Role of amyloid type cross beta-structure in the formation of soluble aggregate and gel in heat-induced ovalbumin

The heat-induced denaturation curve of ovalbumin followed by the ellipticity at 222 nm in circular dichroism spectra was consistent with that monitored by fluorescence with thioflavin T, which is an indication of amyloid fibril formation, while other proteins such as lysozyme and ovotransferrin did not fluoresce with thioflavin T during heat denaturation. The amount of soluble aggregate formed during heat denaturation was proportional to the increase in fluorescence with thioflavin T. The binding of soluble aggregates with thioflavin T was greatly suppressed in heat-denatured ovalbumin in the presence of thioflavin T. The similar inhibition effect of thioflavin T on the gel formation of heat-induced ovalbumin was observed. These results suggest that the amyloidogenic intermolecular beta-structure is involved in the formation of soluble aggregate and gel of heat-induced ovalbumin.     

Methionine oxidation enhances κ-casein amyloid fibril formation

The effects of protein oxidation, for example of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.     

Re-formation of fibrils from hydrolysates of β-lactoglobulin fibrils during in vitro gastric digestion

In this study, in vitro digestion of β-lactoglobulin (β-Lg) fibrils and the re-formation of fibril-like structures after prolonged enzymatic hydrolysis (up to 48 h) were investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), thioflavin T fluorescence photometry, and transmission electron microscopy (TEM). Pure β-Lg fibrils that had been formed by heat treatment at pH 2.0 were rapidly hydrolyzed by pepsin in the simulated gastric fluid (pH 1.2), and some new peptides that were suitable for further fibril formation were produced. TEM showed that the new fibrils were long and straight but thinner than the original fibrils, and both TEM and MALDI-MS indicated that the peptides in the new fibrils were shorter/smaller than the peptides in the original fibrils. The formation of new fibrils was found to be affected more by pH than by enzyme activity or temperature.     

Effect of Specific Mechanical Energy on Protein Bodies and α-Zeins in Corn Flour Extrudates

Zeins, the storage proteins of corn, are located in spherical entities called protein bodies. The disruption of protein bodies and zein release during extrusion may influence the texture of corn-based extruded foods. In this work, chemical and microscopic studies were conducted on corn flour that had been extruded under mild to extreme conditions to determine the specific mechanical energy (SME) required to break apart protein bodies and release α-zein, and to assess changes in protein-protein interactions. Transmission electron microscopy with immunolocalization of α-zein revealed that starch granules and protein bodies remained intact under mild processing conditions (SME 35–40 kJ/kg), but under harsher conditions, protein bodies were disrupted and α-zein was released. At SME ≈100 kJ/kg, protein bodies appeared highly deformed and fused together with the α-zein released, whereas at higher SME, protein bodies were completely disrupted and α-zein was dispersed and may have formed protein fibrils. Protein in extrudates was less soluble in urea and SDS than in unprocessed corn flour, but it was readily extracted with urea, SDS, and 2-ME. This was likely due to protein aggregation upon processing due to a prevalence of hydrophobic interactions and disulfide bonds. This research directly relates SME during extrusion to chemical and structural changes in corn proteins that may affect the texture of corn-based, ready-to-eat food products.     

Characterization of soluble amaranth and soybean proteins based on fluorescence, hydrophobicity, electrophoresis, amino Acid analysis, circular dichroism, and differential scanning calorimetry measurements.

Intrinsic fluorescence (IF), surface hydrophobicity (S(o)), electrophoresis, amino acid analysis, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study folded and unfolded soluble proteins from Amaranthus hypochondriacus (A. h.) and soybean (S). Globulin (Glo) and albumin subfractions (Alb-1 and Alb-2) were extracted from A. h. and S and denatured with urea. Electrophoretic and functional properties indicated a significant correlation between soluble protein fractions from soybean and amaranth. The protein fractions shared some common electrophoretic bands as well as a similar amino acid composition. The larger percent of denaturation in protein fractions, which is associated with enthalpy and the number of ruptured hydrogen bonds, corresponds to disappearance of alpha-helix. The obtained results provided evidence of differences in their secondary and tertiary structures. The most stable was Glo followed by the Alb-2 fraction. Predicted functional changes in model protein systems such as pseudocereals and legumes in response to processing conditions may be encountered in pharmaceutical and food industries. These plants can be a substitute for some cereals.     

Effects of moisture-induced whey protein aggregation on protein conformation, the state of water molecules, and the microstructure and texture of high-protein-containing matrix

Moisture-induced protein aggregation through intermolecular interactions such as disulfide bonding can occur in a high-protein-containing food matrix during nonthermal processing and storage. The present study investigated the effect of moisture-induced whey protein aggregation on the structure and texture of such high-protein-containing matrices using a protein/buffer model system. Whey proteins in the protein/buffer model systems formed insoluble aggregates during 3 months' storage at temperatures varying from 4 to 45 degrees C, resulting in changes in microstructure and texture. The level of aggregation that began to cause significant texture change was an inverse function of storage temperature. The protein conformation and the state of water molecules in the model system also changed during storage, as measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. During storage, the model system that had an initially smooth structure formed aggregated particles (100-200 nm) as measured by scanning electron microscopy, which lead to an aggregation network in the high-protein-containing matrix and caused a harder texture.     

Apricot carotenoids possess potent anti-amyloidogenic activity in vitro

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the progressive accumulation of amyloid β protein (Aβ) in areas of the brain. There has been an increased interest in screening for food-grade anti-amyloidogenic compounds in foodstuffs. The purpose of this study was to screen and identify bioactive compounds with anti-amyloidogenicity in apricot fruits using synthetic Aβ(1-42). The anti-amyloidogenicity was investigated using thioflavin T fluorescence assay, electron microscopy, and dot blotting analysis. The carotenoid fraction from apricot showed strong inhibitory effects against oligomer and fibril formation of Aβ and fibril-destabilizing effects. Among the peaks in the HPLC chromatogram, lutein showed the strongest inhibitory effect on Aβ fibril formation. The inhibitory effect was dependent on the number and portion of hydroxyl groups on both sides of carotenoids. These findings suggest that lutein in fruits may be useful as a preventive agent for amyloid-associated diseases.     

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.     

Physicochemical modifications of high-pressure-treated soybean protein isolates

Changes induced by high pressure (HP) treatment (200-600 MPa) on soybean protein isolates (SPI) at pH 3 (SPI3) and pH 8 (SPI8) were analyzed. Changes in protein solubility, surface hydrophobicity (Ho), and free sulfhydryl content (SH(F)) were determined. Protein aggregation and denaturation and changes in secondary structure were also studied. An increase in protein Ho and aggregation, a reduction of free SH, and a partial unfolding of 7S and 11S fractions were observed in HP-treated SPI8. Changes in secondary structure were also detected, which led to a more disordered structure. HP-treated SPI3 was partially denatured and presented insoluble aggregates. A major molecular unfolding, a decrease of thermal stability, and an increase of protein solubility and Ho were also detected. At 400 and 600 MPa, a decrease of the SH(F) and a total denaturation were observed.     

Heat treatment of beta-lactoglobulin: structural changes studied by partitioning and fluorescence

Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.     

Hypochlorous and peracetic acid induced oxidation of dairy proteins

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.     

Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.     

Interface characterization and aging of bovine serum albumin stabilized oil-in-water emulsions as revealed by front-surface fluorescence

This paper is devoted to the application of front-surface fluorescence to the study of aging and oxidation of oil-in-water emulsions. Emulsions with two oil droplet sizes were stabilized with bovine serum albumin (BSA) and stored at 37 or 47 degrees C. Lipid oxidation was demonstrated by measurement of hydroperoxides and headspace pentane. Front-surface fluorescence spectra (excitation wavelength = 355 nm) revealed gradual formation of oxidized lipid-protein adducts during the 4 weeks of storage. Fluorescence (excitation = 290 nm) of BSA tryptophanyl residues (Trp) declined during the first day of aging and then decreased slightly and linearly. Fourth-derivative Trp spectra exhibited peaks at 316 and 332 nm. Their evolution indicated that the ratio of Trp in hydrophobic environments to total Trp increased in small droplet emulsions. This suggests that, during lipid oxidation, the adsorbed and nonadsorbed protein underwent various degrees of Trp degradations, polymerization, and aggregation. Thus, front-surface fluorescence makes it possible to evaluate, noninvasively, protein modification and lipid oxidation end-products during processing and storage of food emulsions.     

Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of beta-lactoglobulin

The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.     

Rapid fingerprinting of milk thermal processing history by intact protein mass spectrometry with nondenaturing chromatography

Thermal processing of foods results in proteins undergoing conformational changes, aggregation, and chemical modification notably with sugars via the Maillard reaction. This can impact their functional, nutritional, and allergenic properties. Native size-exclusion chromatography with online electrospray mass spectrometry (SEC-ESI-MS) was used to characterize processing-induced changes in milk proteins in a range of milk products. Milk products could be readily grouped into either pasteurized liquid milks, heavily processed milks, or milk powders by SEC behavior, particularly by aggregation of whey proteins by thermal processing. Maillard modification of all major milk proteins by lactose was observed by MS and was primarily present in milk powders. The method developed is a rapid tool for fingerprinting the processing history of milk and has potential as a quality control method for food ingredient manufacture. The method described here can profile milk protein oligomeric state, aggregation, and Maillard modification in a single shot, rapid analysis.     

Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing

During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

Oxidation of skeletal muscle myofibrillar proteins in oil-in-water emulsions: interaction with lipids and effect of selected phenolic compounds

The effect of selected phenolic compounds, namely, gallic acid, cyanidin-3-glucoside, (+)-epicatechin, chlorogenic acid, genistein and rutin (50 and 200 microM), and alpha-tocopherol (50 microM) against the oxidation of oil-in-water emulsions (37 degrees C/10 days) containing 1% myofibrillar proteins (MPs), was investigated. Emulsions containing 1% bovine serum albumin (BSA) were also prepared for comparative purposes. Protein oxidation was assessed by measuring the loss of natural tryptophan fluorescence and the protein carbonyl gain by using fluorescence spectroscopy. Lipid oxidation was concurrently analyzed by measuring the increase of conjugated dienes (CDs) and hexanal. Proteins inhibited lipid oxidation in oil-in-water emulsions, and MPs showed a more intense antioxidant activity than BSA. MPs were also more resistant to oxidative deterioration than BSA. The different antioxidant capacity of MPs and BSA and their susceptibility to suffer oxidative reactions might be derived from their different amino acid composition and three-dimensional structures. The addition of the phenolic compounds resulted in a variety of effects, including both antioxidant and pro-oxidant effects. Gallic acid, cyanidin-3-glucoside, and genistein were the most efficient inhibitors of lipid and protein oxidation. The chemical structure of the phenolic compounds as well as the nature and conformation of the proteins were greatly influential on the overall effect against oxidative reactions.     

Growth kinetics of amyloid-like fibrils derived from individual subunits of soy β-conglycinin

The amyloid-like fibrillation of soy β-conglycinin subunits (α, α', and β) upon heating (0-20 h) at 85 °C and pH 2.0 was characterized using dynamic light scattering, circular dichroism (CD), binding to amyloid dyes (Thioflavin T and Congo red), and atomic force microscopy. The fibrillation of all three subunits was accompanied by progressive polypeptide hydrolysis. The hydrolysis behaviors, fibrillation kinetics, and morphologies of amyloid-like fibrils considerably varied among α, α', and β subunits. Faster hydrolysis rates and special fragments were observed for the α and α' subunits compared to the β subunit. However, the order of the fibrillation rate and capacity to form β-sheets was α' > β > α, as evidenced by CD and Thioflavin T data. Moreover, sequential growth of twisted screw-structure fibrils, leading to macroscopic fibrils with distinct morphological characteristics, was observed for β-conglycinin and individual subunits. The different fibrillation kinetics and morphologies of α, α', and β subunits appear to be associated with the differences in the amino acid composition and typical sequence of peptides. Besides, the disruption of ordered structure of fibrils occurred upon further heating (6-20 h) due to extensive hydrolysis. These results would suggest that all subunits are involved in the fibrillation of β-conglycinin, following multiple steps including polypeptide hydrolysis, assembly to amyloid structure, and growth into macroscopic fibrils with a fibril shaving process.