Rennet gelation properties of milk: Influence of natural variation in milk fat globule size and casein micelle size

Seasonal and farming practises can influence milk composition and functionality. An understanding of changes in milk fat globule (MFG) and casein micelle (CM) size may help to guide the selection of milk on the basis of MFG or CM size for manufacturing of different products and product quality. Milk was obtained from cows known to produce predominantly large or small MFG and CM. The rennet gelation properties of this milk were investigated by measuring the rheological properties during gel development. The structure of the CM and MFG network within the rennet gel were characterised by a series of microscopy techniques. Milk with small CM produced firmer curds, and the combination of large MFG (4.49–5.38 μm) with small CM (164–168 nm) produced the firmest curd of any of the combinations tested. MFG size can influence rennet gel firmness, an effect that is dependent upon the pore structure of the CM network.     

Effects of Size and Stability of Native Fat Globules on the Formation of Milk Gel Induced by Rennet

Rennet‐induced gelation crucially impacts cheese structure. In this study, effects of the size and stability of native fat globules on the kinetics of rennet‐induced coagulation were revealed by determining the caseinomacropeptide release rate and rheological properties of milk. Moreover, the mobility and stability of fat globules during renneting was revealed using diffusing wave spectroscopy and confocal laser scanning microscopy. By use of a 2‐stage gravity separation combined centrifugation scheme, native fat globules were selectively separated into small (SFG, D_4,3 = 1.87 ± 0.02 μm) and large fat globules (LFG, D_4,3 = 5.65 ± 0.03 μm). The protein and fat content of SFG and LFG milk were then standardized to 3.2 g/100 mL and 1.2 g/100 mL, respectively. The milk containing different sized globules were then subjected to renneting experiments in the laboratory. Reduction of globule size accelerated the aggregation of casein micelles during renneting, giving a shorter gelation time and earlier 1/l^* change. The gel produced from LFG milk was broken due to coalescent fat globules and generated coarser gel strands compared to the finer strands formed with SFG milk. Structural differences were also confirmed with a higher final storage modulus of the curd made from SFG milk than that from the LFG. In conclusion, the size of fat globules affects the aggregation of casein micelles. Moreover, fat globule coalescence and creaming during renneting, also affects the structure of the rennet gel. A better understanding of the size of globules effect on milk gelation could lead to the development of cheese with specific properties.     

Serum Protein Aggregates in the High-Heated Milk and Their Gelation Properties in Rennet-Induced Milk Gel

The influence of different heat treatments on the protein aggregates and changes in gelation properties of rennet-induced milk gels were investigated. In the heated milk, a visible difference in milk serum proteins was found resulting from the formation of protein aggregates. Meanwhile, the size of protein aggregates increased from 25 to 170 nm with increasing the intensity of heat treatment. Furthermore, the differences in textural variables of rennet gels were found among the heat treatments using the principal component analysis. The water holding capacity and cheese curd yield of rennet gels obtained from the heated milk were significantly greater than those of raw milk (p < 0.05). It was also found heat treatments above 80°C could endow rennet-induced milk gels with novel textural properties.     

Effect of temperature and pH on the properties of skim milk gels made from a tamarillo (Cyphomandra betacea) coagulant and rennet

Reconstituted skim milk was gelled with a crude protease extract from tamarillo [Cyphomandra betacea or Solanum betacea (syn.)] fruit and compared with gels prepared with calf rennet. The effects of temperature and pH on the gelation of skim milk were investigated by small deformation oscillatory rheology. The tamarillo extract-induced gels had a faster rate of increase in the elastic modulus (G′) at the early stage of gelation than rennet-induced milk gels. This was probably due to the broader proteolytic activity of tamarillo protease extracts as shown by sodium dodecyl sulfate–PAGE analysis. Confocal microscopy also showed that the milk gels resulting from the addition of tamarillo extracts had larger voids than rennet-induced milk gels. The proteolytic activity of tamarillo extracts was found to be optimal at pH 11. For both rennet and tamarillo extracts, the aggregation time was similar between pH 6.7 and 6.5, but the aggregation time of rennet-induced milk gels was lower than that of milk gels obtained by the addition of tamarillo extracts at pH lower than 6.5. An increase in temperature was found to have a significant effect on aggregation time, particularly at 20°C, where rennet did not coagulate milk in 3 h but the tamarillo extracts coagulated milk within 2 h. The results of this study suggest that extracts from tamarillo fruit could be used for milk gelation, particularly under lower temperature or high pH conditions.     

Gelation properties of partially renneted milk

Acid- and rennet-induced gelation properties of milk with modified casein micelles, produced by partial renneting at 4^oC for 15 min, followed by inactivation of enzymes by heat at 60^oC/3 min (referred as low heat treatment milk) and 85^oC/30 min (high heat treatment milk), were investigated to provide a mechanistic understanding of gel formation from partially renneted milk. Acidification of low heat treatment milk gave firmer gel quality, this was reflected by its high elastic modulus (G′) and hardness. In addition, the high heating condition for enzyme inactivation of high heat treatment milk alone increased the elastic modulus of both the control and renneted milk samples. Gel development of the two milk types (low heat treatment and high heat treatment milks) was different. In contrast with acid gelation, rennet-induced gelation of partially pre-rennet treated milk had no impact on the elastic modulus of low heat treatment milk and the rennet gels were very weak. Similarly, the addition of rennet to pre-rennet treated high heat treatment milk did not produce “true gels,” most likely due to the effect of the heat treatment on impairing the rennet coagulation. The findings in this study confirmed that pre-rennet treated milk had positive effects on the end-product acid gels of low heat treatment and high heat treatment milk.     

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.     

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.     

Addition of sodium caseinate to skim milk inhibits rennet-induced aggregation of casein micelles

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl₂ prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α_s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.     

Acid and rennet gelation properties of sheep,goat, and cow milks: Effects of processing and seasonal variation

Gelation is an important functional property of milk that enables the manufacture of various dairy products. This study investigated the acid (with glucono-δ-lactone) and rennet gelation properties of differently processed sheep, goat, and cow milks using small-amplitude oscillatory rheological tests. The impacts of ruminant species, milk processing (homogenization and heat treatments), seasonality, and their interactions were studied. Acid gelation properties were improved (higher gelation pH, shorter gelation time, and higher storage modulus (G′) by intense heat treatment (95°C for 5 min) to comparable extents for sheep and cow milks, both better than those for goat milk. Goat milk produced weak acid gels with low G′ (<100 Pa) despite improvements induced by heat treatments. Seasonality had a marked impact on the acid gelation properties of sheep milk. The acid gels of late-season sheep milk had a lower gelation pH, no maximum in tan δ following gel formation, and 70% lower G′ values than those from other seasons. We propose the potential key role of a critical acid gelation pH that induces structural rearrangements in determining the viscoelastic properties of the final gels. For rennet-induced gelation, compared with cow milk, the processing treatments of the goat and sheep milks had much smaller impacts on their gelation properties. Intense heat treatment (95°C for 5 min) prolonged the rennet gelation time of homogenized cow milk by 8.6 min (74% increase) and reduced the G′ of the rennet gels by 81 Pa (85% decrease). For sheep and goat milks, the same treatment altered the rennet gelation time by only less than 3 min and the G′ of the rennet gels by less than 14 Pa. This difference may have been caused by the different physicochemical properties of the milks, such as differences in their colloidal stability, proportion of serum-phase caseins, and ionic calcium concentration. The seasonal variations in the gelation properties (both acid and rennet induced) of goat milk could be explained by the minor variation in its protein and fat contents. This study provides new perspectives and understandings of milk gelation by demonstrating the interactive effects among ruminant species, processing, and seasonality.     

Rheological properties and microstructure during rennet induced coagulation of UF concentrated skim milk

Rennet induced coagulation of ultrafiltrated (UF) skim milk (19.8%, w/w casein) at pH 5.8 was studied and compared with coagulation of unconcentrated skim milk of the same pH. At the same rennet concentration (0.010 International Milk Clotting Units g⁻¹), coagulation occurred at a slower rate in UF skim milk but started at a lower degree of κ-casein hydrolysis compared with the unconcentrated skim milk. Confocal laser scanning micrographs revealed that large aggregates developed in the unconcentrated skim milk during renneting. Following extensive microsyneresis the protein strands were shorter and thinner in gels from UF skim milk. Moreover, during storage up to 60 days (13 °C), the microstructure and the size of the protein strands of the UF gel changed only slightly. Hoelter–Foltmann plots suggested that the coagulation rate was reduced in the UF skim milk due to a high zero shear viscosity of the concentrate compared with the unconcentrated skim milk.     

Saccharomyces cerevisiae improves rennet-induced gelation of ultra-high temperature milk

Heating milk at high temperatures impairs its renneting properties, but rennet-induced curds can be formed from ultra-high temperature (UHT) milk inoculated with Saccharomyces cerevisiae. Herein, we measured physicochemical indices of UHT milk inoculated with S. cerevisiae before rennet addition, monitored the kinetics of gel formation, and investigated the physicochemical properties and microstructure of rennet-induced curds to explore the mechanisms by which S. cerevisiae influenced rennet-induced gelation of UHT milk. Compared with untreated pasteurized cow milk and UHT milk, the ethanol content was increased, the pH was decreased, the particle size and ζ-potential were increased, the time points at which the elasticity index began to increase were advanced, and the maximum elasticity index was increased for UHT milk inoculated with S. cerevisiae. The number of S. cerevisiae cells affected the structure of rennet-induced curds; with few cells added, the protein network of curds was continuous and tight, the mean square displacement curves showed an asymptotic behavior, and the water retention capacity and curd yield were high; with more cells added, the loosely entangled proteins aggregated, the continuity of the network was destroyed, and the curd yield decreased. In summary, a low number of S. cerevisiae cells (<1.0 × 10⁷ cfu/mL) can increase particle size, ζ-potential, and ethanol content, and decrease pH of S. cerevisiae-inoculated UHT milk, thereby accelerating the aggregation reactions after enzymatic reaction and improving the renneting properties.     

Dextran modulates physical properties of rennet-induced milk gels

Physical properties of rennet-induced milk gels as core intermediates of cheese production are mainly affected by milk composition, type and amount of coagulation enzyme and starter culture activity. We investigated model systems of reconstituted milk and dextran, which triggers effects on milk gels similar to exopolysaccharides from lactic acid bacteria. Furthermore, clotting activity (0.02 or 0.04 IMCU mL⁻¹) and milk pH adjusted prior to renneting (6.5–5.7) were studied. A lower pH at renneting resulted in an earlier gelation onset, a higher gelation velocity and gel stiffness. The addition of dextran stabilised the gels especially at higher pH, and microstructural analysis revealed larger, more interconnected protein aggregates. However, at pH 5.7, a reverse effect was observed, indicating a destabilisation of the casein network. The current study indicates that altering milk pH and addition of polysaccharides gives the potential to change textural properties of cheese by affecting rennet-induced gelation.     

Effects of ultrasonication on the physicochemical properties of milk fat globules of Bubalus bubalis (water buffalo) under processing conditions: A comparison with shear-homogenization

Ultrasonication has been widely studied in bovine milk but the effects of ultrasound (US) on the buffalo's milk fat globules (MFG) are not well known yet. In this study, buffalo's milk samples were ultrasonicated at 20 kHz and physicochemical properties were assessed under different processing conditions. Shear homogenization was performed with 1188 J/mL energy density for the comparison. Results show that ultrasonication reduced the average volume-weighted mean diameter (D[4,3]) of MFG by 93% and increased the surface area by a factor of 8.5 compared to the native counterparts. The zeta-potential (ZP) of MFG are increased by ultrasonication compared to fresh milk (−26.37 vs. −18.22 mV) indicating better stability. Changing the pH of ultrasonicated milk to the isoelectric pH (pI) reduced the zeta-potential (ZP) by −19.8 mV and increased the (D[4,3]) > 0.4 mm indicating the gelation. The size of milk particles increased up to 76–586 μm and the ZP was reduced by 3.1 mV with the increase of ionic strength from 50 to 200 mM. Heating of milk at 90 °C for 30 min increased D[4,3] in US homogenized milk by 22%. Both ultrasonication and shear-homogenization increased the free saturated fatty acids by 2.75–3 g/100 g fat compared to raw buffalo's milk. Ultrasonication increased the gel hardness by 98% compared and shear-homogenized milks. The results of this study indicate that the ultrasonication reduced the size of buffalo's MFG up to sub-micron level with superior stability while improving (P < 0.05) the gels strength compared to shear-homogenization.Industrial relevanceBuffalo set-yoghurts made with unhomogenized milk exhibit higher syneresis and poor stability upon shear-induced breakdown, which are mainly due to the porous gel structure containing a large number of bigger fat globules. Under the large scale production, buffalo set-yoghurts are often fortified with dairy/non-dairy solids and stabilizers in order to prevent this problem. However, the use of some inexpensive, non-food grade alternatives are also reported in some parts of the world for the cottage or medium level production of buffalo yoghurts. In this work, it was shown that ultrasonication with the energy density of 1188 J/mL can be used to produce buffalo set-yoghurts with superior gel strength and therefore, can be used as an unconventional approach to improve the product quality.     

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.     

Modelling of rheological,microbiological and acidification properties of a fermented milk product containing a probiotic strain of Lactobacillus paracasei

The simultaneous effect of fermentation temperature (FT, 36.7–43.4°C), milk total solid level (TS, 11.3–14.7%, w/v) and total inoculum concentration (TI, 2.16–3.84 v/v) on the acidification process and the rheological properties of fermented milk products with Lactobacillus paracasei ssp paracasei B117, Lactobacillus delbrueckii ssp bulgaricus Y 6.15 and Streptococcus thermophilus Y 4.10 was explored by means of response surface methodology. Maximum storage modulus (G′_max), minimum loss tangent (tan δ_min), rate of gelation (I_E) and onset of gelation were the rheological parameters studied. Maximum acidification rate (V_m), time at which maximum acidification rate was observed (T_m), and time to reach the end of fermentation (T_e) characterized the kinetics of acidification, whereas the increase in the number of the three bacteria at the end of fermentation was chosen as the microbiological parameter of the system. The growth/survival of microorganisms and the organic acid profile during cold storage as well as the overall product acceptability by a consumer panel were also assessed. TS strongly affected G′_max and tan δ_min; high TS resulted in large increase in G′_max and decrease in tan δ_min. Increasing fermentation temperature gave a decrease in the onset of gelation, V_m, T_m and T_e, and an increase in the gelation rate (I_E). Under conditions of relatively low FT (37–40°C), high TS (about 14%) and high TI (3–4%), relatively high gelation and low acidification rates were observed, fermentation took a longer time to finish, but the formed gels were firmer, showing higher G′_max and lower tan δ_min values. Low FT (36–38°C) enabled higher increase in the number of L. paracasei B 117. The probiotic strain showed good compatibility with the S. thermophilus Y 4.10 and L. bulgaricus Y 6.15, and satisfactory levels of all bacteria were found during fermentation and storage at 4°C for 21 days. No major differences in lactic and uric acid contents were seen between the control (probiotic strain-free product) and the probiotic fermented milk, whereas the latter contained slightly higher amounts of citric, pyruvic and orotic acids. Moreover, the probiotic fermented milk was graded by the consumer panel with a similar acceptability score as the control product.     

Nutrient enrichment of dairy curd by incorporation of whole and ruptured microalgal cells (Nannochloropsis salina)

This work investigated incorporation of Nannochloropsis salina into renneted dairy gels and curd. Whole and ruptured microalgal cells did not impair κ-casein macropeptide cleavage by the rennet enzyme. However, insoluble components of ruptured cells impeded gelation, presumably by hindering interactions between renneted casein micelles. Confocal imaging showed that whole cells were retained and homogenously distributed within the protein network of the gels and cooked curd, whereas ruptured algae formed large aggregates that altered the protein matrix. Eicosapentaenoic acid (EPA) in the whole microalgal cells was incorporated within the curds, with considerably less EPA retained for ruptured cells. Soluble algal debris did not impair gelation, however EPA wasn't retained in the curd. The study demonstrates that nutrient enrichment of renneted dairy products is possible by incorporating whole microalgal cells to displace milk fat with protein and the beneficial long-chain omega-3 fatty acid EPA. Future research into the optimisation of product organoleptic properties is required.     

Atlantic Cod Gastric Protease. Characterization with Casein and Milk Substrate and Influence of Sepharose Immobilization on Salt Activation, Temperature Characteristics and Milk Clotting Reaction

The temperature coefficient for the enzymic phase of milk clotting was lower for cod gastric protease than for calf rennet and various other rennet substitutes. Immobilization of cod pepsin on Sepharose resulted in an increase in Arrhenius activation energy for hemoglobin hydrolysis from 8.5 Kcal/mole to 12.8 Kcal/mole. Sepharose-cod protease did not catalyze the enzymic phase of milk clotting. Cold renneting of milk substrate with cod gastric protease at 0°C resulted in continued formation of nonprotein nitrogen (NPN) after the enzymic phase of milk clotting was complete. Initiation of milk clotting by raising the temperature to 39°C prevented the subsequent formation of NPN.     

Physicochemical Properties of Milk Fat Emulsions Stabilized with Bovine Milk Fat Globule Membrane

Milk fat globules (MFG) were reconstituted with milk fat globule membrane (MFGM) and milk fat (MF). Viscosity of the reconstituted MFG was highest at pH 5.0 and 4 min emulsifying, and rose with an increase of MFGM between 40–80 mg/g fat. Adsorbed protein/unit fat increased at acid pH with increase of MFGM. The composition of proteins adsorbed on the surface of MFG was not influenced by factors of reconstitution. The size and specific surface area of globules were influenced by emulsifying time, MFGM and MF concentrations, and pH. The size range of MFG prepared by standard method was 0.9–17 μm in diameter. Median diameter was 5 μm and specific surface area was 15,600 cm²/cm³ of emulsion.     

Rheological properties of milk gels formed by a combination of rennet and glucono-delta-lactone

The effects of heat treatment of milk, and a range of rennet and glucono-delta-lactone (GDL) concentrations on the rheological properties, at small and large deformation, of milk gels were investigated. Gels were made from reconstituted skim milk at 30 degrees C, with two levels each of rennet and GDL. Together with controls this gave a total of sixteen gelation conditions, eight for unheated and eight for heated milk. Acid gels made from unheated milks had low storage moduli (G') of < 20 Pa. Heating milks at 80 degrees C for 30 min resulted in a large increase in the G' value of acid gels. Rennet-induced gels made from unheated milk had G' values in the range approximately 80-190 Pa. However, heat treatment severely impaired rennet coagulation: no gel was formed at low rennet levels and only a very weak gel was formed at high levels. In gels made with a combination of rennet and GDL unusual rheological behaviour was observed. After gelation, G' initially increased rapidly but then remained steady or even decreased, and at long ageing times G' values increased moderately or remained low. The loss tangent (tan delta) of acid gels made from heated milk increased after gelation to attain a maximum at pH approximately 5.1 but no maximum was observed in gels made from unheated milk. Gels made by a combination of rennet and GDL also exhibited a maximum in tan delta, indicating increased relaxation behaviour of the protein-protein bonds. We suggest that this maximum in tan delta was caused by a loosening of the intermolecular forces in casein particles caused by solubilization of colloidal calcium phosphate. We also suggest that in combination gels made from unheated milk a low value for the fracture stress and a high tan delta during gelation indicated an increased susceptibility of the network to excessive large scale rearrangements. In contrast. combination gels made from heated milk formed firmer gels crosslinked by denatured whey proteins and underwent fewer large scale rearrangements.