Effects of High Intensity Ultrasound on Physiochemical and Structural Properties of Goat Milk ß-Lactoglobulin.

This study aimed to compare the effects of high intensity ultrasound (HIU) applied at various amplitudes (20~40%) and for different durations (1~10 min) on the physiochemical and structural properties of goat milk ß-lactoglobulin. No significant change was observed in the protein electrophoretic patterns by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deconvolution and second derivative of the Fourier transform infrared spectra (FTIR) showed that the percentage of ß-sheet of goat milk ß-lactoglobulin was significantly decreased while those of α-helix and random coils increased after HIU treatment The surface hydrophobicity index and intrinsic fluorescence intensity of samples was enhanced and increased with increasing HIU amplitude or time. Differential scanning calorimetry (DSC) results exhibited that HIU treatments improved the thermal stability of goat milk ß-lactoglobulin. Transmission electron microscopy (TEM) of samples showed that the goat milk ß-lactoglobulin microstructure had changed and it contained larger aggregates when compared with the untreated goat milk ß-lactoglobulin sample. Data suggested that HIU treatments resulted in secondary and tertiary structural changes of goat milk ß-lactoglobulin and improved its thermal stability.

Structure and allergenicity assessments of bovine ß-lactoglobulin treated by sonication-assisted irradiation.

Bovine ß-lactoglobulin (ß-LG) is recognized as a major allergen in milk. This study aimed to investigate ultrasound-assisted irradiation for reducing the allergenicity of ß-LG, since irradiation can reduce the allergenicity of cow milk proteins and ultrasound can improve the quality of milk. The structural changes induced in high purity ß-LG, treated by irradiation, with or without sonication, were characterized by native PAGE, circular dichroism spectroscopy, and fluorescence spectroscopy. The changes in allergenicity were measured by IgE binding capacity to, and inflammatory mediator secretion by, human basophil KU812 cells. Surface hydrophobicity was reduced and aggregation of ß-LG increased after treatment by irradiation, both with and without sonication. The IgE binding capacity and release of inflammatory mediators were reduced significantly and the reduction induced by irradiation before sonication was the greatest, suggesting that irradiation after sonication can be a safe and effective method to reduce the allergenicity of ß-LG in dairy processing.

Protection of resveratrol against the photodecomposition of folic acid and photodecomposition-induced structural change of beta-lactoglobulin.

Folic acid is a synthetic form of the B-group vitamin known as folate and essential for a variety of physiological processes. However, it decomposes under UV irradiation, causing indirect oxidation and structural change of protein. In this study, the protective effect of resveratrol against the photodecomposition of folic acid and its caused protein structural change was investigated by using fluorescence and absorbance spectroscopy, high performance liquid chromatography and ABTS assay. It was found that resveratrol could inhibit the folic acid decomposition and control the decomposition process, depending on the polyphenol concentration and addition time. Transformation of resveratrol was accelerated by photodecomposition of folic acid. Antioxidant activity of resveratrol was important for the protective effect. Moreover, resveratrol could also inhibit the unfolding of beta-lactoglobulin caused by the folic acid decomposition by using circular dichroism.

Improved digestibility of ß-lactoglobulin by pulsed light processing: a dilatational and shear study.

Modifying the protein conformation appears to improve the digestibility of proteins in the battle against allergies. However, it is important not to lose the protein functionality in the process. Light pulse technology has been recently tested as an efficient non-thermal process which alters the conformation of proteins while improving their functionality as stabilizers. Also, in order to rationally design emulsion based food products with specific digestion profiles, we need to understand how interfacial composition influences the digestion of coated interfaces. This study has been designed to investigate the effects of pulsed light (PL) treatment on the gastrointestinal digestion of protein covered interfaces. We have used a combination of dilatational and shear rheology which highlights inter and intra-molecular interactions providing new molecular details on protein digestibility. The in vitro digestion model analyses sequentially pepsinolysis, trypsinolysis and lipolysis of ß-lactoglobulin (BLG) and pulsed light treated ß-lactoglobulin (PL-BLG). The results show that the PL-treatment seems to facilitate digestibility of the protein network, especially regarding trypsinolysis. Firstly, PL treatment just barely enhances the enzymatic degradation of BLG by pepsin, which dilutes and weakens the interfacial layer, due to increased hydrophobicity of the protein owing to PL-treatment. Secondly, PL treatment importantly modifies the susceptibility of BLG to trypsin hydrolysis. While it dilutes the interfacial layer in all cases, it strengthens the BLG and weakens the PL-BLG interfacial layer. Finally, this weakening appears to slightly facilitate lipolysis as evidenced by the results obtained upon addition of lipase and bile salts (BS). This research allows identification of the interfacial mechanisms affecting enzymatic hydrolysis of proteins and lipolysis, which demonstrates an improved digestibility of PL-BLG. The fact that PL treatment did not affect the functionality of the protein makes it a valuable alternative for tailoring novel food matrices with improved functional properties such as decreased digestibility, controlled energy intake and low allergenicity.

Magnetic-responsive hybrids of Fe3O4 nanoparticles with ß-lactoglobulin amyloid fibrils and nanoclusters.

We report on the synthesis and magnetic-responsive behavior of hybrids formed by dispersing negatively charged iron oxide (Fe3O4) magnetic nanoparticles in positively charged ß-lactoglobulin protein solutions at acidic pH, followed by heating at high temperatures. Depending on the pH used, different hybrid aggregates can be obtained, such as nanoparticle-modified amyloid fibrils (pH 3) and spherical nanoclusters (pH 4.5). We investigate the effect of magnetic fields of varying strengths (0-5 T) on the alignment of these Fe3O4-modified amyloid fibrils and spherical nanoclusters using a combination of scattering, birefringence and microscopic techniques and we find a strong alignment of the hybrids upon increasing the intensity of the magnetic field, which we quantify via 2D and 3D order parameters. We also demonstrate the possibility of controlling magnetically the sol-gel behavior of these hybrids: addition of salt (NaCl, 150 mM) to a solution containing nanoparticles modified with ß-lactoglobulin amyloid fibrils (2 wt % fibrils modified with 0.6 wt % Fe3O4 nanoparticles) induces first the formation of a reversible gel, which can then be converted back to solution upon application of a moderate magnetic field of 1.1 T. These hybrids offer a new appealing functional colloidal system in which the aggregation, orientational order and rheological behavior can be efficiently controlled in a purely noninvasive way by external magnetic fields of weak intensity.

Structural changes and allergenic properties of ß-lactoglobulin upon exposure to high-intensity ultrasound.

SCOPE: The aim of this work was to investigate the effects of high-intensity ultrasound (sonication), on the structure and allergenicity of the major cow's milk allergen, beta-lactoglobulin (BLG). METHODS AND RESULTS: Structural changes upon sonication of BLG were monitored by circular dichroism spectroscopy, tryptophan emission fluorescence, hydrophobic dye and retinol binding, as well as digestibility and phenol-oxidase cross-linking capacity. Allergenicity was monitored in individual patients' sera, basophil activation test, and skin prick testing in 41 cow's milk allergy patients. Uncontrolled local temperature changes induced modifications in BLG secondary structure accompanied by formation of dimers, trimers, and oligomers of BLG that were more digestible by pepsin and had reduced retinol binding. Controlled temperature conditions induced changes in secondary structure of BLG without causing formation of oligomers, or changing protein's capacity to bind retinol. Both sonicated forms of BLG had more exposed hydrophobic surfaces than native BLG and underwent facilitated cross-linking reaction with phenol-oxidase. Sonication had a minor effect on IgE-binding properties of BLG. CONCLUSION: Sonication-induced structural changes in major whey allergen were not clinically significant in cow's milk allergy patients. Ultrasound can be a safe procedure for dairy processing as it maintains the nutritional value and does not increase allergenic potential of BLG.

Photoinduced unfolding of beta-lactoglobulin mediated by a water-soluble porphyrin.

We investigated the effects that the irradiation of a tetra-anionic porphyrin (mesotetrakis(sulfonatophenyl)porphyrin) noncovalently bound to beta-lactoglobulin (BLG) produces on the conformation of the protein. Although BLG is not a potential target for the biomedical applications of porphyrins, it is a useful model for investigating the effects of photoactive ligands on small globular proteins. We show in this paper that irradiation causes a large unfolding of the protein and that the conformational change is not mediated by the formation of reactive oxygen species. Instead, our data are consistent with an electron-transfer mechanism that is capable of triggering structural changes in the protein and causes the Trp19 residue to undergo chemical modifications to form a derivative of kynurenine. This demonstrates that protein unfolding is prompted by a type-III photosensitizing mechanisms. Type-III mechanisms have been suggested previously, but they have been largely neglected as useful mediators of biomolecular damage. Our study demonstrates that porphyrins can be used as mediators of localized protein conformational changes and that the biomedical applications as well as the mechanistic details of electron transfer between exogenous ligands and proteins merit further investigation.

Impact of gamma-radiation on antigenic properties of cow's milk beta-lactoglobulin.

This study evaluated the effects of gamma-radiation on the antigenic properties of beta-lactoglobulin in cow's milk. Liquid and lyophilized samples of cow's milk and whey were irradiated with gamma-cells (60Co) at dose levels of 3, 5, and 10 kGy, at room temperature in the presence of air. Effects of treatment on proteins were monitored by Lowry's method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. Radiation did not affect the molecular-weight distributions of proteins, but it did reduce their solubility. Furthermore, results showed that irradiation at 10 kGy increased the recognition of milk and whey powders by anti-beta-lactoglobulin (beta-Lg) rabbit immunoglobulin G, with the other samples remaining antigenically stable. These results indicate that gamma-rays do not reduce cow's milk beta-lactoglobulin antigenicity.

Effects of gamma radiation on beta-lactoglobulin: oligomerization and aggregation.

The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.

Electric birefringence study of an amyloid fibril system: the short end of the length distribution.

In this article, a system of amyloid fibrils, based on the protein beta-lactoglobulin, is studied by transient electric birefringence. Single pulses of an electric field were applied to the solution, and the initial rise and subsequent decay of birefringence analysed. The decay takes place on a range of relaxation times, and therefore contains information about the length distribution of fibrils in the system. The information can be extracted using theories of the electric polarisability of polyelectrolyte rods, since the fibrils are an example of these. Despite the long-standing complications of such theories, useful quantitative information about the system can still be obtained. Using the Fixman model of polyelectrolyte polarisability, we obtain a measurement of the short end of the length distribution which shows the fibril concentration as a function of length rising linearly from 0.02-2 microm. The short end of the length distribution was unobtainable in our previous study using rheo-optics (S.S. Rogers et al., Macromolecules 38, 2948 (2005)), but reasonable agreement between the two techniques shows they are complementary.

Effects of gamma radiation on the allergenic and antigenic properties of milk proteins.

This study was carried out to evaluate the application of food irradiation technology as a method for reducing milk allergies. Bovine alpha-casein (ACA) and beta-lactoglobulin (BLG) were used as milk proteins. Using milk-hypersensitive patients' immunoglobulin E (IgE) and rabbit IgGs individually produced to ACA and BLG, the changes of allergenicity and antigenicity of irradiated proteins were observed by competitive indirect enzyme-linked immunosorbent assay. Allergenicity and antigenicity of the irradiated proteins were changed with different slopes of the inhibition curves. The disappearance of the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increase of the turbidity showed that solubility of the proteins decreased by radiation, and this decrease might be caused by agglomeration of the proteins. These results indicated that epitopes on milk allergens were structurally altered by gamma irradiation.

Microwave enhanced kinetics observed in ORD studies of a protein.

Microwaves are shown to affect the kinetics of conformational changes of the protein beta-lactoglobulin. Microwaves can accelerate conformational changes in the direction towards the equilibrium state. This applies both for the folding and the unfolding processes. Cold denaturing thermal unfolding of the proteins is accelerated by negative temperature gradients. Microwave irradiation of the protein solution heated it by about 0.3 degree, and hence the observed acceleration of denaturing is therefore non-thermal.

The conformational status of a protein influences the aerobic photolysis of its tryptophan residues: melittin, beta-lactoglobulin and the crystallins.

We have studied the aerobic photolysis of the tryptophan residues of the proteins melittin and beta-lactoglobulin when the proteins are in ordered conformations and when they are in randomly coiled states. The results suggest that the conformational status of the protein is a factor that influences the photolysis of the constituent tryptophan residues. This point appears to be of relevance to the photo-oxidation of the tryptophan residues of the eye lens proteins crystallins.

Oxidative radioiodination damage to human lactoferrin.

Oxidative iodination of human lactoferrin (Lf) as commonly performed by using the chloramine-T, the Iodogen or the lactoperoxidase method produces an unreliable tracer protein because of excessive and heterogeneous polymer formation. Before iodination a minor tetramer fraction may be demonstrable in iron-saturated Lf only. Iodination-induced polymerization of iron-poor as well as iron-saturated Lf occurs independently of the presence or absence of 10 mM-EDTA and the 125I-/Lf molar ratio used for iodination. 125I-Lf polymers are mainly covalently linked, as suggested by the lack of substantial dissociation in SDS/polyacrylamide-gel electrophoresis. Damage to the 125I-Lf monomer may be another consequence of oxidative iodination. This is demonstrated in SDS/polyacrylamide-gel electrophoresis where 50% of the radioactivity of apparently normal monomer (Mr 75,000) is displaced to a lower-Mr region (30,000-67,000) after reduction with dithiothreitol. Non-oxidative iodination by the Bolton-Hunter technique produces an antigenetically stable tracer that is not being subjected to polymerization and monomer degradation as judged by high-performance gel chromatography and SDS/polyacrylamide-gel electrophoresis with and without dithiothreitol treatment. It is concluded that oxidation in itself leads to covalent non-disulphide cross-linking between human Lf molecules and, possibly, to intramolecular peptide-bond breaking becoming unmasked under reducing conditions. In biological experiments with human 125I-Lf this problem should be carefully considered.

[Changes in the total protein and protein fractions in cow's milk irradiated with ultraviolet rays]. / Promeni v obshtiia beltuk i beltuchnite fraktsii v krave mliako, oblucheno s ultravioletovi luchi

Comparative experimental electrophoretic studies were carried out to establish the effect of ultraviolet treatment on cow's milk at various rates of irradiation (5, 10 and 15 min) with a view to raising and regulating the content of the total protein and the protein fractions in milk intended for the feeding of calves. Results showed that there were no substantial changes in the level of the total protein, the albumins having a rising trend that was manifested best at a 15-minute treatment. Most characteristic were the changes taking place in the immunoglobulin fraction which rose by 4.7 per cent. The beta-lactoglobulin fraction dropped by 4.5 per cent, on an average, at all rates of treatment, and the level of alfa-lactoglobulin at 5- and 10-minute irradiation underwent almost no changes, it rising by about 2.09 per cent, on an average, at 15-min treatment. Similar was the trend manifested by the proteoso-pepton fraction showing a rise as early as the first ten minutes of treatment.

Photolysis accompanying peptide absorption in proteins. Electrophoresis and optical rotary dispersion studies.

Exposure of proteins and polypeptides to ultraviolet radiation below 240 nm produces peptide cleavage which may or may not be accompanied by observable changes in conformation and optical rotary dispersion (ORD) properties, depending on the stability of the secondary and tertiary structure of the macromolecule under the experimental conditions. Helical and coiled forms of poly-L-glutamic acid undergo degradation at similar rates but only the helical form shows a significant change in rotatory properties. The helical form of poly-L-lysine, but neither the coiled nor beta forms, shows a change in [alpha](233) on irradiation at 233 nm. beta-Lactoglobulin shows essentially no change in [alpha](233) on irradiation in either dilute salt solution or 4 M urea at room temperature; however, in 4 M urea at 56 degrees C a large change occurs. A model is developed which shows that studies of the effect of radiation on ORD properties may be useful in providing information on possible intermediate steps in protein denaturation. The method is illustrated with results on bovine plasma albumin. A quantum yield, 4.3 x 10(-3) moles/einstein, was obtained for peptide cleavage in this protein at 225 nm. These studies, based on gel electrophoresis, also showed that the fragments produced are essentially random, suggesting that transfer of energy from aromatic residues is not an important contributor to the peptide photolysis. Possible errors which could arise in ORD and other studies involving intense ultraviolet radiation are considered.