Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein :true protein ratio were found.

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.

Optimization of centrifugal separation of Ó-lactalbumin and ¿̐ư-lactoglobulin

Selostus: Ó-lactalbumiinin ja ¿̐ư-lactoglobuliinin sentrifugointierotuksen optimointi

Effects of composite casein and [beta]-lactoglobulin genotypes on renneting properties and composition of bovine milk by assuming an animal model

Selostus: Kaseiinien yhdistelmägenotyyppien ja [beta]-laktoglobuliinin genotyyppien vaikutus maidon juoksettumisominaisuuksiin ja koostumukseen

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 µg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Beta-lactoglobulin fibers under capillary flow

We describe the capillary flow behavior of gels of beta-lactoglobulin (beta-lg) containing droplets of fibrils and the shear flow alignment of beta-lg fibers in dilute aqueous solutions. Polarized optical microscopy and laser scanning confocal microscopy are used to show that capillary shear flow does not affect the fibril droplet sizes in the beta-lg gels, the system behaving in this respect as a solution of compact colloidal particles under shear flow. Small-angle X-ray scattering (SAXS) on dilute aqueous solutions indicates that the fibers can be initially aligned under capillary shear, but this alignment is lost after 18 min of shear. Transmission electron microscopy experiments on the samples studied by SAXS suggest that the loss of orientation is due to a shear-induced breakup of the swollen fibril network. Dynamic and static light scattering on dilute beta-lg fibril aqueous solutions are used to show that before shear beta-lg fibrils behave as strongly interacting semiflexible polymers, while they behave as weakly interacting rods after 18 min of capillary shear.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions (a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SIDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to similar to 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

Production of novel ACE inhibitory peptides from beta-lactoglobulin using Protease N Amano

Potent angiotensin l-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of beta-lactoglobulin (beta Lg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to beta Lg f(36-42) and had an IC50 value of 8 mu m, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine beta Lg reported so far. (C) 2008 Elsevier Ltd. All rights reserved.

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M-CTAB/M-TP) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M-CTAB/M-TP = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate. (c) 2005 Wiley Periodicals, Inc.

Production of angiotensin converting enzyme inhibitory peptides from β-lactoglobulin and casein derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin and casein derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N ‘Amano’ in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50= 285 μg/mL. In this hydrolysate we identified the well known potent ACE-I and anti-hypertensive tri-peptide Ile-Pro-Pro (IPP) and another novel octa-peptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=128 µg/mL. This hydrolysate contained a tetra-peptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC50 value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Deactivation of Ferrylmyoglobin by Vanillin as Affected by Vanillin Binding to beta-Lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = S7 +/- 1 L mol(-1) s(-1) for reduction to metmyoglobin with Delta H(double dagger) = 58.3 +/- 0.3 kJ mol(-1) and Delta S(double dagger) = -14 +/- 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 degrees C. Binding to beta-lactoglobulin (AG) was found to affect the reactivity of vanillin at 25 degrees C only slightly to k(2) = 48 +/- 2 L mol(-1) s(-1) (Delta H(double dagger) = 68.4 +/- 0.4 kJ mol(-1) and Delta S(double dagger) = 17 +/- 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to beta LG was found to have a binding stoichiometry vanillin/beta LG > 10 with K(A) = 6 x 10(2) L mol(-1) and an apparent total Delta H degrees of approximately -38 kJ mol(-1) and Delta S degrees = -S5.4 +/- 4J mol(-1) K(-1) at 25 degrees C and Delta C(p), (obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for beta LG by vanillin, approximately one vanillin was found to bind to each beta LG far stronger with K(A) = 5 x 10(4) L, mol(-1) and a Delta H degrees = 10.2 kJ mol(-1) and Delta S degrees = 55J mol(-1) K(-1) at 25 degrees C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to beta LG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the beta LG surface with the phenolic group pointing toward the solvent, in effect making both Delta H(double dagger) and Delta S(double dagger) more positive. The more strongly bound vanillin capable of tryptophan quenching in the fiLG calyx seems less or nonreactive.

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein :true protein ratio were found.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.