Plasticizer effect on oxygen permeability of beta-lactoglobulin films

Plasticizer effect on oxygen permeability (OP) of beta-lactoglobulin (beta-Lg) films was studied. Propylene glycol (PG), glycerol (Gly), sorbitol (Sor), sucrose (Suc), and polyethylene glycol at MW 200 and 400 (PEG 200 and PEG 400, respectively) were studied due to their differences in composition, shape, and size. Suc-plasticized beta-Lg films gave the best oxygen barrier (OP < 0.05 cm3 x microm/m2 x day x kPa). Gly- and PG-plasticized films had similar OP values, and both had higher OP than Sor-plasticized films. PEG 200- and PEG 400-plasticized films were the poorest oxygen barriers. Empirical equations including plasticizer efficiencies for OP were employed to elucidate the relationships between OP of plasticized beta-Lg films and plasticizer type and content. Plasticizer efficiency ratios between mechanical and OP properties of beta-Lg films show the relative efficiency of plasticizers in modifying mechanical and OP properties. A large ratio is desirable.     

Changes in chymotrypsin hydrolysis of beta-lactoglobulin A induced by high hydrostatic pressure

Beta-lactoglobulin (beta-Lg) was subjected to high pressures up to 400 MPa and proteolysis with chymotrypsin. The hydrolysates were analyzed by SDS-PAGE and RP-HPLC, and the fragments obtained were identified by ESI-MS/MS. The results obtained showed that beta-Lg was hydrolyzed by chymotrypsin in a "progressive proteolysis" manner at either atmospheric or high pressure. Proteolysis during or after high-pressure treatment showed longer and more hydrophobic peptides than proteolysis at atmospheric pressure. Chymotrypsin showed a behavior similar to that of trypsin, with some differences, probably related to the orientation of the target residues specific for each enzyme. The similarities between proteolytic fragments produced by the two enzymes support that proteolysis enhancement under high pressure depends on the substrate changes rather than the enzyme. High pressure seemed to accelerate the first steps of proteolysis, probably through dimer dissociation, while leaving portions of the molecule more resistant to the enzyme.     

Ethanol effect on the structure of beta-lactoglobulin b and its ligand binding

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.     

Tryptophan-mediated denaturation of beta-lactoglobulin A by UV irradiation

beta-Lactoglobulin A, a genetic variant of one of the main whey proteins, was irradiated at 295 nm for 24 h. After irradiation, 18% of the protein was denatured (determined by reverse-phase chromatography). The fluorescence spectrum of the irradiated protein was red-shifted compared to that of the native protein, indicating a change in protein folding. Sulfhydryl groups, which are buried in native beta-lactoglobulin, were exposed following irradiation and became available for quantification using the Ellman assay. The quantity of exposed sulfhydryls increased, but the number of total sulfhydryl groups decreased. Gel permeation chromatography showed that some protein aggregation occurred during irradiation. Fourier transform infrared (FTIR) spectroscopy of irradiated beta-lactoglobulin revealed changes in the secondary structure, comparable to that of early events during heat-induced denaturation. There was evidence for some photo-oxidation of tryptophan.     

Inhibitory effect of low partial pressures of acetylene on nitrification

The inhibiting effect of C₂H₂ on nitrification was investigated in two agricultural soils. Nitrification was totally inhibited at 10 Pa partial pressure of C₂H₂ which is lower than previously reported for soils. There were no differences in rates of nitrate production between flasks without C₂H₂ and flasks with C₂H₂ at 0.01 Pa, while there was an effect on nitrification at 0.1 PaC₂H₂. At 0.1 Pa no inhibition was noted during the first 3 days; after this period nitrification was partially inhibited. The inhibiting effect did not cease until 7 days after removal of C₂H₂. The sensitivity of nitrification to low concentrations of C₂H₂ should be noted when denitrification rates are determined by the use of the acetylene inhibition method (usually C₂H₂ at 10kPa).     

Role of lysine epsilon-amino groups of beta-lactoglobulin on its activating effect of Kluyveromyces lactis beta-galactosidase

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.     

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH,KIO(3) and N-ethylmaleimide

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.     

Antiviral activity of esterified alpha-lactalbumin and beta-lactoglobulin against herpes simplex virus type 1. Comparison with the effect of acyclovir and L-polylysines

The antiviral activity of methylated alpha-lactalbumin (Met-ALA), methylated and ethylated beta-lactoglobulins (Met- and Et-BLG) was evaluated against acyclovir (ACV)-sensitive and -resistant strains of herpes simplex virus type 1 (HSV-1) and compared to that of ACV and L-polylysines (4-15 kDa) using fixed or suspended Vero cell lines. Esterified whey proteins and their peptic hydrolyzates displayed protective action against HSV-1, which was relatively lower than that induced by ACV or L-polylysines. The higher activity of L-polylysines was maintained against an ACV-resistant strain of HSV-1, whereas ACV lost much of its activity. The mean 50% inhibitory concentration (IC50) was about 0.8-0.9 microg/mL for L-polylysines against ACV-sensitive and -resistant strains of HSV-1 when using two concentrations of virus (50% and 100% cytopathic effect, CPE). The IC50 values of ACV against the sensitive strain of HSV-1 were 3 and 15 microg/mL when using the low and high concentrations of virus, respectively. When using 50% CPE, IC50 values for esterified whey proteins ranged from 20 to 95 microg/mL, depending on the nature of the ester group, the degree of esterification, and the nature of the protein. Using the real-time PCR technique, it was shown that Met-ALA inhibited HSV-1 replication.     

Glycation and phosphorylation of beta-lactoglobulin by dry-heating: effect on protein structure and some properties

Beta-lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation.     

河西走廊富钾土壤钾肥效应及钾素平衡的长期定位研究

带状间作4年定位试验表明，增施化学K肥平均增产17．0％－20．3％，土粪基础上K肥增产时限推迟2年。连续18年小麦、玉米定位轮作试验表明，土壤K素能保证2个轮作期（每轮3年）作物K素需求，第3－6轮作期施K平均增产10．3％－30．7％，增效与试验历程呈正相关，增施土粪土壤K素平均每年亏缺135．0－335．5kg/hm^2；增施化学K肥平均每年亏缺97．4－335．7kg/hm^2；等量土粪和化学K肥配合施用年均亏缺76．5－268．0kg/hm^2。无K投入时，连续种植作物土壤速效钾年均减少2．6－17mg/kg，施土粪带田速效钾年均下降7.5mg/kg，轮作田略有提高。在土粪基础上增施化学K肥能保持和提高土壤速效钾，土壤缓效钾下降速率是速效钾的6.5-8.2倍。     

Use of catalyst in a 3D-QSAR study of the interactions between flavor compounds and beta-lactoglobulin

This paper reports a 3D-QSAR study using Catalyst software to explain the nature of interactions between flavor compounds and beta-lactoglobulin. A set of 35 compounds, for which dissociation constants were previously determined by affinity chromatography, was chosen. The set was divided into three subsets. An automated hypothesis generation, using HypoGen software, produced a model that made a valuable estimation of affinity and provided an explanation for the lack of correlation previously observed between the hydrophobicity of terpenes and the affinity for the protein. On the basis of these results, it appears that aroma binding to beta-lactoglobulin is caused by both hydrophobic interactions and hydrogen bonding, which plays a critical role. Catalyst appears to be a reliable tool for the application of 3D-QSAR study in aroma research.     

Effects of caseins on thermal stability of bovine beta-lactoglobulin

Casein fractions have been shown to act as molecular chaperones and inhibit aggregation of whey proteins in dilute solutions (< or =1% w/v). We evaluated if this approach would stabilize protein solutions at higher concentration and thermal processing temperatures desired for beverage applications. Mixtures of beta-lactoglobulin (BLG) (6% w/v) with either beta-casein (BCN) (0.01-2% w/v) or alpha s-casein (ACN) (2% w/v) were adjusted to pH 6.0 and heated (70-90 degrees C) for 20 min, cooled, and then analyzed to determine the degree of aggregation. Aggregation was determined by solution turbidity as optical density (OD) at 400 or 600 nm. The addition of 0.05% (w/v) BCN or greater caused a drop in turbidity for solutions heated at 70-90 degrees C. In contrast, inhibition was observed in BLG-ACN mixtures at 70 degrees C but not at > or =75 degrees C. Moreover, prolonged heating (90 min) of BLG with 2% (w/v) BCN (pH 6.0) at 90 degrees C produced a clear solution while BLG-ACN solutions formed translucent gels after heating for 15 min. The weight-averaged molar mass and root-mean-square (rms) radius of soluble aggregates were determined by size exclusion chromatography in conjunction with multiangle laser light scattering (SEC-MALS). SEC-MALS confirmed the turbidity results by showing that the BLG-BCN mixture (8% w/v protein) produced aggregates with lower molar mass and smaller rms radius (majority 20-40 nm). These results showed that BCN is a feasible component to stabilize higher concentrations of whey proteins in beverages.     

Spectrofluorometric characterization of beta-lactoglobulin B covalently labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonate.

Bovine beta-lactoglobulin, genetic variant B, has been labeled with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid through covalent attachment through the Cys-121 thiol group for the study of stepwise pressure denaturation of this whey protein by fluorescence spectroscopy. The labeling was performed under nondenaturing conditions with a factor of 5 excess of the fluorophore in dimethylformamide/water (1:10) to yield the whey protein highly labeled after chromatographic separation. MALDI-TOF mass spectroscopy confirmed labeling. The emission from the fluorophore, which is sensitive to the microenvironment, has been characterized for the labeled protein (aqueous pH 7.4 solution, 25 degrees C) and has a lambda(em,max) = 410 nm (lambda(ex,max) = 318 nm) with a fluorescence lifetime of 6.1 +/- 0.2 ns. Fluorescence anisotropy increases and fluorescence quantum yield (Phi(f) = 0.103 at 320 nm) decreases with increasing excitation wavelength. For increasing hydrostatic pressure, fluorescence quantum yield showed a minimum at approximately 50 MPa, corresponding to the pre-denatured "pressure-melted" state in which thiol reactivity previously was found to increase prior to reversible protein unfolding.     

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

Pressure-induced denaturation of monomer beta-lactoglobulin is partially irreversible: comparison of monomer form (highly acidic pH) with dimer form (neutral pH)

This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.     

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.     

Effect of pH on retention of aroma compounds by beta-lactoglobulin

Interactions between volatile compounds and BLG in aqueous solution were studied using static and dynamic headspace techniques (exponential dilution). The intensity of interactions between methyl ketones (C7-C9), ethyl esters (C6-C9), limonene, myrcene, and beta-lactoglobulin (BLG) were estimated by determination of the relative infinite dilution activity coefficients (gamma(r)). For a constant pH value, the methyl ketones retention by BLG increased significantly with the hydrophobicity of the volatiles, whereas the retention reached a maximum for ethyl octanoate in the ester series, indicating a possible steric hindrance. For limonene and myrcene an unexpected increase in headspace concentration or "salting out" effect was noticed for acid pH. The variations of the retention according to the pH increase of the medium from pH 3 to pH 11 could be related to structural modifications of the BLG. The retention increase observed between pH 3 and pH 9 resulted from the flexibility modification of the protein, allowing better accessibility to the primary or the secondary hydrophobic sites, whereas the dramatic decrease observed at pH 11 was the consequence of the alkaline denaturation of BLG. Electrostatic interactions occurring at pH 7.5 could also explain the observed retention increase.     

Binding of the pepper alkaloid piperine to bovine beta-lactoglobulin: circular dichroism spectroscopy and molecular modeling study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.