Sodium caseinate-stabilized fat globules inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave Spectroscopy

Sodium caseinate (NaCas)-stabilized oil-in-water emulsions were added to skim milk and the rennet-induced aggregation was observed in situ using light scattering and dynamic oscillatory rheology. The gelation of the recombined milk was greatly inhibited by the addition of the oil droplets, at volume fractions >0.025. The development of the turbidity parameter, 1/l*, and the apparent hydrodynamic radius during renneting were determined using diffusing wave spectroscopy. Although the recombined milk samples contained two scattering particles, namely, casein micelles and fat globules, the latter overwhelmingly contributed to the overall light-scattering signal. This made possible to follow the behaviour of NaCas-stabilized fat globules during the gelation process. The enzymatic reaction associated with the hydrolysis of micellar κ-casein was not significantly affected by the presence of the NaCas-stabilized fat globules. However, the emulsion droplets impeded the aggregation of rennet-altered casein micelles preventing the formation of a gel network. The inability of renneted casein micelles to develop a gel network can be attributed in part to an altered equilibrium between soluble and micellar calcium phosphate, caused by the association of soluble Ca²⁺ with casein molecules, but mostly can be attributed to the effect of non-adsorbed caseins on the surface of the casein micelles.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

Effect of storage time and temperature of milk protein concentrate (MPC85) on the renneting properties of skim milk fortified with MPC85

This study investigated the effect of storage temperature (20–50 °C) and time (0–60 days) on the renneting properties of milk protein concentrate with 85% protein (MPC85). Reconstituted skim milk was fortified with the MPC85 (2.5% w/w) and the renneting properties of the skim milk/MPC85 systems were investigated using rheology. It was found that the final complex modulus (final G∗) and the yield stress of the rennet-induced skim milk/MPC85 gels decreased exponentially with storage time of the MPC85 for storage temperatures greater than 20 °C, with a greater effect at the higher storage temperatures. Changes in the solubility of MPC85 with storage time were correlated with the rheological properties. The primary phase of renneting (cleavage of κ-casein) was not affected by the storage of the MPC85; hence the effect was related to the secondary stage of renneting (aggregation/coagulation of rennet-treated casein micelles). Using a temperature–time superposition method, a master curve was formed from the final G∗, yield stress and solubility results. This suggested that the same physical processes affected the solubility and rennet gelation properties of the milks. It is proposed that the MPC85 protein in rennet-treated skim milk/MPC85 solutions may transform from an interacting material, when solubility is high, to an inert or weakly interacting material, when solubility is low, and that this results in the reduced final G∗ and yield stress of the rennet gels when MPC85 is stored at elevated temperatures for long periods.     

Effect of pH adjustment at heating on the rheological properties of acid skim milk gels with added potato starch

The rheological properties of acid skim milk gels, prepared from milk with added potato starch and pH adjusted (pH 6.5–7.1) prior to heat treatment and acidification, were investigated. The storage modulus, G′, of the final acid gels was increased by heating the milk at higher pH and further increased by adding starch. The effect of pH at heating and addition of starch appeared to be additive and independent of each other up to a starch addition level of 1%. Above this starch level, the pH at heating had a lesser effect. This may have been due to the increased viscosity of the aqueous phase as a result of starch gelatinization or to direct contributions of the starch to the gel network structure. Confocal microscopy showed that milk proteins developed fewer but broader protein clusters at higher pH than at lower pH. Starch addition resulted in an increased density of the protein network.     

Coagulation properties of ultrafiltered milk retentates measured using rheology and diffusing wave spectroscopy

The effect of concentration of milk by ultrafiltration on renneting has been widely studied as it is of great interest in dairy technology. Although a number of reports are available on the texture and microstructure of the milk gels formed at various concentrations, very little is understood on the effect of concentration on the stages preceding aggregation, or how concentration may affect the interactions between micelles. This study aims to investigate the renneting behavior of milk concentrated by ultrafiltration (without diafiltration) to 3× and 5× (v/v) and compare it to that of skim milk. The scattering properties of the casein micelles under quiescent conditions suggest that they deviate from hard-sphere behavior at 5× concentration (micelle volume fraction, ? = 0.5). The release of the caseinomacropeptide during renneting was not significantly different amongst the three different casein concentrations tested (1×, 3×, and 5×). No significant differences were also noted in the rennet coagulation time as detected by both diffusing wave spectroscopy and rheology. Concentrated milk samples formed significantly (p-value < 0.05) stiffer gels than regular milk due to an increased number of bonds in the network. The level of milk concentration also accelerated a change in the spatial distribution and rate of change of turbidity of the micelles because of a decrease in the overall inter-particle distance and increased collision frequencies. This in situ investigation of concentrated milk samples suggested that the changes in rennet coagulation with concentration are merely a cause of crowding effects.     

Gelation of casein micelles in ��-casein reduced milk prepared using membrane filtration

Pasteurized skim milk was subjected to membrane filtration using a molecular weight cut-off of 80 kDa and a plate and frame pilot scale system at temperatures below 10 °C. Via this process, transmission of whey proteins and ??-casein through the membrane was achieved. The milk was concentrated to two times (based on volume reduction), and whey protein-free permeate was added to return to the original volume fraction of casein micelles in milk. This diafiltration process was carried out four times, and the retentate obtained was nearly free of whey proteins and with approximately 20% of ??-casein removed. The same membrane filtration was also carried out at 25 °C to achieve transmission of whey protein but not of ??-casein, and to obtain whey protein-depleted milk without depletion of ??-casein.The gelling behaviour of these samples, reconstituted to the original casein volume fraction, was examined using rheology and diffusing wave spectroscopy. When compared to the original skim milk it was found that there were no statistically significant differences in gelation behaviour during acidification, but differences were noted in gelation time and final stiffness modulus for samples undergoing renneting. These differences were attributed mostly to the changes in ionic composition, as when the serum composition of the retentates was re-equilibrated against the original skim milk by dialysis; the gelation behaviour of the samples was comparable to that of skim milk. The results clearly indicate the importance of the milk's overall ionic balance in the early stages of aggregation of rennet-induced gelation of milk.     

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.     

Comparison of casein micelles in raw and reconstituted skim milk

During the manufacture of skim milk powder, many important alterations to the casein micelles occur. This study investigates the nature and cause of these alterations and their reversibility upon reconstitution of the powders in water. Samples of skim milk and powder were taken at different stages of commercial production of low-, medium-, and high-heat powders. The nature and composition of the casein micelles were analyzed using a variety of analytical techniques including photon correlation spectroscopy, transmission electron microscopy, turbidity, and protein electrophoresis. It was found that during heat treatment, whey proteins are denatured and become attached to the casein micelles, resulting in larger micelles and more turbid milk. The extent of whey protein attachment to the micelles is directly related to the severity of the heat treatment. It also appeared that whey proteins denatured during heat treatment may continue to attach to casein micelles during water removal (evaporation and spray-drying). The process of water removal causes casein and Ca in the serum to become increasingly associated with the micelles. This results in much larger, denser micelles, increasing the turbidity while decreasing the viscosity of the milk. During reconstitution, the native equilibrium between colloidal Ca and serum Ca is slowly reestablished. The reequilibration of the caseins and detachment of the whey proteins occur even more slowly. The rate of reequilibration does not appear to be influenced by shear or temperature in the range of 4 to 40°C.     

Effect of ultrasonication on the properties of skim milk used in the formation of acid gels

Skim milk was ultrasonicated for times up to 30 min either with or without temperature control. Ultrasonication (US) without temperature control resulted in the generation of considerable heat, with the milk reaching  95 °C within 15 min of treatment. The whey proteins were denatured. Changes to the casein micelle size were observed, with decreases during the early stages of US and increases (because of aggregation) on prolonged treatment. Significant κ-casein dissociated from the micelles. Acid gels prepared from these ultrasonicated samples increased in firmness (final G′) up to a maximum final G′ after  15 min of US, followed by a decrease from this maximum on prolonged treatment. US with temperature control demonstrated that the denaturation of the whey proteins was entirely due to the heat generated during US, although the casein micelle size was still reduced. Acid gels prepared from ultrasonicated skim milk in which the temperature remained below the denaturation temperature of the whey proteins had low final G′, although a small increase was observed with increasing US time. Acid gels prepared from the samples that were ultrasonicated at temperatures above the denaturation temperature of the whey proteins had higher final G′, which could reach values similar to those obtained by the conventional heating of milk. The results of this study indicate that, in skim milk, most of the effect of US can be related to the heat generated from the treatment, with US itself having only a small effect on the milk when the temperatures are controlled.

Industrial relevance

The control and the manipulation of the firmness of acid skim milk gels are important in many dairy food applications such as yoghurts and some types of cheese. US is an emerging technology that could be used to process skim milk for use in acid gelled products. This study has demonstrated that acid gel firmness can be substantially manipulated when skim milk is ultrasonically treated before acidification; however, most of the effect is due to the heat generated during US treatment. As the effects of US are similar to those obtained through conventional heating processes, and as US can control spoilage microorganisms, using US under controlled temperature conditions could be an alternative to conventional heating to give desired functional properties and storage stability to milk products. However, the temperature/denaturation/aggregation would need to be carefully controlled to minimize the detrimental effects of excessive heating.     

Oil-in-water emulsions stabilized by sodium caseinate: Influence of pH,high-pressure homogenization and locust bean gum addition

The effect of pH, addition of a thickening agent (locust bean gum) or high-pressure homogenization on the stability of oil-in-water emulsions added by sodium caseinate (Na-CN) was evaluated. For this purpose, emulsions were characterized by visual analysis, microstructure and rheological measurements. Most of the systems were not stable, showing phase separation a few minutes after emulsion preparation. However, creaming behavior was largely affected by the pH, homogenization pressure or locust bean gum (LBG) concentration. The most stable systems were obtained for emulsions homogenized at high pressure, containing an increased amount of LBG or with pH values close to the isoelectric point (pI) of sodium caseinate, which was attributed to the size reduction of the droplets, the higher viscosity of continuous phase and the emulsion gelation (elastic network formation), respectively. All the studied mechanisms were efficient to decrease the molecular mobility, which slowed down the phase separation of the emulsions. In addition, the use of sodium caseinate was also essential to stabilize the emulsions, since it promoted the electrostatic repulsive interactions between droplets.     

Relationship between physical properties of casein micelles and rheology of skim milk concentrate

The properties of casein micelles in milk concentrates are of interest for the use of ultrafiltered (UF) skim milk concentrates in dairy products, and for the general understanding of colloidal stability and behavior of the casein micelle. The rheological behavior of UF skim milk concentrate with a casein concentration of 19.5% (wt/wt) was investigated at different pH and NaCl concentrations by analyzing flow viscometry and small amplitude oscillatory shear measurements. Viscometric flow curves were fitted to the Carreau-Yasuda model with the aim of determining values for the viscosity at infinite high shear rates and thereby estimate the voluminosity of the casein micelles (ν_casein) in the UF concentrate. The voluminosity of the casein micelles increased with addition of NaCl and decreased when pH was decreased from 6.5 to 5.5. At pH 5.2, ν_casein increased because of acid-induced aggregation of the casein micelles. The changes in ν_casein could be interpreted from transmission electron microscopy of freeze-fractured samples of the UF concentrate and partly from dynamic light scattering measurements. Altered interactions between casein micelles due to different pH and NaCl concentrations are proposed to occur due to collapse of the κ-casein layer, changed ionic strength, and altered distance between casein micelles.     

Effect of added calcium chloride on the physicochemical and rheological properties of aqueous mixtures of sodium caseinate/sodium alginate and respective oil-in-water emulsions

Changes induced by addition of calcium chloride in particle size distribution and electrokinetic potential were determined in sodium caseinate/sodium alginate mixtures dissolved in water or acetate buffer at ambient temperature. Rheological properties of aqueous mixtures and respective oil-in-water emulsions (30% oil w/w) were evaluated using a low-stress rheometer. Stability and particle diameter of emulsions were measured. Caseinate and alginate solutions were negatively charged and showed negative electrokinetic potential; however values of mixtures were between those of the values for the individual hydrocolloids. When calcium ions were added the electrokinetic potential diminished while the negative charge was preserved. Aqueous mixtures of caseinate and alginate showed average particles size between of those of caseinate or alginate samples. We observed low viscosity values and Newtonian behavior for both caseinate (1 and 2%) and alginate (0.1%). Addition of 5 mM CaCl₂ to alginate solutions induced shear-thinning behavior as well as the development of viscoelasticity. Both the viscosity and the elastic modulus of these polysaccharide solutions were attenuated by the presence of protein or dispersed oil in mixtures or emulsions, respectively. High average particle diameter of emulsions prepared was obtained (close to 10 μm), however, stability of emulsions was possible only with the addition of CaCl₂ to the mixtures, in both water and acetate buffer. In these cases elastic behavior predominated to viscosity in the formation of emulsions, confirming the prevalence of aqueous phase rheology on emulsions.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

Effect of microbial transglutaminase, sodium caseinate and non-fat dry milk on quality of salt-free, low fat turkey döner kebab

Effects of microbial transglutaminase (MTGase) and its combinations with sodium caseinate (SC) or non-fat dry milk (NFDM) on quality of salt-free, low fat turkey döner kebab were investigated. The purpose of this study was to prevent quality deteriorations (e.g. cooking loss, textural problems) caused by a lack of salt in the product. The results of this study indicated that the use of MTGase can minimize textural quality loss caused by a lack of salt in turkey döner manufacture (p < 0.05). When the enzyme was combined with SC, or NFDM, its effect on texture was enhanced (p < 0.05) and improved cooking yield, moisture and sensory properties compared to the corresponding control manufactured without the enzyme, salt, SC and NFDM. (p < 0.05), however, there was no significant effect on pH and color As a result, the possible quality problems which may occur in salt free, low fat turkey döner can be overcome by the use of combinations of MTGase with SC or NFDM.     

葡萄糖对脱脂乳酪蛋白胶束稳定性的影响研究

研究了葡萄糖添加量、体系pH、热处理温度、热处理时间对脱脂牛乳酪蛋白胶束稳定性的影响。研究表明,在接近中性pH条件下,少量葡萄糖分子可降低脱脂乳酪蛋白胶束的稳定性,大量葡萄糖分子可增强其稳定性。添加葡萄糖后,在高温热处理的诱导下,酪蛋白沉淀的pH升高。随着热处理时间的延长,脱脂乳中酪蛋白胶束表现出聚集行为,致使粒径增大,浊度和沉淀率总体呈现上升趋势。该研究结论可为乳品加工提供参考依据。      

Effect of alkalinization and ultra-high-pressure homogenization on casein micelles in raw and pasteurized skim milk

Mechanical and physicochemical treatments of milk induce structural modifications of the casein (CN) micelles, affecting their techno-functional properties in dairy processing. Here, we studied the effect of alkalinization and ultra-high-pressure homogenization (UHPH) on CN micelles in raw skim milk (rSM) and pasteurized skim milk (pSM). The pH of both skim milks (approximately 6.7) was adjusted to 8.5 and 10.5 before UHPH at 100, 200, and 300 MPa. The structural changes of the CN micelles during the treatments were assessed using laser diffraction, transmission electron microscopy, and turbidity measurements. Finally, ultracentrifugation (70,000 × g for 1 h at 20°C) was carried out to evaluate the protein's distribution between the supernatant (serum phase) and the pellet (colloidal phase) by gel electrophoresis and protein concentration measurement. Alkalinization of both skim milks induced a significant reduction in turbidity, whereas an increase of the average particle size was observed, the effect being more severe in pSM than rSM. At alkaline pH, more proteins were recovered in the serum phase, which suggested that the CN underwent major rearrangements into nonsedimentable CN forms of various sizes, as confirmed by transmission electron microscopy. The amount of CN found in the serum phase at pH 8.5 also increased with the UHPH pressure. Although UHPH did not influence the average CN micelle size at pH 6.7 and 8.5, a pressure-dependent decrease was observed at pH 10.5 for both skim milks. The structural changes of the CN micelles observed in this study throughout the combination of alkalinization and UHPH could be of interest for developing new dairy ingredients with improved functionality.     

Modelling of heat stability and heat‐induced aggregation of casein micelles in concentrated skim milk using a Weibullian model

Heat stability is known to be limited in concentrated skim milk leading to severe coagulation and sediment formation during storage. It has often been found to be in conflict with the extension of the shelf life by thermal treatment. A Weibullian model was used to describe the course of coagulation of casein micelles in concentrated skim milk of different total solids and at different heating temperatures. A maximum heating temperature–time–total solids relationship was calculated based on a certain maximum allowance of sediment formation. Optimal heat treatment conditions for concentrated skim milk of different total solids content could be defined.     

Heat and/or high-pressure treatment of skim milk: changes to the casein micelle size, whey proteins and the acid gelation properties of the milk

Skim milk was subjected to heat, pressure or combined processes. In general, higher levels of whey protein denaturation were observed for milk subjected to combined processes than those heat- or pressure-treated only. Heat treatment caused small changes to the casein micelle size. Pressure treatment decreased the casein micelle size; however, the effect was less marked when heat and pressure treatments were combined. Acidification of the skim milks produced gels with a range of firmness, yield stresses and yield strains depending on the treatments applied. These changes in acid gel properties were not related only to whey protein denaturation levels in the milks.