Heat-Induced Aggregation and Denaturation of Egg White Proteins in Acid Media

Heat-induced aggregation and denaturation of egg white proteins adjusted to pH 5.5, 4.5, 3.5 and 2.5 were investigated by vertical flat-sheet polyacrylamide gel electrophoretic and differential scanning calorimetric methods. The fractional and step-wise aggregation of egg white proteins was caused by heating. As the acidity was increased from pH 5.5 to 2.5, ovotransferrin, ovomacroglobulin, globulin G3A, globulins A1 and A2, and ovalbumin became much more unstable to heat. However, ovomucoid and ovoinhibitor did not aggregate in the acidified egg white under heat treatments of 3 min at 90°C or 20 min at 74°C. The heat-induced aggregation of flavoprotein was slightly greater at pH 4.5 and 3.5 than at pH 5.5 and 2.5.     

Heat resistance of Escherichia coli O157:H7 in cook‐in‐bag ground beef as affected by pH and acidulant†

Summary The heat resistance of a four‐strain mixture of Escherichia coli O157:H7 was tested. The temperature range was 55–62.5 °C and the substrate was beef at pH 4.5 or 5.5, adjusted with either acetic or lactic acid. Inoculated meat, packaged in bags, was completely immersed in a circulating water bath and cooked to an internal temperature of 55, 58, 60, or 62.5 °C in 1 h, and then held for pre‐determined lengths of time. The surviving cell population was enumerated by spiral plating meat samples on tryptic soy agar overlaid with Sorbitol MacConkey agar. Regardless of the acidulant used to modify the pH, the D ‐values at all temperatures were significantly lower (P < 0.05) in ground beef at pH 4.5 as compared with the beef at pH 5.5. At the same pH levels, acetic acid rendered E. coli O157:H7 more sensitive to the lethal effect of heat. The analysis of covariance showed evidence of a significant acidulant and pH interaction on the slopes of the survivor curves at 55 °C. Based on the thermal‐death–time values, contaminated ground beef (pH 5.5/lactic acid) should be heated to an internal temperature of 55 °C for at least 116.3 min and beef (pH 4.5/acetic acid) for 64.8 min to achieve a 4‐log reduction of the pathogen. The heating time at 62.5 °C, to achieve the same level of reduction, was 4.4 and 2.6 min, respectively. Thermal‐death–time values from this study will assist the retail food processors in designing acceptance limits on critical control points that ensure safety of beef originally contaminated with E. coli O157:H7.     

Dynamic rheological behavior of meat proteins during acid‐induced gelation

Glucono‐δ‐lactone (gdl) is used to promoting gelification of muscle proteins in restructured foods. Protein gelification can be achieved by lowering enough the pH to promote interactions between protein molecules. The gdl was added to a muscle extract (1.2 % w/w) at 4, 12, 20 and 28°C and pH and rheological behavior were observed using a controlled stress rheometer. Changes in pH induced by gdl hydrolysis were more noticeable at 28°C (final pH 4.40) than at 4°C (final pH 5.02). The gdl hydrolysis rate depends on temperature, being lower at low temperature and increasing with temperature. This temperature influence on pH decrease was reflected on muscle protein gels viscoelastic behavior, where the proteins cross‐linking were caused by the acidification, enough to form a gel structure. Maximum storage (G') and loss (G”) modules were achieved at pH values between 4.5‐5.5, near to myofibrillar proteins isoelectric point. Samples presented a liquid‐like behavior (G'>G") during the acid‐induced gelation. Stronger gels were formed at high temperatures. At this point, loss of solubility and structure were caused by an excess of acid in the medium.     

Short communication: Stabilization of milk proteins at pH 5.5 using pectic polysaccharides derived from potato tubers

Potato pectin has unique molecular characteristics that differentiate it from commercially available pectins sourced from citrus peels or apple pomace, including a higher degree of branching and a higher acetyl content. The objective of this study was to evaluate the ability of potato pectin to stabilize milk proteins at an acidic pH above their isoelectric point, pH 5.5, at which no citrus- or apple-derived pectins are functional. Potato pectin was extracted from raw potato tubers by heating at pH 4.5 and 120°C for 30 min after removing starch solubilized using a dilute HCl solution adjusted to pH 2. The potato pectin was found to have a galacturonic acid content of 17.31 ± 3.29% (wt/wt) and a degree of acetylation of 20.20 ± 0.12%. A portion of the potato pectin was deacetylated by heating it in an alkaline condition. The deacetylation resulted in a galacturonic acid content of 19.12 ± 4.64% (wt/wt) and a degree of acetylation of 3.03 ± 0.03%. Particle size distributions in acidified milk drink (AMD) samples adjusted to pH 5.5 demonstrated that the acetylated and deacetylated potato pectins were capable of inhibiting the aggregation of milk proteins to the largest degree at a pectin concentration of 1.0 and 0.25% (wt/wt), respectively. Pectin molecules that were not bound to milk proteins in these AMD samples were quantified after centrifugally separating milk proteins and pectin bound to them from the serum. We found that, for the acetylated and deacetylated potato pectins, all or approximately half of the pectin molecules were bound to milk proteins at a pectin concentration of 0.25 or 1.0% (wt/wt), respectively. These results suggest that the presence of acetyl groups is a critical factor that determines how potato pectin molecules bind electrostatically to milk protein surfaces, form 3-dimensional structures there, and function as a stabilizer. The present results demonstrate that potato pectin can stabilize milk proteins at pH 5.5 and potentially enable the development of novel AMD products with improved functionality for casein-containing products with moderately acidic pH profiles.     

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     

Impact of process conditions on the rheological detectable structure of UHT treated milk protein–carrageenan systems

UHT treated dairy based drinks containing carrageenan to produce a weak gel type of structure are often found to vary considerably in textural properties. Target of this study was to investigate the effect of the heating temperature in the range of 120–139 °C and the filling temperature after cooling down to 4–14 °C prior to storage for 24 and 96 h at 4 °C on a system comprising milk and 400 ppm kappa-2 type of carrageenan. The textural properties and the stability of such systems mainly depend on the interaction of the carrageenan and the casein micelle surface which was assessed by means of the hysteresis loop area between the upward and downward flow curves upon variation of the shear stress. The loop area was used as a dimension for the energy required breaking down the systems’ textural structure. The hysteresis loop area was found to be significantly increased the higher the heating temperature and the lower the cooling temperature was. During storage, a further influence of the process parameters on structural development between casein and carrageenan was observed. Structure point analysis, small angle oscillatory rheology and particle size measurements were used for further explanation of the results of the hysteresis loop area. Particle size measurements indicated an aggregation phenomenon in the micellar casein dominated microdomains at increasing UHT heating temperatures. Variation of the carrageenan concentration enlightened the participation of casein dominated microdomains in the mixed system’s texture.     

Whey protein aggregation under shear conditions – effects of pH-value and removal of calcium

The effects of pH-value and a reduction in calcium content on the kinetics of whey protein denaturation and the aggregation behaviour, under shear in a scraped surface heat exchanger, were examined. The denaturation rate of β-lactoglobulin at 80 °C is considerably retarded as the pH-value decreases from pH 6.7 to 4.5. Aggregates which are produced under shear between pH 4 and 5.5 reveal a small particle size (<5 μm) irrespective of the lactose content and the heating temperature. This is attributed to the low reactivity of the thiol groups and the small net charge of the proteins in this pH-range. At a reduced calcium concentration the heat- and shear-treatment resulted in a gritty structure with large rubber-like particles. These are not to be taken as primary whey protein aggregates but as fragments of a fine-stranded gel.     

Stability of protein‐stabilised β‐carotene nanodispersions against heating,salts and pH

BACKGROUND: Milk proteins are used in a wide range of formulated food emulsions. The stability of food emulsions depends on their ingredients and processing conditions. In this work, β‐carotene nanodispersions were prepared with selected milk‐protein products using solvent‐displacement method. The objective of this work was to evaluate the stability of these nanodispersions against heating, salts and pH. RESULTS: Sodium caseinate (SC)‐stabilised nanodispersions possessed the smallest mean particle size of 17 nm, while those prepared with whey‐protein products resulted in larger mean particle sizes (45–127 nm). Formation of large particles (mean particle size of 300 nm) started after 1 h of heating at 60 °C in nanodispersions prepared with SC. More drastic particle size changes were observed in nanodispersions prepared with whey protein concentrate and whey protein isolate. The SC‐stabilised nanodispersions were fairly stable against Na⁺ ions at concentrations below 100 mmol L^?1, but drastic aggregation occurred in ≥ 50 mmol L^?1 CaCl₂ solutions. Aggregation was also observed in whey protein‐stabilised nanodispersions after the addition of NaCl and CaCl₂ solutions. All sample exhibited the smallest mean particle size at neutral pH, but large aggregates were formed at both ends of extreme pH and at pH around the isoelectric point of the proteins. CONCLUSION: The nanodispersions prepared with SC were generally more stable against thermal processing, ionic strength and pH, compared to those prepared with whey proteins. The stable β‐carotene nanodispersions showed a good potential for industrial applications. Copyright © 2008 Society of Chemical Industry     

Stability of casein micelle subjected to reversible CO2 acidification: Impact of holding time and chilled storage

Casein micelle stability and reactivity were assessed on milk subjected to reversible acidification by carbonation. Pressurised CO₂ was injected at 4 °C, leading to controlled acidification from 6.63±0.02 to a target pH (5.5 or 5.2). After holding the pressurised milk under these conditions for 15 or 60 min, the pressure was released and the milk pH returned to its initial value under stirring and vacuum degassing. Upon CO₂ treatment, calcium and protein partition, zeta potential and size of casein micelles were restored directly after neutralisation. The rheological properties of the gel obtained by acid coagulation of CO₂-treated milk did not change as a result of carbonation. Micelle hydration increased after neutralisation and during storage. Milk buffering capacity in the pH range of 4.5–5.5 decreased after neutralisation of milk acidified by carbonation, but increased during chilled storage of this milk. Holding time of carbonated milk at low pH was found to have no impact on the physicochemical characteristics of casein micelles and the rheological properties of the gel obtained by acid coagulation of this milk.     

The effect of pH and calcium ion on rheological behaviour of β‐lactoglobulin‐basil seed gum mixed gels

Effect of pH (4.5–7.5) and Ca²⁺ (0.01–0.5 m ) on gelation of single and mixed systems of 10% β‐lactoglobulin (BLG) and 1% basil seed gum (BSG) was investigated. The gelling point of BLG and BSG gels was strongly pH‐dependent, and stiffer gels formed at higher pH. The BLG gels were formed upon heating to 90 °C and reinforced on cooling to 20 °C; however, the gelation of BSG occurred at temperatures below 70 °C. By increasing Ca²⁺ concentration, storage modulus of BLG and BSG gels were increased, although pH had a greater effect than Ca²⁺. In contrast, mixed systems showed two distinct types of behaviour: BLG gel formation and BSG network, suggesting that phase‐separated gels were formed. In addition, higher strength was obtained for BLG‐BSG mixture at higher Ca²⁺ concentration.     

Heat Induced Gelation of Pea (Pisum sativum) Mixed Globulins, Vicilin and Legumin

Mixed globulins (MG) were extracted from ground dry peas (Pisum sativum, B-160) with 0.5M NaCl, 50 mM potassium phosphate, pH 7.2, and isolated by precipitation at pH 4.5. Crude vicilin and legumin were fractionated from the MG by dialysis against 0.2M NaCl, pH 4.8, and centrifugation, then further purified using DEAE-cellulose chromatography. Conditions for maximum gel hardness of heat induced MG gel, as determined with an Instron Universal Testing Machine, were heating for 20 min at pH 7.1 at 87°C. Purified vicilin, but not legumin, formed heat induced gels. The relationship was linear between protein (globulin) concentration and log gel hardness. At all protein concentrations studied, as proportion of legumin decreased, gel hardness increased.     

Swelling Characteristics of Native and Chemically Modified Wheat Starches as a Function of Heating Temperature and Time

The effects of hydroxypropylation (molar substitution, MS 0.05, 0.12, and 0.18) and cross‐linking (0.03%, 0.1%, and 0.2%) on swelling properties of wheat starch granules at several temperatures and heating times were investigated by laser diffraction particle size analysis. Starch samples were dispersed in water at temperatures ranging from 30 to 90°C, for 1 to 360 min. All starch granules exhibited distinct bimodal size distributions: small B‐granules with mean diameter of 2.3 μm and large A‐granules with mean diameter of 20.4 μm. As temperature increased, the B‐granules swelled more than A‐granules. Swelling of A‐granules sharply increased at 60°C. Swelling was more pronounced with increasing molar substitution of hydroxypropyl groups, while increased swelling was not observed in cross‐linked starches. The dependence of swelling capacity on heating time was different at 60 and 80°C as well as amongst modified starches. As heating time was prolonged, mean granule sizes for native, control, and hydroxypropylated starches at 80°C decreased after reaching maximum size due to loss of granule integrity, while those at 60°C showed no significant change.     

Changes of Molecular Forces During Thermo-Gelling of Protein Isolated from PSE-Like Chicken Breast by Various Isoelectric Solubilization/Precipitation Extraction Strategies

Changes in protein intermolecular interactions during thermo-gelling were measured to compare the gelation properties of isoelectric solubilization/precipitation (ISP)-isolated protein extracted (solubilized at 3.5 and 11.0, precipitated at 5.5 and 6.2) from pale, soft, and exudative (PSE)-like chicken breast meat with raw meat paste. The solubility of both in water and salt (0.6 M NaCl) decreased significantly after ISP treatments. Protein profile analysis revealed that precipitation pH showed little influence on protein profile. Under pH 3.5 and pH 11.0 solubility conditions, the recovered protein at pH 6.2 showed significantly higher gel hardness than that at pH 5.5, which can be induced by hydrophobic change. Surface hydrophobicity (SH₀) and hydrophobic interactions at 25 °C presented similar results, indicating that the soluble protein at pH 11.0 exhibited a higher value after precipitation at pH 6.2 than that at pH 5.5, and the hydrophobicity of pH 3.5 isolates was higher than that of the pH 11.0 groups. However, the maximum hydrophobicity upon heating was inconsistent with initial tendencies. Given that the hydrophobic residues were exposed sufficiently during the ISP process, the ISP-treated proteins, particularly the samples extracted at pH 3.5, might be less susceptible to heat-induced exposure. The gelation behavior of the ISP-treated proteins had been modified on the basis of the intermolecular bonds during heating. In conclusion, the precipitation condition demonstrated excellent relevance for product development based on functionality.     

Effect of preheating temperature on the microstructure of walleye pollack surimi gels under the inhibition of the polymerisation and degradation of myosin heavy chain

BACKGROUND: The physical attribute of heat‐induced gel texture is highly dependent on the microstructure of the gel. In this study the microstructures of walleye pollack surimi gels preheated at various temperatures with and without inhibitors (ethylenediamine‐N,N,N′,N′‐tetraacetic acid, iodoacetamide and leupeptin) were observed with a natural scanning electron microscope. RESULTS: Without inhibitors, gels preheated at 30 °C showed a fine and uniform network structure together with the highest polymerisation of myosin heavy chain (MHC) and the highest gel strength. At 60 °C, gels exhibited a broken, disrupted and loose cluster‐like structure together with the highest degradation of MHC and the lowest gel strength. Under the inhibition of polymerisation and degradation of MHC a fine network was observed up to 40 °C during preheating. However, after a second step of heating at 80 °C the microstructures were disrupted and resembled each other regardless of the preheating temperature. CONCLUSION: Heat‐induced gel formation is related to the polymerisation and degradation of MHC and the microstructure of the gel during preheating. Gelation occurred during setting even under the inhibitory condition, and the formation of covalent bonding by transglutaminase is not essential to the formation of a three‐dimensional network during setting but is essential to the gel strength enhancement effect of setting by subsequent heating at 80 °C. Copyright © 2010 Society of Chemical Industry     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Pilot-scale formation of whey protein aggregates determine the stability of heat-treated whey protein solutions—Effect of pH and protein concentration

Denaturation and consequent aggregation in whey protein solutions is critical to product functionality during processing. Solutions of whey protein isolate (WPI) prepared at 1, 4, 8, and 12% (wt/wt) and pH 6.2, 6.7, or 7.2 were subjected to heat treatment (85°C × 30 s) using a pilot-scale heat exchanger. The effects of heat treatment on whey protein denaturation and aggregation were determined by chromatography, particle size, turbidity, and rheological analyses. The influence of pH and WPI concentration during heat treatment on the thermal stability of the resulting dispersions was also investigated. Whey protein isolate solutions heated at pH 6.2 were more extensively denatured, had a greater proportion of insoluble aggregates, higher particle size and turbidity, and were significantly less heat-stable than equivalent samples prepared at pH 6.7 and 7.2. The effects of WPI concentration on denaturation/aggregation behavior were more apparent at higher pH where the stabilizing effects of charge repulsion became increasingly influential. Solutions containing 12% (wt/wt) WPI had significantly higher apparent viscosities, at each pH, compared with lower protein concentrations, with solutions prepared at pH 6.2 forming a gel. Smaller average particle size and a higher proportion of soluble aggregates in WPI solutions, pre-heated at pH 6.7 and 7.2, resulted in improved thermal stability on subsequent heating. Higher pH during secondary heating also increased thermal stability. This study offers insight into the interactive effects of pH and whey protein concentration during pilot-scale processing and demonstrates how protein functionality can be controlled through manipulation of these factors.     

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.     

HEAT-INDUCED TRANSITIONS IN THE PROTEIN-PROTEIN INTERACTION OF BOVINE NATURAL ACTOMYOSIN1

Protein-protein interaction of bovine natural actomyosin (NAM) was studied by means of optical density changes resulting from discrete particle formation in the temperature range of4°C to 70°C. From Arrhenius plots, the apparent heat of activation (ΔH_a) at pH 5.5 (17.1 kcallmole) was significantly (P<0.05) lower than activation energies in the pH range of 6.0 to 7.5. The lower Δ H_a resulted in initiation of protein-protein interaction at a temperature near 16°C at pH 5.5, whereas interaction did not proceed until the temperature approached 37°C at pH 6.0 and above. Derivative curves (dOD/dT) at pH 5.5 and 6.0 showed two distinct NAM thermal transition regions. Tm₁ occurred at 43.0°C at pH 5.5 and 48.5°C at pH 6.0, with the 5.5°C difference possibly arising from effects of proton binding in altering protein conformation. Only a 1.5°C difference in Tm₂ (56.0°C at pH 5.5 versus 57.5°C at pH 6.0) was found. Although the overall heat-mediated NAM aggregation (in dilute solution) was found to follow first order kinetics by two evaluation methods, the existence of two thermal transitions supports a two-step reaction mechanism proposed for the formation of protein gels (in higher concentration solutions).     

Thermal Stability of Cathepsin D

The effect of pH and temperature on Cathepsin D stability was examined using a hemoglobin assay following preincubation of the enzyme at various pH and temperature combinations. The results of the study showed that the enzyme retained 87% of its activity at 45°C (pH 3.5) when compared to a control at 37°C (pH 3.5). Further increases in temperature and pH resulted in decreased enzyme activity and approximately 43% remained at 55°C (pH 5.5). The amount of activity remaining at higher temperatures decreased but suggests that the enzyme could contribute to textural changes in meat at temperatures up to 60°C.     

Biopolymer Nanoparticles from Heat-Treated Electrostatic Protein–Polysaccharide Complexes: Factors Affecting Particle Characteristics

ABSTRACT: Biopolymer nanoparticles can be formed by heating globular protein–ionic polysaccharide electrostatic complexes above the thermal denaturation temperature of the protein. This study examined how the size and concentration of biopolymer particles formed by heating β-lactoglobulin–pectin complexes could be manipulated by controlling preparation conditions: pH, ionic strength, protein concentration, holding time, and holding temperature. Biopolymer particle size and concentration increased with increasing holding time (0 to 30 min), decreasing holding temperature (90 to 70 °C), increasing protein concentration (0 to 2 wt/wt%), increasing pH (4.5 to 5), and increasing salt concentration (0 to 50 mol/kg). The influence of these factors on biopolymer particle size was attributed to their impact on protein–polysaccharide interactions, and on the kinetics of nucleation and particle growth. The knowledge gained from this study will facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes.