and the change in the polarity and hydrophobicity of the microenvironment around Tyr residues of the protein. Modeling docking predicted that osthole bound to the hydrophobic cavity of BLG through stable hydrogen bonds primarily with the amino acid residues Lys75 and Thr76.

Studies on the interactions between bile salts and food emulsifiers under in vitro duodenal digestion conditions to evaluate their bile salt binding potential.

During the last decade a special interest has been focused on studying the relationship between the composition and structure of emulsions and the extent of lipolysis, driven by the necessity of modulate lipid digestion to decrease or delay fats absorption or increase healthy fat nutrients bioavailability. Because bile salts (BS) play a crucial role in lipids metabolism, understanding how typical food emulsifiers affect the structures of BS under duodenal conditions, can aid to further understand how to control lipids digestion. In the present work the BS-binding capacity of three emulsifiers (Lecithin, Tween 80 and ß-lactoglobulin) was studied under duodenal conditions. The combination of several techniques (DLS, TEM, ζ-potential and conductivity) allowed the characterization of molecular assemblies resulting from the interactions, as modulated by the relative amounts of BS and emulsifiers in solution.

Changes in the content of immunostimulating components of colostrum obtained from dairy cows at different levels of production.

BACKGROUND: Recent data indicate that almost 60% of colostrum samples do not have enough antibodies to provide adequate protection for the calf. Thus, the aim of this study was to determine the influence of the level of production of cows on the content of immunostimulating components of protein fraction of colostrum. The observations were conducted on two farms that keep dairy cows. One of the farms was selected to represent a typical performance level for the mass population of dairy cows in Poland, about 5500 kg of milk per cow per year (LI) and the second one was characterized by a higher level of production, about 8000 kg (HI). RESULTS: Based on the data obtained, 100% of the samples collected from LI had an immunoglobulin G (IgG) content >50 gL-1 , in the first collection. In the case of HI, 63% of the samples had an IgG content >50 gL-1 , with a distribution in the range of 10.7 to 72.7 gL-1 . The colostrum of LI cows was characterized by a higher content of lactoferrin, lysozyme, α-lactalbumin, and ß-lactoglobulin compared to the milk of HI cows. CONCLUSIONS: The immunostimulating components of colostrum declined rapidly in subsequent milkings or with the increased productivity of the cows. The concentration of IgG was negatively correlated with the performance level of cows, which means that high-producing cows had colostrum with a low concentration of IgG. © 2018 Society of Chemical Industry.

A mixture of milk and vegetable lipids in infant formula changes gut digestion, mucosal immunity and microbiota composition in neonatal piglets.

PURPOSE: Although composition of infant formula has been significantly improved during the last decade, major differences with the composition and structure of breast milk still remain and might affect nutrient digestion and gut biology. We hypothesized that the incorporation of dairy fat in infant formulas could modify their physiological impacts by making their composition closer to that of human milk. The effect of milk fat and milk fat globule membrane (MFGM) fragments in infant formulas on gut digestion, mucosal immunity and microbiota composition was evaluated. METHODS: Three formulas containing either (1) vegetable lipids stabilized only by proteins (V-P), (2) vegetable lipids stabilized by a mixture of proteins and MFGM fragments (V-M) and (3) a mixture of milk and vegetable lipids stabilized by a mixture of proteins and MFGM fragments (M-M) were automatically distributed to 42 newborn piglets until slaughter at postnatal day (PND) 7 or 28, and compared to a fourth group of sow's suckling piglets (SM) used as a breast-fed reference. RESULTS: At both PND, casein and ß-lactoglobulin digestion was reduced in M-M proximal jejunum and ileum contents compared to V-P and V-M ones leading to more numerous ß-Cn peptides in M-M contents. The IFNγ cytokine secretion of ConA-stimulated MLN cells from M-M piglets tended to be higher than in V-P ones at PND 7 and PND 28 and was closer to that of SM piglets. No dietary treatment effect was observed on IL-10 MLN cell secretion. Changes in faecal microbiota in M-M piglets resulted in an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes phyla compared to V-P ones. M-M piglets showed higher abundances of Parabacteroides, Escherichia/Shigella and Klebsiella genus. CONCLUSIONS: The incorporation of both milk fat and MFGM fragments in infant formula modifies protein digestion, the dynamic of the immune system maturation and the faecal microbiota composition.

Short communication: Variation of total immunoglobulin G and ß-lactoglobulin concentrations in colostrum and milk from Canadian Holsteins classified as high, average, or low immune responders.

The objective of this study was to evaluate IgG and ß-lactoglobulin (ß-LG) concentrations in colostrum and milk of Canadian Holsteins (n=108) classified as high (H), average (A), or low (L) for antibody-mediated (AMIR) or cell-mediated immune responses (CMIR) based on estimated breeding values. It was hypothesized that H-AMIR and H-CMIR cows produce colostrum (first milking) and milk (d 5 postcalving) with higher concentrations of IgG and ß-LG. Data for IgG and ß-LG in colostrum and milk were analyzed independently using mixed linear models. Least squares means were compared using Tukey's test. Cows classified as H-AMIR had higher IgG and ß-LG concentrations in colostrum compared with A- and L-AMIR cows; 84% of H-AMIR, 69% of A-AMIR, and 68% of L-AMIR cows had over 5,000 mg/dL IgG in colostrum. No differences in IgG and ß-LG concentrations in colostrum were noted among cows ranked on CMIR or in milk of cows ranked on AMIR. ß-Lactoglobulin and IgG concentrations were positively correlated in colostrum. Breeding cows for H-AMIR status may reduce failure of passive transfer of IgG in their calves; ß-LG may play a role in bovine immune defenses. Colostrum from H-AMIR cows may serve as a more economical feedstock source for manufacturing natural health products.

Effect of supplementation of cows diet with linseed and fish oil and different variants of ß-lactoglobulin on fatty acid composition and antioxidant capacity of milk.

BACKGROUND: The aim of this study was to determine the influence of polymorphic variants of ß-lactoglobulin in cows supplemented with linseed and fish oil on the fatty acid composition and antioxidant capacity of milk. From the herd of 320 Polish Holstein Friesian cows three groups of cows were selected according to the variants of ß-LG (ß-LGAA, ß-LGBB, ß-LGAB). During the first 7 days (the initial period) all the cows were fed the same total mixed ration (TMR) diet. From day 8 to 28,150 g fish oil and 250 g linseed (FOL) was added to the TMR diet of each cow. RESULTS: The results showed that the diet supplemented with FOL was effective in reducing atherogenic and thrombogenic indices. Introducing supplementation improved the antioxidant capacity: higher concentration of C18:2cis-9 trans-11, C20:5 n-3, C22:6 n-3, bioactive whey proteins and vitamin soluble in fat has been recorded. The results showed that ß-LGAA was associated with lower levels of atherogenic and thrombogenic indices and higher concentration of C22:5 n-6, phospholipids and ß-carotene. ß-LGBB favours a higher content of C18:1trans-11, C18:2cis-9 trans-11 and lactoferrin. ß-LGAB was associated with higher concentrations of C20:5 n-3, Lysozyme, α-retinol, α-tocopherol and total antioxidant status. CONCLUSION: Modification of the diet of cows with fish oil and linseed significantly influenced fatty acid composition and antioxidant properties of milk. The effect of ß-LG phenotype on the fatty acid composition and antioxidant capacity of milk is variable, which could partly be the result of a ß-LG phenotype × diet interaction.

Structural mechanism of complex assemblies: characterisation of beta-lactoglobulin and pectin interactions.

Knowledge of how proteins and polysaccharides interact is the key to understanding encapsulation and emulsification in these composite systems and ultimately to understanding the structures of many biological network systems. As a model system we have studied ß-lactoglobulin A (ßLgA) interacting with pectins of various amounts and distribution patterns of charge. The studies were conducted at pH 4 at minimal ionic strength, where the ßLgA and the pectins are oppositely charged, resulting in an electrostatic attraction to each other. Isothermal titration calorimetry (ITC) experiments were performed to determine the thermodynamics associated with ßLgA-pectin titration. It was found that ßLgA only interacted with pectins with an adequate amount of charge, and that the complexation between ßLgA and pectin was a two-step process initially involving binding of the protein to available sites on the pectin, and subsequently binding of the protein onto the bound protein that has previously adsorbed. Circular dichroism (CD) and intrinsic tryptophan fluorescence were also measured of ßLgA during its interaction with the pectin samples, and show that the binding leads to significant conformational changes in ßLgA. An increase in the turbidity of the solution of the resultant complexes indicates the formation of large-scale interpolymer associations of the primary complexes mediated by protein-rich domains.

Se metallomics during lactic fermentation of Se-enriched yogurt.

Selenium biotransformation by lactic acid bacteria during the preparation of Se-enriched yogurt was evaluated. The study focused on the distribution of selenium in the aqueous soluble protein fraction and the detection of selenoamino acids. Screening of selenium in Tris-buffer-urea soluble fraction was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis after pre-fractionating with asymmetric field flow fractionation using inductively coupled plasma-mass spectrometry as the detector. Selenium-containing fractions were identified by peptide mapping using nano LC-ESI/LTQMS. Proteins such as thioredoxin, glutaredoxin, albumin, ß-lactoglobulin, and lactoperoxidase were identified in the selenium-containing fraction. All these proteins were detected in both the control and the selenium-enriched yogurt except chaperones, which were only detected in the control samples. Chaperones are heat-shock proteins expressed in response to elevated temperature or other cellular stresses. Selenium may have an effect on chaperones expression in Lactobacillus. For the amino acids analysis, selenocysteine was the primary seleno-containing species.

Multifunctional 8-hydroxyquinoline-appended cyclodextrins as new inhibitors of metal-induced protein aggregation.

Mounting evidence suggests a pivotal role of metal imbalances in protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target. In this context, the synthesis of chelators that also contain complementary functionalities to combat the multifactorial nature of neurodegenerative diseases is a highly topical issue. We report two new 8-hydroxyquinoline-appended cyclodextrins and highlight their multifunctional properties, including their Cu(II) and Zn(II) binding abilities, and capacity to act as antioxidants and metal-induced antiaggregants. In particular, the latter property has been applied in the development of an effective assay that exploits the formation of amyloid fibrils when ß-lactoglobulin A is heated in the presence of metal ions.

Investigation of molecular interactions between ß-lactoglobulin and sugar beet pectin by multi-detection HPSEC.

Molecular interactions between ß-lactoglobulin (ß-LG) and sugar beet pectin (SBP) were studied using online multi-detection high performance size exclusion chromatography (HPSEC) at neutral pH and 50mM ionic strength. The hydrodynamic properties of various interacting polymer fractions were characterized in detail and compared with those of ß-LG and SBP. Results showed that ∼6.5% (w/w) of native dimeric ß-LG molecules formed complexes with over 35% SBP molecules of varying sizes, 800, 110 and 75 kDa. Although the ß-LG molecules bind to SBP molecules of all sizes and shapes, they tend to favor the intermediate (110 kDa) and small sized (75 kDa) SBP molecules. All resulting complexes possess altered shapes and hydrodynamic properties when compared to unbound SBP and ß-LG. About half of the interacting ß-LG (∼3.5%) molecules were thought to bind to a small amount of non-covalently bound feruloyl groups, possibly present in SBP. When pre-heat treated ß-LG and SBP were combined, more than 16% of ß-LG formed complexes with at least 45% of SBP molecules of varying sizes, Mw∼750-800, 110, and 55-80 kDa. The complexes formed between ß-LG aggregates and/or oligomers and the large SBP molecules (750-800 kDa) adopt the shape of ß-LG aggregates, random coil. Both groups of complexes formed between ß-LG intermediate (110 kDa) and small sized (55-80 kDa) SBP take on the shape of rigid rod. It was speculated that half of the interacting heat-treated ß-LG molecules (∼8%) are complexed with non-covalently bound feruloyl groups in SBP.

In vitro iron absorption of α-lactalbumin hydrolysate-iron and ß-lactoglobulin hydrolysate-iron complexes.

To study the feasibility of promoting iron absorption by peptides derived from α-lactalbumin and ß-lactoglobulin, the present work examined the transport of iron across Caco-2 monolayer cell as in vitro model. Caco-2 cells were seeded in bicameral chambers with α-lactalbumin hydrolysate-Fe (α-LAH-Fe) complex and ß-lactoglobulin hydrolysate-Fe (ß-LGH-Fe) complex, α-LAH and iron mixture, ß-LGH and iron mixture, FeSO4 and ascorbic acid mixture, and FeSO4. In addition, the cytotoxicity of α-LAH-Fe and ß-LGH-Fe complexes were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The iron absorption and ferritin content were assessed using the coupled in vitro digestion/Caco-2 cell model. Results support that peptide-iron complexes can promote ferritin formation and it is possible to apply ß-LGH-Fe complexes as iron-fortified supplements with high iron absorbability.

Expression of the mammary gland-specific tammar wallaby early lactation protein gene is maintained in vitro in the absence of prolactin.

Marsupial ELP (early lactation protein) and its eutherian orthologue, CTI (colostrum trypsin inhibitor) are expressed in the mammary gland only for the first 100 days postpartum (Phase 2A) in the tammar wallaby and during the bovine and canine colostrogenesis period 24-36h postpartum respectively. The factors which regulate temporal ELP and CTI expression are unknown. A tammar mammary gland explant culture model was used to investigate ELP gene regulation during pregnancy and early- and mid-lactation (Phase 1, 2A and 2B respectively). Tammar ELP expression could only be manipulated in explants in vitro if the gene was already expressed in vivo. ELP expression was maximal in Phase 1 explants treated with lactogenic hormones (insulin, hydrocortisone and prolactin), but unlike LGB (ß-lactoglobulin), ELP expression was maintained in insulin or insulin and hydrocortisone over a 12-day culture period. In contrast, ELP was down-regulated when cultured without hormones. ELP could not be induced in explants cultured from mid-lactation which suggested that transcriptional repressors may prevent ELP expression during this period.

Buffalo colostrum ß-lactoglobulin inhibits VEGF-induced angiogenesis by interacting with G protein-coupled receptor kinase.

ß-lactoglobulin (ß-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as ß-lg. The sequences IIVTQ f[1-5] and LSFNPTQLEEQCHV f(149-162) of m/z 933(+) and 851(2+) were found to match N- and C-extreme of ß-lg while IDALNENK f(84-91) and TPEVDDEALEKFDK f(125-138) sequences deduced for m/z 916(+) and 818(2+) were in compliance to buffalo milk ß-lg. Considering the sequence similarity of ß-lg to glycodelin, a proven angiogenic protein, similar role for ß-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled ß-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.

The age of cows as a factor shaping the antioxidant level during a nutritional experiment with fish oil and linseed supplementation for increasing the antioxidant value of milk.

BACKGROUND: So far, in research studies, the age of cows has not been considered as a factor that may influence the changes in the content of milk ingredients with antioxidant properties modified by the feed supplementation. The aim of this study was to determine the influence of supplementation on the content of ingredients having antioxidant properties and to determine the influence of the age of cows taking part in the experiment on these changes. The experiment was conducted using 20 Polish Holstein Friesian cows, 10 primiparous and 10 multiparous. The combined supplementation of fish oil and linseed constituted the experimental factor. RESULTS: The milk of primiparous cows after 21 days of supplementation was characterised by a higher content of C18:1 trans-11, C18:2 cis-9, trans-11, α-retinol, α-tocopherol and ß-lactoglobulin compared to the milk of multiparous cows, in which a higher level of lactoferrin, C20:5 and ß-carotene was recorded. In both groups an increase in the total antioxidant status was noted (a higher level in the milk of primiparous cows). CONCLUSIONS: Modification of the diet of cows with fish oil and linseed significantly influenced antioxidant properties of their milk; however, the response of multiparous and primaparous cows was noticeably different to the supplement introduced.

Binding between bixin and whey protein at pH 7.4 studied by spectroscopy and isothermal titration calorimetry.

Bixin is the major coloring component of annatto used in manufacturing colored cheeses, but its presence in liquid whey causes undesirable quality of the recovered whey protein ingredients. The objective of this work was to study molecular binding between bixin and three major whey proteins (ß-lactoglobulin, α-lactalbumin, and bovine serum albumin) at pH 7.4 using UV-vis absorption spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and circular dichroism. These complementary techniques illustrated that the binding is a spontaneous complexation process mainly driven by hydrophobic interactions. The complexation is favored at a lower temperature and a higher ionic strength. At a lower temperature, the binding is entropy-driven, while it changes to an enthalpy-driven process at higher temperatures. The binding also increases the percentage of unordered secondary structures of proteins. Findings from this work can be used to develop whey protein recovery processes for minimizing residual annatto content in whey protein ingredients.