Emulsification Capacity of Microgels Assembled from β-Lactoglobulin and Pectin

Microgels formed from beta-lactoglobulin were used to prepare oil-in-water emulsions in order to examine their emulsifying capacity. Corn oil emulsions prepared with microgels of pure beta-lactoglobulin at pH 5.8 were initially stable, but a fraction of the droplets quickly flocculated to form a creamed layer that could not be dispersed by shear, which was attributed to hydrophobic attractions between the microgels on adjoining droplets. Emulsions prepared from microgels of beta-lactoglobulin and pectin at pH 4.75 possessed greater droplet sizes at lower concentrations, yet all emulsions were relatively stable to irreversible flocculation. Increased stability of emulsions stabilized by BP-gels was attributed to the presence of pectin on the surface of microgels, which increased repulsions between adjoining droplets. Stable corn oil emulsions were still prepared from microgels that were previously dialyzed to remove non-aggregated protein, which verified that the microgels were responsible for stabilizing emulsion droplets. Equilibrium surface pressure of corn oil droplets was similar between microgels and the unheated beta-lactoglobulin and pectin, yet the dynamic surface pressure was reduced at intermediate times and indicated a slow relaxation and deformation of the microgels at the interface. Microgels formed with pectin stabilized emulsions containing 90 % limonene for up to 5 days of room temperature storage, demonstrating the capacity of such protein microgels to stabilize flavor oil emulsions.     

Enzyme-Induced Formation of β-Lactoglobulin Fibrils by AspN Endoproteinase

This paper describes a low temperature, enzymatic route to induce fibrillar structures in a protein solution. The route comprises two steps. First, β-lactoglobulin was hydrolyzed into peptides at pH 8 and 37 °C with the enzyme AspN endoproteinase, which resulted in the formation of random aggregates. After hydrolysis, the pH was lowered to 2. As a result, long fibrillar aggregates were formed which was observed using transmission electron microscopy and Thioflavin T fluorescence measurements.     

Stability of Biopolymer Particles Formed by Heat Treatment of β-lactoglobulin/Beet Pectin Electrostatic Complexes

The purpose of this study was to examine the stability of biopolymer particles formed by heating electrostatic complexes of β-lactoglobulin and sugar beet pectin together (pH 5, 80 °C for 15 min). The effects of electrostatic interactions on the formation and stability of the particles were investigated by incorporation of different salt levels (0 to 200 mM NaCl) during the preparation procedure. Biopolymer particles were characterized by turbidity, electrophoretic mobility, dynamic light scattering, and visual observance. Salt inclusion (≥25 mM) prior to heating β-lactoglobulin/pectin complexes led to the formation of large biopolymer particles (d > 1,000 nm) that rapidly sedimented, but salt inclusion after heating (0 to 200 mM) led to the formation of biopolymer particles that remained relatively small (d < 350 nm) and were stable to sedimentation. The biopolymer particles formed in the absence of salt remained stable over a wide range of pH values (e.g., pH 3 to 7 in the presence of 200 mM NaCl). These biopolymer particles may therefore be suitable for application in a number of food products as delivery systems, clouding agents, or texture modifiers.     

Formation of β-Lactoglobulin Nanofibrils by Microwave Heating Gives a Peptide Composition Different from Conventional Heating

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of β-lactoglobulin (β-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of β-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact β-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of β-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of β-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.     

Interaction of Curcumin and Diacetylcurcumin with the Lipocalin Member β-Lactoglobulin

The binding of curcumin (CUR) and diacetylcurcumin (DAC) to bovine beta-lactoglobulin (BLG) genetic variant B was investigated by fluorescence and circular dichroism techniques. The binding parameters including number of substantive binding sites and the binding constants have been evaluated by fluorescence quenching method. The distance (r) between donor (BLG) and acceptor (CUR and DAC) was obtained according to the Förster’s theory of non-radiative energy transfer. The far-UV circular dichroism spectra were used to investigate the possible changes in the secondary structure of BLG in the presence of CUR and DAC and showed that these two ligands change the α-helix and random coil contents of this protein to some extent. The visible circular dichroism spectra indicated that the optical activity during the ligand binding was observed due to the induced-protein chirality. All of the achieved results suggested the important role of the phenolic OH group of CUR in the binding process resulted in more affinity of CUR than DAC for binding to BLG.     

Interactions Between Esterified Whey Proteins (α-Lactalbumin and β-Lactoglobulin) and DNA Studied by Differential Spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated β-lactoglobulin and of methylated α-lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated β-casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Improvement of the Surface Functional Properties of β-Lactoglobulin and α-Lactalbumin by Heating in a Dry State

Controlled heating in a dry state greatly improved the surface functional properties of whey proteins (β-lactoglobulin and α-lactalbumin). Although whey proteins were completely insolubilized by heating at 80°C in an aqueous solution, their solubility was kept even after heating at 80°C in a dry state (7.5% moisture content) for 5 days. The surface hydrophobicity of α-lactalbumin was increased during the dry-heating, while that of β-lactoglobulin was decreased. In addition, the fluorescence spectra excited at 280 nm of dry-heated whey proteins suggested the significant conformational changes. High-performance gel chromatography showed that a considerable amount of soluble aggregates was formed in the dry-heated β-lactoglobulin, while a small amount of soluble aggregate was observed in the dry-heated α-lactalbumin. The foaming properties of dry-heated whey proteins were increased to about 3 times that of untreated proteins. The emulsifying properties of dry-heated whey proteins were also increased, compared to untreated proteins, although a slight decrease in the emulsion stability was observed in dry-heated β-lactoglobulin. The improvement of the surface properties seemed to come from the partial unfolding suitable for the formation of foam film and the entrapment of oil droplets.     

The Antigenic Properties of β-Lactoglobulin Examined with Mouse IgE Antibody

The effects of enzymic or chemical fragmentations and of chemical modifications on the antigenic properties of bovine β-Mactoglobulin were examined using specific mouse IgE antibody. The antigenic reactivity of β-Mactoglobulin derivatives was represented in terms of their ability to neutralize specific IgE antibodies assayed by passive cutaneous anaphylaxis in rat. The tryptic, chymotryptic or peptic hydrolysate free of native β-Mactoglobulin had no antigenic reactivity but the fragments obtained after CNBr cleavage retained the ability to bind the antibody. Modification of the sulfhydryl group, arginine or tryptophan residues and amino groups had no effect on antigenic reactivity but a little decrease in the reactivity was observed on the cleavage of the two disulfide bridges. These results suggest that one sulfhydryl group, two arginine and tryptophan residues and most of the amino groups are out of antigenic sites in β-Mactoglobulin and that the antigenicity depends on the conformation maintained by the disulfide bridges.     

Heat Treatment and β-Carotene Incorporation Effect in the Interaction of β-Lactoglobulin and Carboxymethylcellulose System

Encapsulation technologies using proteins or polysaccharides can be employed with the purpose of solubilizing and protecting carotenoids. However, information on the role of protein and polysaccharide interactions is still slightly limited. The aim of this work was to investigate the effect of β-carotene linked to protein β-lactoglobulin (BLG) in the interaction carboxymethylcellulose (CMC) using isothermal titration calorimetry (ITC). Firstly, BLG and CMC interaction was assessed by means of turbidity analysis. Based on the results of turbidity, the thermodynamic profile of BLG-CMC complexes at pH 4.0 was obtained using ITC analysis at 25 °C. Afterward, it was evaluated the effect of a thermal treatment applied to the BLG (68 °C for 50 min) in the interaction with CMC also using ITC and circular dichroism (CD). ITC and CD analysis showed that the heat treatment applied on BLG did not cause changes in molecular interactions. The binding isotherm of BLG-CMC complexes incorporated with β-carotene showed an increase in the molar ratio and a slight decrease in enthalpy of the system. Incorporation of β-carotene in the system did not significantly affect the BLG and CMC interaction, suggesting this system can be applied in food application as encapsulation.     

Competition between Folding, Native-State Dimerisation and Amyloid Aggregation in β-Lactoglobulin

We show that a series of peptides corresponding to individual β-strands in native β-lactoglobulin readily form amyloid aggregates and that such aggregates are capable of seeding fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are reduced. By contrast, preformed fibrils corresponding to only one of the β-strands that we considered, βA, were found to promote fibril formation by a full-length form of β-lactoglobulin in which the disulfide bonds are intact. These results indicate that regions of high intrinsic aggregation propensity do not give rise to aggregation unless at least partial unfolding takes place. Furthermore, we found that the high aggregation propensity of one of the edge strands, βI, promotes dimerisation of the native structure rather than misfolding and aggregation since the structure of βI is stabilised by the presence of a disulfide bond. These findings demonstrate that the interactions that promote folding and native-state oligomerisation can also result in high intrinsic amyloidogenicity. However, we show that the presence of the remainder of the sequence dramatically reduces the net overall aggregation propensity by negative design principles that we suggest are very common in biological systems as a result of evolutionary processes.     

Genetic Polymorphism of the β-Lactoglobulin Gene in Native Sheep from India

The genetic polymorphism of the β-lactoglobulin (β-LG) gene was determined in 638 animals belonging to 15 native Indian sheep breeds reared in different agroecological regions for various production traits. Variants of β-LG were found using PCR–RFLP of genomic DNA. Rsa1 restriction enzyme digestion of a 120-bp PCR fragment of exon 2 of β-LG revealed two genetic variants, A (0.37) and B (0.63), and the three genotypes AA (0.175), AB (0.389), and BB (0.436). The differences in allelic frequency were not significant across the breeds, irrespective of their geographic origin and utility (χ² test, P > 0.05). The pattern of occurrence of allelic variants revealed that the B allele was more frequent in the majority of the Indian breeds than in breeds reported from countries of Southwest Asia, Eastern and Central Europe, and the Mediterranean. A higher level of heterozygosity (0.422) was discerned, despite the declining status of several of the Indian breeds. These findings revealed that Indian sheep are predominantly of the β-LG B type.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Chemical Cleavage of Bovine β-Lactoglobulin by BNPS-Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine β-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1–19, 20–61, 62–162) and incomplete cleaved fragments (1–61, 20–162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47°C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1–19 and 62–162. The chemical side reactions identified are discussed.     

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.     

Characterization of β-Lactotensin,a Bioactive Peptide Derived from Bovine β-Lactoglobulin,as a Neurotensin Agonist

β-Lactotensin (β-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine β-lactoglobulin. The ileum-contracting activity of β-LT was blocked by the NT₁ antagonist SR48692. β-LT was selective for the neurotensin NT₂ receptor while neurotensin was selective for the NT₁ receptor. β-LT is the first natural ligand showing selectivity for the NT₂ receptor. β-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of β-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT₂ antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of β-LT. These results suggest that the hypertensive activity of β-LT is mediated by the NT₂ receptor. It was concluded that the NT₁ and NT₂ receptors mediate the opposite effect on blood pressure.     

Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum)

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.     

Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases

p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H₂ ¹⁸O to liberate galactose containing ¹⁸O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D ¹H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.     

Selective inhibition of β-N-acetylhexosaminidases by thioglycosyl–naphthalimide hybrid molecules

To develop selective inhibitors for β-N-acetylhexosaminidases which are involved in a myriad of physiological processes, a series of novel thioglycosyl–naphthalimide hybrid inhibitors were designed, synthesized and evaluated for inhibition activity against glycosyl hydrolase family 20 and 84 (GH20 and GH84) β-N-acetylhexosaminidases. These compounds which incorporate groups with varied sizes and lengths at the linker region between thioglycosyl moiety and naphthalimide moiety are designed to improve the selectivity and stacking interactions. The GH84 human O-GlcNAcase (hOGA) was sensitive to the subtle changes in the linker region and the optimal choice is a small size linker with six atoms length. And the GH20 insect β-N-acetylhexosaminidase OfHex1 could tolerate compounds with a hydrophobic bulky linker. Especially, the compound 5c (hOGA, K_i?=?3.46?μM; OfHex1, K_i?>?200?μM) and the compound 6f (hOGA, K_i?>?200?μM; OfHex1, K_i?=?21.81?μM) displayed high selectivity. The molecular docking results indicated that the inhibition mechanism was different between the two families due to their different structural characteristics beyond the active sites. These results provide some promising clues to improve selectivity of potent molecules against β-N-acetylhexosaminidases.     

Splitting of naphthol AS-BI β-galactopyranoside by acid β-galactosidase

Summary -Galactosidase hydrolyses naphthol AS-BI -galactopyranoside in a variety of rat and mouse organs using freeze-dried cryostate sections, hexazonium-p-rosaniline for simultaneous coupling and long time incubation. In comparison with the indolyl and naphthyl derivate the splitting rate of the naphthol AS compound is far lower.