Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Effect of pH at pressure treatment on the acid gelation of skim milk

Skim milk at pH between 6.4 and 7.3 was pressure treated at 200–600 MPa for 30 min and then slowly acidified with glucono-δ-lactone to form acid gels. Milks at low pH produced acid gels with low elastic moduli (final G′) and yield stresses and those at higher pH produced acid gels with higher final G′ and yield stresses. Pressure treatment disrupted the casein micelles at all pH and transferred high levels of casein to the serum phase. Denaturation of α-lactalbumin occurred at a pressure of 600 MPa only, and the level of denaturation increased with increasing pH. Denaturation of β-lactoglobulin (β-LG) occurred at all pressures, with the level of denaturation increasing with the magnitude of the pressure treatment and with pH. The denaturation of the whey proteins and the disruption of the casein micelles could not entirely account for the changes in the rheological properties of the acid gels, as denaturation of up to 50% of the whey proteins produced acid gels with very low final G′ and yield stresses. It is proposed that the pH and the magnitude of the pressure treatment affect the interactions of the denatured β-LG with the casein proteins in the pressure-treated milks, and that this affects the ability of the denatured β-LG to participate in the acid gel structures.Industrial relevanceThe control and manipulation of the firmness of acid skim milk gels is important in many dairy food applications such as yogurts and some types of cheeses. This study has demonstrated that acid gel firmness can be substantially manipulated when the milk is pH adjusted and pressure treated before acidification, and that these effects are different to those obtained through heating. The commercial uptake of high pressure processing in the dairy industry is dependent on this technology producing unique functional properties in milk when compared with traditional processing. The results of this study indicates that high pressure processing of milk may offer unique functional properties in acid gel applications which could be used for the development of new or improved dairy products.     

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 pressure treatment and heat treatment of skim milk with added starch on subsequent acid gelation of milk

Skim milk with added starch (waxy rice starch or potato starch at levels of 0-1.5 g/100 g) was either pressure-treated (500 MPa, 20 °C, 30 min) or heat-treated (80 °C, 30 min) and subsequently acidified (using glucono-δ-lactone) to form acid milk gels. In the second part of the study, the pH of the skim milk samples was adjusted from the natural condition (pH 6.64) to pH 6.5, 6.6 or 6.9 before the pressure or heat treatment and re-adjusted back to pH 6.64 after the respective treatment. The rheological properties of the samples during acidification and of the final acid gels were studied. The storage modulus, G′, of the final acid milk gels increased as more waxy rice starch was added to milk before pressure or heat treatment. However, acid milk gels made from pressure-treated milk with added potato starch did not show significant changes in the G′ of the final acid gels whereas those made from the heat-treated counterparts showed a marked increase in the final G′ as the potato starch level increased. Waxy rice starch was gelatinised in milk by both pressure treatment and heat treatment whereas potato starch was gelatinised by heat treatment only. Increasing the pH of milk before pressure or heat treatment increased the final G′ of the acid milk gel produced on subsequent acidification of the milk and the final G′ was increased further by the addition of waxy rice starch before the pressure or heat treatment.     

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.     

Properties of milk protein gels formed by phosphates

We investigated the properties of gels that were formed by adding emulsifying salts, such as tetrasodium pyrophosphate (TSPP), to reconstituted milk protein concentrate solution. The pH of a 51 g/L milk protein concentrate solution was adjusted to 5.8 after adding TSPP. Milk protein concentrate solutions were placed in glass jars and allowed to stand at 25°C for 24 h. Gels with the highest breaking force were formed when TSPP was added at a concentration of 6.7 mM, whereas no gel was formed when TSPP was added at concentrations of ≤2.9 or ≥10.5 mM. Several other phosphate-based emulsifying salts were tested but for these emulsifying salts, gelation only occurred after several days or at greater gelation temperatures. No gelation was observed for trisodium citrate. Gelation induced by TSPP was dependent on pH, and the breaking force of gel was greatest at pH 6.0. Furthermore, when the concentration of milk protein concentrate in solution was increased to 103 g/L, the breaking force of the gel increased, and a clearly defined network between caseins could be observed by using confocal scanning laser microscopy. These results suggest that TSPP-induced gelation occurs when the added TSPP acts with calcium as a cross-linking agent between dispersed caseins and when the balance between (a reduced) electrostatic repulsion and (enhanced) attractive (hydrophobic) interactions becomes suitable for aggregation and eventual gelation of casein molecules.     

Effect of potato starch addition on the acid gelation of milk

Potato starch was added to skim milk at levels of 0–1.5%. The milks were heated and then acidified to form acid milk gels. The properties of the milks during acidification and the final properties of the acid gels were studied. The addition of starch resulted in a higher storage modulus, G′, in the final acid gels, and increasing the level of starch caused a linear increase in the final G′. Compared with acid gels prepared with no starch, the gelation time was reduced and the gelation pH was increased. However, the temperature and frequency dependences of the acid gels were not affected by the addition of starch. Furthermore, the breaking strain of the acid gels was not markedly affected by the addition of starch, whereas the breaking stress was dependent on the level of starch added. Confocal microscopy showed that the acid gels contained swollen starch granules embedded in a protein network, and that the protein network increased in density as the level of starch added increased.     

Excessive cross-linking of caseins by microbial transglutaminase and its impact on physical properties of acidified milk gels

By varying cross-linking intensity, the effect of microbial transglutaminase on acid gels made from casein solution and raw milk was studied. To avoid any impact of heating, N-ethylmaleimide was used for enzyme inactivation after appropriately checking its efficiency. Up to a specific degree of oligomerisation gel stiffness and firmness increased and tan δ, time at gelation onset and syneresis decreased. Above approximately 70% and 25% of cross-linked protein in casein solution and raw milk, respectively, these parameters showed an opposite behaviour, and weak gels with high syneresis were obtained. Substrate differences, such as preferred cross-linking of adjoining κ-caseins on the surface of the micelle enhanced the effect of steric hindrance in raw milk and impaired proper rearrangements upon acidification at a much lower level of oligomerised protein. It is mainly dimeric and trimeric casein that successfully contributed to the enhanced properties of milk protein gels.     

Physical properties of acid milk gels prepared at 37°C up to gelation but at different incubation temperatures for the remainder of fermentation

We investigated the effect of altering temperature immediately after gels were formed at 37°C. We defined instrumentally measurable gelation (IMG) as the point at which gels had a storage modulus (G′) ≥5 Pa. Gels were made at constant incubation temperature (IT) of 37°C up to IMG, and then cooled to 30 or 33.5, or heated to 40.5 or 44°C, at a rate of 1°C/min and maintained at those temperatures until pH 4.6. Control gel was made at 37°C (i.e., no temperature change during gelation/gel development). Gel formation was monitored using small strain dynamic oscillatory rheology, and the resulting structure and physical properties at pH 4.6 were studied by fluorescence microscopy, large deformation rheology, whey separation (WS), and permeability (B). A single strain of Streptococcus thermophilus was used to avoid variations in the ratios of strains that could have resulted from changes in temperature during fermentation. Total time required to reach pH 4.6 was similar for samples made at constant IT of 37°C or by cooling after IMG from 37 to either 30 or 33.5°C, but gels heated to 40 or 44°C needed less time to reach pH 4.6. Cooling gels after IMG resulted in an increase in G′ values at pH 4.6, a decrease in LT_max, WS, and B, and an increase in the area of protein aggregates of micrographs compared with the control gel made at constant IT of 37°C. Heating gels after IMG resulted in a decrease in G′ values at pH 4.6 and an increase in LT_max values and WS. The physical properties of acid milk gels were dominated by the temperature profile during the gel-strengthening phase that occurs after IMG. This study indicates that the final properties of yogurt greatly depend on the environmental conditions (e.g., temperature, time/rate of pH change) experienced by the casein particles/clusters during the critical early gel development phase when bonding between and within particles is still labile. Cooling of gels may encourage inter-cluster strand formation, whereas heating of gels may promote intra-cluster fusion and the breakage of strands between clusters.     

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.     

Effects of gelation temperature on Mozzarella-type curd made from buffalo and cows’ milk. 1: Rheology and microstructure

The rheology and microstructure of Mozzarella-type curds made from buffalo and cows’ milk were measured at gelation temperatures of 28, 34 and 39 °C after chymosin addition. The maximum curd strength (G′) was obtained at a gelation temperature of 34 °C in both types of bovine milk. The viscoelasticity (tan δ) of both curds was increased with increasing gelation temperature. The rennet coagulation time was reduced with increase of gelation temperature in both types of milk. Frequency sweep data (0.1–10Hz was recorded 90 min after chymosin addition, and both milk samples showed characteristics of weak viscoelastic gel systems. When both milk samples were subjected to shear stress to break the curd system at constant shear rate, 95 min after chymosin addition, the maximum yield stress was obtained at the gelation temperatures of 34 °C and 28 °C in buffalo and cows’ curd respectively. The cryo-SEM and CLSM techniques were used to observe the microstructure of Mozzarella-type curd. The porosity was measured using image J software. The cryo-SEM and CLSM micrographs showed that minimum porosity was observed at the gelation temperature of 34 °C in both types of milk. Buffalo curd showed minimum porosity at similar gelation temperature when compared to cows’ curd. This may be due to higher protein concentration in buffalo milk.     

Effect of transglutaminase and acidification temperature on the gelation of reconstituted skim milk

Effects of transglutaminase (TG), acidification temperature and total milk solids level on the acid gelation of skim milk were investigated. Despite similar acidification kinetics, TG-treated milk acidified at ≥35 °C showed differences in elastic modulus (Gʹ) with acidification time, particularly by inhibiting the formation of the peculiar shoulder (Gʹ_shoulder) observed at an early stage of gelation in the control milk. Regardless of the milk solids content, the G'_shoulder was absent in both types of milk at 30 °C. However, control milk above 2.5% (w/w) milk solids showed the G'_shoulder at 45 °C. G'_shoulder is proposed as the transition from the first increase in Gʹ, due to aggregation of soluble protein complexes at the early stage of acidification, to the second, due to further aggregation of casein micelles (and aggregated soluble complexes) as acidification progresses. The G'_shoulder was absent in acidified TG-treated milk due to the lack of soluble protein complexes containing caseins.     

Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A,B and C variants of β-lactoglobulin

The effect of fortification of reconstituted skim milk with different levels of a whey protein mixture containing a 1:2 ratio of α-lactalbumin (α-la) and different genetic variants of β-lactoglobulin (β-LG) on the rheological properties of acid milk gels, formed by acidification with glucono-δ-lactone, was investigated. Milk samples were either unheated or heated at 80°C for 30 min before acidification. Acid gels prepared from unheated skim milk had very low G′ values, long gelation times and low gelation pH. Samples prepared from heated milk had markedly higher G′ values, a reduced gelation time and an increased gelation pH. The addition of increasing levels of whey protein mixtures containing β-LG B or β-LG C to the milk prior to heating and acidification caused an almost linear increase in the G′. In contrast, whey protein mixtures containing β-LG A caused a progressive increase in the G′ with added protein levels up to about 0.7% (w/w) but little further change at higher addition levels. A mixture of the A and B variants of β-LG gave an intermediate behaviour between those of the A and B variants. In all samples, the G′ value at 5°C was approximately twice that at 30°C so that the relative differences as a result of the β-LG genetic variants were similar for the two temperatures.     

Are disulphide bonds formed during acid gelation of preheated milk?

The role of thiol/disulphide exchanges during acid gelation of preheated milk was studied with milk samples with or without N‐ethylmaleimide (NEM), a thiol‐blocking agent, and acidified to pH 4 by the addition of glucono‐delta‐lactone at 20 °C. Active or total thiol groups, particle size with light scattering measurements in a dissociating solvent or by SDS‐agarose electrophoresis were determined on acidified milk samples. Diffusing wave spectroscopy and rheology in low strain were applied during acidification of sample, while rheology in large strain was applied on final acid gels. The only effect of the presence of NEM was a reduced firmness of acid gels as measured at large strain and a reduced tendency to form large aggregates at pH<5.5. In conclusions, thiol/disulphide exchanges during acidification of milk played only a minor role in the building of acid gel networks from heated milk.     

Comparative study on acid-induced gelation of myosin from Atlantic cod (Gardus morhua) and burbot (Lota lota)

Physicochemical and rheological properties of myosin from Atlantic cod and burbot during acid-induced gelation at room temperature (22–23 °C) by d-gluconic acid-δ-lactone (GDL) were monitored. Turbidity and particle size of both myosins increased and salt soluble content decreased when pH decreased, suggesting the formation of protein aggregates caused by acidification. The formation of disulphide bonds in myosin gelation was induced by acid. Ca²⁺-ATPase activity of myosin decreased (p < 0.05), while surface hydrophobicity increased during acidification (p < 0.05). Furthermore, the decreases in maximum transition temperature (T_max) and the denaturation enthalpies (ΔH) were found in both myosins. During acidification, the increases in storage modulus (G′) and loss modulus (G″) of myosin were observed (p < 0.05), revealing the formation of elastic gel matrix. Thus, gelation of myosin from Atlantic cod and burbot could take place under acidic pH via denaturation and aggregation. However, myosin from Atlantic cod was generally more favourable to gelation than was burbot myosin.     

Effect of insoluble calcium concentration on rennet coagulation properties of milk

Rennet-induced gels were made from milk acidified to various pH values or milk at pH 6.0 that had added EDTA. The objective was to examine the effect of removing insoluble Ca (INS Ca) from casein micelles (CM) on rennet gelation properties. For the pH trial, diluted lactic acid was added to reconstituted skim milk to decrease the pH to 6.4, 6.0, 5.8, 5.6, and 5.4. For the EDTA trial, EDTA was slowly added (0, 2, 4, and 6 mM) to reconstituted skim milk, and the final pH values were subsequently adjusted to pH 6.0. Dynamic low amplitude oscillatory rheology was used to monitor gel development. The Ca content of CM and rennet wheys made from these milks was measured using inductively coupled plasma spectroscopy. The INS Ca content of milk was altered by the acidification pH values or level of EDTA added. In all samples, the storage modulus (G′) exhibited a maximum (GM), with a decrease in G′ during longer aging times. Gels made at pH 6.4 had higher GM compared with gels made at pH 6.7 probably due to the reduction in electrostatic repulsion, whereas the INS Ca content only slightly decreased. The highest GM value of gels was observed at pH 6.4 and the GM value decreased with decreasing pH from 6.4 to 5.4. This was due to an excessive loss of INS Ca from CM. There was a decrease in GM with the increase in the concentration of added EDTA, which was probably due to the loss of colloidal calcium phosphate, which weakens the integrity of CM. Loss tangent (LT) values at GM increased with a reduction in milk pH and the addition of EDTA to milk. Rennet gels at the point of the GM were subjected to constant low shearing to fracture the gels. With a reduction in INS Ca content, the yield stress decreased, whereas LT values increased indicating a weaker, more flexible casein network. Microstructure of rennet-induced gels near the GM point and 2 to 10 h after this point was studied using fluorescence microscopy. At GM, gels made from milk acidified to pH 6.4 exhibited more branched, interconnected networks, whereas strands and clusters became larger with a reduction in milk pH to 5.4. Gels made from milk with EDTA added had more finely dispersed protein clusters compared with gels made from milk with no EDTA added. These microscopic observations supported the effect of loss of INS Ca on GM and LT. There was a decrease in apparent interconnectivity between strands in gel microstructure during aging, which agreed with the decrease in G′ after GM. It can be concluded that low levels of solubilization of INS Ca and the decrease in milk pH resulted in an increase in GM. With greater losses of INS Ca there was excessive reduction in cross-linking within CM, which resulted in weaker, more flexible rennet gels. This complex behavior cannot be explained by adhesive hard sphere models for CM or rennet gels made from these CM.     

Gelation upon long storage of milk drinks with carrageenan

The carrageenan-induced stabilization and gelation of ultra-high-temperature-treated milk was studied during long storage. Severe heating (causing increased protein denaturation), lowering of the pH, or the use of κ-carrageenan (instead of ι-carrageenan) led to excessive gelation. It is suggested that the balance between carrageenan-carrageenan interactions and carrageenan-protein interactions determines the gel strength. If the interactions between carrageenan and proteins are decreased, more carrageenan is available for carrageenan-carrageenan interactions, leading to a stronger gel. This is the case if κ-carrageenan is used instead of ι-carrageenan because the former forms weaker interactions with proteins than the latter. Also, heating and pH influence the attachment of whey proteins to the casein micelle surface, hindering the attachment of carrageenan to the casein proteins. Upon storage, gel strength increased. Particle size and rheology measurements indicated that upon storage, tenuous carrageenan-protein aggregates are formed. The firming of the gel was probably related to slow structural arrangements of the gel and not related to slowly changing calcium equilibria or age gelation.     

Influence of heat treatment of goat milk on casein micelle size,rheological and textural properties of acid gels and set type yoghurts

Acid gels and yoghurts were made from goat milk that was heated at 72°C/30 s, 85°C/5 min, and 95°C/5 min, followed by acidification with starter culture at 43C until pH 4.6. The rheological and textural properties of acid gels and yoghurts were analyzed using dynamic low amplitude oscillatory rheology and back extrusion texture analysis, respectively. The effect of goat milk heat treatment on the mean casein micelle diameter and protein profile was also determined by dynamic light scattering and SDS PAGE electrophoresis, respectively. The shortest gelation and fermentation time was recorded for yoghurt prepared from milk heated at 85°C/5 min. Also, the pH of gelation, the storage moduli (G′) and yield stress were higher for this yoghurt, compared with the other two. Textural properties of goat milk yoghurts such as firmness and consistency were strongly affected by milk heat treatment, and the highest values were recorded for yoghurt produced from milk preheated at 85°C/5 min, as well. The largest casein micelles were measured after 85°C/5 min treatment and their size decreased at higher temperature, despite higher denaturation of whey proteins at the most intense heat regime, indicating the structure changes that influence on the acid gelation.     

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.