Aggregation behaviour of bovine serum albumin as a cause of sauce liquid separation by heating

Heating white sauce with bovine serum albumin (BSA) at 90 °C caused the sauce to separate into aggregates and liquid phase, although this did not occur at 75 °C. Data from differential scanning calorimetry of BSA solution suggested that heat denaturation of BSA was insufficient at 75 °C but was complete at about 90 °C. Larger aggregates of BSA were formed by heating at 90 °C compared with 75 °C, as shown by gel permeation chromatography combined with a multi‐angle laser light‐scattering detector. Dynamic viscoelasticity measurement showed a higher storage modulus of BSA solution formed by heating at 90 °C than at 75 °C. Scanning electron microscopy revealed a random agglomerate structure of aggregates (spherical aggregates) obtained by 75 °C heating, while a well‐developed network structure containing voids was observed after heating at 90 °C. These findings suggest that sauce liquid separation induced by heating at 90 °C is due to encapsulation and restriction of white sauce components by large structured aggregates of BSA containing voids. Aggregates generated at 75 °C were not sufficiently developed and structured; therefore there was no sauce liquid separation due to encapsulation and restriction of components. © 2000 Society of Chemical Industry     

Effects of added whey protein aggregates on textural and microstructural properties of acidified milk model systems

Non-fat milk model systems containing 5% total protein were investigated with addition of micro- or nanoparticulated whey protein at two levels of casein (2.5% and 3.5%, w/w). The systems were subjected to homogenisation (20 MPa), heat treatment (90 °C for 5 min) and chemical (glucono-delta-lactone) acidification to pH 4.6 and characterised in terms of denaturation degree of whey protein, particle size, textural properties, rheology and microstructure. The model systems with nanoparticulated whey protein exhibited significant larger particle size after heating and provided acid gels with higher firmness and viscosity, faster gelation and lower syneresis and a denser microstructure. In contrast, microparticulated whey protein appeared to only weakly interact with other proteins present and resulted in a protein network with low connectivity in the resulting gels. Increasing the casein/whey protein ratio did not decrease the gel strength in the acidified milk model systems with added whey protein aggregates.     

Microstructural changes in casein supramolecules during acidification of skim milk

Pasteurized skim milk was acidified using different levels of glucono-δ-lactone at 10, 20, 30, and 40°C to give slow, medium, and fast rates of acidification. Milk coagulation was monitored by measuring turbidity and curd firmness, and microstructural changes during acidification were observed on glutaraldehyde-fixed, agar-solidified milk samples using transmission electron microscopy. Rate of acidification had little influence on changes observed during acidification, except at 10°C. At 40°C, the casein supramolecules were spherical throughout acidification, whereas at lower temperatures they became progressively more ragged in appearance. All of the milks gelled at the same pH (pH 4.8), as measured by curd firmness, whereas increases in turbidity, assumed to be brought about by an increase in number of light-scattering particles, were observed to start at about pH 5.2 to 5.4. As the milk was acidified, aggregates of loosely entangled proteins were observed, presumably originating from proteins that had dissociated from the casein supramolecules. These aggregates were often as large as the casein supramolecules, particularly as the pH of the milk approached the isoelectric point of the caseins. Larger aggregates were observed at 40°C than at the lower temperatures, suggesting the involvement of hydrophobic interactions between the proteins. A 3-phase model for acid-induced gelation of milk is proposed in which the first phase involves temperature-dependent dissociation of proteins from the casein supramolecules, with more dissociation occurring as temperature is decreased. Dissociation continues as milk pH is lowered, with the released proteins forming into loosely entangled aggregates, some as large as the casein supramolecules. The second phase of acid gelation of milk occurs between pH 5.3 and pH 4.9 and involves a reassociation of proteins with loosely entangled protein aggregates forming into more-compact colloidal particles or combining with any remaining casein supramolecules. The third and final phase involves rapid aggregation of the colloidal casein supramolecules into a gel network at about pH 4.8. Different gel structures were formed based on temperature of acidification, with a coarse-stranded gel network formed at 40°C and a fine-stranded gel network at 10°C.     

Survival and Recovery of Thermally Stressed Yeast in Orange Juice

Injury and survival of thermally stressed yeast in orange juice were assessed by plating on plate count agar, acidified potato dextrose agar, and orange serum agar. Thermally stressed yeast were found to have reduced plate counts on both orange serum agar and acidified potato dextrose agar in comparison to plate count agar. Severely injured populations were not able to repair injury in orange juice held at 25°C but survived storage at 25, 6, and -18°C.     

Controlling heat induced aggregation of whey proteins by casein inclusion in concentrated protein dispersions

Formation of soluble, heat stable whey protein (WP) particles may be achieved via caseins and their chaperone like activity. The aim of this study was to establish effects of caseins on the properties of heat induced WP aggregates. Concentrated dairy protein mixtures (10% total-solids) were prepared with various casein:WP ratios (5:95–30:70) at adjusted pH (5.7–7.5) and heated to 80–120 °C, under a constant shear (500 s⁻¹). Increasing casein fraction at higher pH reduced the average size of WP aggregates and the surface hydrophobicity with simultaneous reduction in apparent viscosity at pH 6.7. WP denaturation was minimal when 30% casein was included in the dispersions. Inhibition of WP denaturation at elevated pH with inclusion of caseins may be attributed to a chaperon like activity of the casein. Thus, by increasing pH and the casein content in the system it was possible to form nano-size whey protein particles.     

Optimization of Carrot Juice Color and Cloud Stability

Investigations were conducted on the effects of heating, acidification, and enzyme treatment on carrot juice color and cloud stability. Heating whole carrots to 93°C prior to milling and pressing improved juice color, but reduced juice yield compared to heating milled carrots to 93°C. Juice color was improved by acidification of milled carrots to pH 5 or 4 with citric acid prior to pressing. Juice from carrots heated before milling clarified quickly if not acidified before pressing. Acidification after juice extraction did not stabilize cloud. A commercial pectinase/hemicellulase preparation improved juice color, but not juice yield. Only 20% of potential β-carotene was extracted from carrots during pressing, and β-carotene was extracted to a greater extent than α-carotene.     

Precipitation of proteins from potato juice with bentonite

The recovery of proteins from potato juice by treatment with bentonite has been investigated. All proteins can be precipitated from potato juice by acidification and addition of bentonite. The acid-coagulatable protein fraction is adsorbed less by bentonite than the acid-soluble protein fraction. Maximum adsorption of the acid-soluble fraction occurs at pH 5.0. The working conditions recommended for obtaining a protein-free potato juice are acidification to pH 4.5 and addition of bentonite to obtain a weight ratio of soluble protein: bentonite of 0.9. At the natural pH of potato juice (pH 5.8-6.0), adsorption of potato proteins on bentonite is irreversible. About 62% of the adsorbed protein can be recovered by alkali treatment at pH 13.     

The influence of bovine serum albumin on β-lactoglobulin denaturation,aggregation and gelation

The effect of bovine serum albumin (BSA) on the heat-induced denaturation, aggregation and subsequent acid-induced gelation of β-lactoglobulin (β-lg) was investigated in this work. Changes in the denaturation kinetics of β-lg during heating at 78 °C were determined by monitoring the disappearance of the native protein by reverse-phase chromatography. Replacing β-lg with increasing amounts of BSA, while keeping the total protein concentration constant at 5% (w/w), significantly increased the denaturation rate of β-lg from 2.57±0.30×10⁻³(g L⁻¹)⁽¹⁻ⁿ⁾s⁻¹ to 5.07±0.72×10⁻³(g L⁻¹)⁽¹⁻ⁿ⁾s⁻¹ (β-lg: BSA ratio of 3:1 w/w). The reaction order for β-lg was 1.40±0.09. Partial replacement of β-lg with BSA (β-lg: BSA ratio of 3:1 w/w) significantly increased the reaction order to 1.67±0.13. Heat-induced aggregates between β-lg and BSA were studied by dynamic light scattering, two-dimensional electrophoresis and size exclusion chromatography. The partial replacement of β-lg with BSA significantly changed the gelling properties of the acid-induced gels. A rapid rate of acidification resulted in a significant decrease, while a slow acidification rate resulted in a significant increase in gel strength. Size exclusion chromatography demonstrated that intermolecular disulphide bond formation occurred during both heat-induced denaturation/aggregation and subsequent acid-induced gelation. Results clearly indicate that BSA contributed to the formation of these disulphide bonds.     

Effects of ph and neutral salts on the formation and quality of thermal aggregates of ovalbumin. A study on thermal aggregation and denaturation

The effect of NaCl and CaCl₂ on the aggregation and denaturation temperature of ovalbumin was examined in the pH interval 3-11. It was found that ovalbumin had maximal thermal stability between pH values of 6-10. NaCl did not affect the denaturation temperature, while a small decrease was observed with CaCl₂ even at the low concentration range investigated. Both salts extended the pH interval of thermal aggregation, but at the alkaline side of the isoelectric point CaCk suppressed the aggregation temperature more than did NaCl. The aggregation properties of S-ovalbumin were also investigated and found to be similar to those of ovalbumin, although aggregation occurred at a higher temperature level. The quality of the aggregates was described by their dry matter content. Aggregates appeared as transparent gels, opaque gels, gel-like precipitates and precipitates. The addition of NaCl gave predominantly gel aggregates at the alkaline side of the isoelectric point, while precipitates dominated when CaCl₂ was added. Gelling is an intermediate phenomenon which occurs between the two extremes of precipitating and non-aggregating conditions. Special conditions, in terms of electrostatic repulsion between the protein molecules, are required for gelling, which could be achieved by manipulating with pH, type of salt, salt concentration or added detergent. The difference between aggregation and denaturation temperature correlated with the dry matter content of the aggregates formed. When aggregation temperature was well below denaturation temperature, the resulting aggregates had a high dry matter content (precipitates). Gelling, on the other hand, was observed at an aggregation temperature equal to or above the denaturation temperature.     

Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice

The purpose of this work was to develop a process enabling the quick inactivation of the polyphenol oxidase and pectin methylesterase enzymes, which are present in cloudy or unclarified apple juice; These enzymes are respectively responsible for enzymatic browning and opalescence instability. In order to fulfill this objective, acidification of the apple juice to pH 2.0 was conducted by electrodialysis (bipolar–anionic membranes) followed by mild heat treatment at temperature of 40, 45 and 50 °C for a duration of 0–60 min. Then, juice pH was readjusted to its initial value by electrodialysis with bipolar–anionic membranes. It was shown that a mild heat treatment at 45 °C for 5 min performed on the acidified juice represents an appropriate condition to quickly inactivate the enzymes. Furthermore, the organoleptic properties of the juice after treatment were found to be preserved and the adjusted juice (pH readjusted to its initial value) shows a better color than an untreated apple juice. Opalescence of the adjusted juice was also more stable than for an untreated cloudy apple juice, when stored at 4 °C for 3 months.     

Influence of high isostatic pressure on structural and functional characteristics of potato protein

Potato protein possesses promising nutritional and techno-functional properties, but distinct heat sensitivity. Therefore, the potential of high isostatic pressure as an alternative preservation and modification method was investigated. Pressures of 200, 400 and 600 MPa were applied at isothermal conditions of 20 and 40 °C to dispersions made of potato protein concentrate and isolated patatin for dwell times of 10 min. Process induced changes in solubility, foaming properties and selected structural characteristics were compared to results of pure thermal treatments from 20 to 80 °C. Potato protein solubility in neutral solutions made of concentrate was reduced to 21% after heating to 70 and 80 °C whereas it only decreased to 74% after pressurization at 600 MPa. Processing of isolated patatin at pH 6 and pH adjustment from 7 to 6 after processing reduced protein solubility to 12% for heat treatments and to 55 and 89%, respectively, for pressure treatments indicating different denaturation or aggregation mechanisms. Hydrogen bonds and hydrophobic interactions were involved in pressure induced aggregation, whereas aggregates formed during heat treatments were primarily stabilized by hydrophobic interactions. The surface hydrophobicity of soluble protein increased by factor 2.5 to 4.5 after heat treatments and by factor 1.3 at maximum after pressure treatments. High pressure processing provides therefore a good alternative to conventional heat pasteurization as initial potato protein quality may be preserved to a higher extent. Foam stability was increased to 177% by pressure treatments, but this modification was not long-term stable. Applying high pressure with the aim of a functional modification therefore requires further investigations.     

Stability of oil‐in‐water emulsions as influenced by thermal treatment of whey protein dispersions or emulsions

Properties of whey protein concentrate stabilised emulsions were modified by protein and emulsion heat treatment (60–90 °C). All liquid emulsions were flocculated and the particle sizes showed bimodal size distributions. The state and surface properties of proteins and coexisting protein/aggregates in the system strongly determined the stability of heat‐modified whey protein concentrate stabilised emulsions. The whey protein particles of 122–342 nm that formed on protein heating enhanced the stability of highly concentrated emulsions. These particles stabilised protein‐heated emulsions in the way that is typical for Pickering emulsions. The emulsions heated at 80 and 90 °C gelled due to the aggregation of the protein‐coated oil droplets.     

Effect of transglutaminase on rheological properties and microstructure of chemically acidified sodium caseinate gels

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.     

Whey protein aggregation under shear conditions – effects of lactose and heating temperature on aggregate size and structure

Summary Whey protein concentrates with different lactose contents were heat- and shear-treated in a scraped surface heat exchanger at various temperatures. The properties of the resulting protein aggregates are closely correlated with the denaturation kinetics of β-lactoglobulin and the different mechanisms – unfolding and aggregation – which determine the overall reaction rate. At temperatures below 85 °C, unfolding is slowed down especially if there is a high content of lactose. A loose, porous aggregate structure is formed and the particle size and the serum binding capacity increase. The smallest aggregates are produced when heating takes place between 85 and 95 °C. In the temperature range above 100 °C aggregation is the rate-limiting step and the aggregate structure is very dense and compact. The particle size increases and is no longer dependent on the concentration of lactose.     

RELATIONSHIPS OF COLOR, VISCOSITY, ORGANIC ACID PROFILES AND ASCORBIC ACID CONTENT TO ADDITION OF ORGANIC ACIDS AND SALT IN TOMATO JUICE

Malate and citrate acidified juices produced statistically similar results for pH and titratable acidity (TA), but these acidified juices were significantly different from nonacidified juice when comparing pH and TA. During storage time, the pattern of the pH and TA curves, regardless of acidification, were similar. Salted juice exhibited a significantly higher ascorbic acid content and greater viscosity than did the unsalted juice during storage. The organic acids citric, lactic, malic, and pyrocarboxylic increased immediately after heat processing for commercial sterility, while the presence of salt in this juice lowered these same organic acids. There was minimal correlation of the organic acids or the summation of the acids equivalents of all the organic acids to the titratable acidity or pH of the juices. Juices acidified with malate or citrate did not consistently display an increased level of that specific acid after processing and storage.     

Film-forming Mechanism and Heat Denaturation Effects on the Physical and Chemical Properties of Pea-Protein-Isolate Edible Films

Pea‐protein isolate solutions were heat‐denatured and dried to form stand‐alone edible films. Heat treatment at 90 °C over 5 min increased tensile strength and elongation‐at‐break, and decreased the elastic modulus. No significant differences were found in the initial contact angle of water and surface energies of heat‐denatured films from those of nonheated films except for the 20 min heat‐treated film. Additionally, heat denaturation reduced the water absorption rate of the films to 19 to 40% of the nonheated film. FTIR spectroscopy showed that more water existed in the nonheated films as compared to the heat‐denatured films. Electrophoresis studies suggested that intermolecular disulfide bonds were created during heat denaturation, which resulted in increased film integrity.     

Calcium sulphate‐induced soya bean protein tofu‐type gels: influence of denaturation and particle size

Soya bean protein isolate (SPI) dispersions (7.25%, w/v) were heated at 65, 75, 85 or 90 °C for different time periods to produce SPI aggregates with diverse degrees of denaturation and particle size to investigate the effects on calcium sulphate (CaSO₄)‐induced tofu‐type gel. The results revealed that gel hardness and water‐holding capacity correlated positively with the degree of denaturation of glycinin (11S) and the particle size of the SPI aggregates. The formed gels showed more uniform and denser network structures with increasing degrees of denaturation and particle size of SPI. Hydrophobic interaction was speculated to be the crucial factor for the retention of gels prepared by SPI whose degree of denaturation by 11S was lower than 4.35%. However, disulphide bonds probably played a more important role in the retention of gels generated by SPI with the 11S denaturation degree of >84.47%. Moreover, the bulk density of the protein aggregates might determine the gel structures to a certain extent.     

The protective effect of sodium dodecylsulphate on the thermal precipitation of conalbumin. A study on thermal aggregation and denaturation

The thermal precipitation and denaturation of conalbumin was studied separately at different pH values, low concentrations of an anionic detergent and salt concentrations relevant to food products. Sodium dodecylsulphate (SDS) was used as a model detergent and was found to lower the limit for thermal precipitation from pH 10–11 to pH 6–8 leaving a neutral pH region open for heat processing without any decrease in solubility. This protection against precipitation took place at the expense of a reduced thermal stability of conalbumin. Analysis at linearly increasing temperatures was shown to be a productive method to study the coupling between the thermal precipitation and denaturation processes. This coupling was examined at two heating rates, 10 and 1.25°C/min, respectively. Two types of precipitation behaviour were identified, a rapidly and a slowly sedimenting one. At the lower heating rate and under conditions where rapidly sedimenting precipitates were formed there was a close correlation between precipitation and denaturation. The precipitation was, however, always completed prior to denaturation. When approaching conditions where no precipitation took place, which also implied a successive transition towards slowly sedimenting precipitates, the precipitation process became delayed compared to denaturation. Precipitation could thus be registered at temperatures far above those of complete denaturation. The precipitates formed showed a considerable variation in particle size and water holding capacity.     

Kinetics of Formation and Physicochemical Characterization of Thermally-Induced β-Lactoglobulin Aggregates

Abstract: The kinetics of heat denaturation and aggregation for β-lactoglobulin dispersions (5% w/v) were studied at 3 pHs (6, 6.4, and 6.8) and at a heating temperature of 80 °C. Protein aggregates were characterized for hydrodynamic diameter, microstructure, and molecular weight by means of dynamic light scattering, transmission electron microscopy, and polyacrylamide gel electrophoresis, respectively. Concentration of native β-lactoglobulin decreased with holding time and with a decrease in the pH. Apparent rate constants were calculated for β-lactoglobulin denaturation applying the general kinetic equation solved for a reaction order of 1.5. Values of the apparent reaction rate constant k = 7.5, 6.3 and 5.6 × 10⁻³ s⁻¹ were found for pH 6, 6.4, and 6.8, respectively. Decreasing the pH of the dispersions produced higher aggregate sizes. After a holding time of 900 s, average hydrodynamic diameters for β-lactoglobulin aggregates at pH 6, 6.4, and 6.8 were 96, 49, and 42 nm, respectively. These results were confirmed by transmission electron microscopy images, where a shift in the size and morphology of aggregates was found, from large and spherical at pH 6 to smaller and linear aggregates at pH 6.8. β-Lactoglobulin formed disulfide-linked intermediates (dimers, trimers, tetramers) and so on) which then formed high molecular weight aggregates. From the results obtained by DLS, TEM, and SDS-PAGE a mechanism for β-lactoglobulin aggregation was proposed. This study shows that heat treatment can be used to produce protein aggregates with different sizes and morphologies to be utilized as ingredients in foods.     

Factors Influencing Ellagic Acid Precipitation in Muscadine Grape Juice During Storage

Ellagic acid sedimentation in white muscadine grape juice was monitored following different processing treatments and storage temperatures. High storage temperatures (40°C) greatly accelerated sediment formation, and pasteurization (100°C, 10 min) resulted in a faster sediment formation than sterile filtration. Sediment also increased substantially after juice was hydrolyzed (121°C and pH 2 for 10 min). Ultrafiltration of juice through a 10,000 or 30,000 dalton membrane resulted in significantly less sediment formation in the juice. Treatment of juice with polyvinylpolypyrrolidone (0.1– 0.2 g/L juice), egg albumen (6–10 mg/L juice) or gelatin (0.05 – 0.4 g/L juice) resulted in a significant reduction of juice phenolics and sediment formation. A commercial pectinase added to the grapes increased sediment formation and total phenolics in the juice. Levels of ellagic acid in juice were very low and did not correlate well with amount of sediment formed.