Rennet Gels Research Articles

The heat treatment and the gelation are strong determinants of the kinetics of milk proteins digestion and of the peripheral availability of amino acids

This study aimed to determine the kinetics of milk protein digestion and amino acid absorption after ingestion of four dairy matrices by six minipigs: unheated or heated skim milk and corresponding rennet gels. Digestive contents and plasma samples were collected over a 7h-period after meal ingestion. Gelation of milk slowed down the outflow of the meal from the stomach and the subsequent absorption of amino acids, and decreased their bioavailability in peripheral blood. The gelled rennet matrices also led to low levels of milk proteins at the duodenum. Caseins and β-lactoglobulin, respectively, were sensitive and resistant to hydrolysis in the stomach with the unheated matrices, but showed similar digestion with the heated matrices, with a heat-induced susceptibility to hydrolysis for β-lactoglobulin. These results suggest a significant influence of the meal microstructure (resulting from heat treatment) and macrostructure (resulting from gelation process) on the different steps of milk proteins digestion.

GELATION OF MIXTURES OF SOYMILK AND RECONSTITUTED SKIM MILK SUBJECTED TO COMBINED ACID AND RENNET

ABSTRACTThe present work investigated the formation of a mixed gel containing soymilk and reconstituted milk. The mixtures contained 1.4 and 2% (w/v) milk and soymilk protein, respectively. When gelation was induced by addition of glucono‐delta‐lactone, the mixtures showed a gelation point well above the isoelectric point of the milk proteins, suggesting that soy proteins play a major role in the formation of the network. When rennet was added in combination with acidification, the gels showed an earlier onset of aggregation and a higher storage modulus than the gels prepared only with acid. Confocal microscopy showed networks with mixed acid–rennet gels having more branches and compact structures with denser clusters than acid‐induced gels. These results demonstrated that by fine‐tuning the gelation of mixed soymilk and reconstituted milk, it is possible to obtain gels with unique microstructure and texture, where both proteins are contributing to the network structure.PRACTICAL APPLICATIONSMixed protein gels are increasingly employed to develop novel high‐protein products. This work illustrates the potential to employ a mixed gelation to induce the formation of protein matrices containing aggregates of soy and milk proteins. At the ratio used in this work (2% soy protein and 1.4% milk protein), soymilk proteins determined the gelation behavior of the mixtures. The gels showed an onset of gelation at pH around 6, and in the presence of rennet the skim milk proteins also participated in the network.

HEAT‐INDUCED PROTEIN AGGREGATES AND DIFFERENCE IN THE TEXTURAL PROPERTIES OF WHOLE MILK GEL

ABSTRACTThe effects of heat on milk protein aggregates and the changes in the textural properties of whole milk gels induced by acid or rennet were investigated. Milk samples were heated at 70, 80 and 90C for 15, 30, 45 and 60 s. Acid gels prepared from heating whole milk showed larger values for hardness, adhesiveness and viscosity than the gels from unheated milk, while the corresponding rennet gels showed lower values when compared with the gels from unheated milk. These results reflect the changes in the heat‐induced protein aggregates, which were examined by size exclusion chromatography using Sephacryl S‐500 High Resolution (GE, Piscataway, NJ). It was also demonstrated that the protein aggregates were an important factor for milk gelation by active influence on acid gels and negative influence on rennet gels.PRACTICAL APPLICATIONSHeat‐induced whey protein‐rich cheeses are gaining in popularity for various reasons such as reducing the risk of pathogens and improving the cheese yield. However, the heat treatment can lead to changes in texture. As a part of the whole study on whey protein‐rich cheese, the research analyzed the effects of high pasteurization on textural properties of the whole milk gels induced by rennet or acid. The results observed should guide the study on modification of protein aggregates, such as the use of an acidifier, and enable manufacturer to make rennet gel with textural properties close to raw milk.

EFFECT OF VARYING LEVELS OF ADDED CALCIUM ON THE BIOCHEMISTRY, TEXTURE AND MICRO-STRUCTURE OF FRESH CHEESE CONTAINING SESAME PROTEIN ISOLATE

This article investigates the impact of sesame protein isolate (SPI) and increased calcium in milk on the biochemistry, rheology and micro-structure of cheese. The characteristics of two sets of cheeses made from raw milk and milk of 8% protein substituted by SPI (SPI milk) with 0, 5, 10 and 20 mM calcium addition were investigated. Adding SPI to milk resulted in rennet gel formation with longer coagulation time and lower storage modulus (G′) at 60 min in cheesemaking. The synergistic influence of SPI and calcium resulted in increased hardness, cohesiveness, adhesiveness and meltability during 21 days of cheese aging. Adding calcium to SPI milk resulted in a more compact and regular micro-structure in cheese. Scanning electron microscopy indicated the associative effect of sesame protein and calcium generated greater aggregation in complex cheese micelles, which was key in SPI cheese property formation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gel electrophoretograms of the wheys and caseins indicated the interaction of sesame protein and casein with calcium. This study is of benefit in the manufacture of novel cheese with traditional orient food to meet with the taste of Chinese people. PRACTICAL APPLICATION This research is focused on the influence of sesame protein and calcium ions on the properties of fresh cheese. In recent years, cheese has become increasingly popular in China and its market has increased more than 50% year upon year. To further promote cheese consumption, its nutritional benefits have been emphasized and various efforts have been made to promote cheese and to make it more acceptable to Chinese tastes. The addition of typical Chinese food ingredients into cheese could provide sensory familiarity to local consumers, and at the same time could also help to improve nutritional balance. The result of this research is of great value to the application of plant proteins as they may serve as novel ingredients added to cheese to meet the requirements of this new and fast-growing market.

Effects of Milk Composition Proportioning on the Texture Properties of Rennet Gels Produced by Reconstituted Milk Powder

The texture properties of rennet gels made with different concentrations of milk fat and protein were investigated. The textural variables, water-holding capacity and apparent viscosity of rennet gels were closely correlated with the reduced fat ratio in the reconstituted milk powder. The demonstrable difference in textural variables was found using the principal component analysis. The apparent viscosity of these gels significantly decreased and the water-holding capacity showed less change compared to the rennet gel made with raw milk. Totally, the gel produced by reconstituted milk powder had less hardness and adhesive, low gumminess, chewiness and viscosity, and more cohesiveness.

Simultaneous use of transglutaminase and rennet in milk coagulation: Effect of initial milk pH and renneting temperature

The objective of the present study was to assess competitive interactions between transglutaminase (TGase) and rennet during rennet coagulation of skim milk. Rennet coagulation was achieved at two different renneting temperatures (30 °C and 34 °C), three initial milk pH levels (6.5, 6.3 and 6.1) and two TGase concentrations (0.6 U g−1 protein and 1.8 U g−1 protein). Results of the relative caseinomacropeptide in serum and degree of polymerization revealed that TGase influenced both the primary and secondary phases of rennet coagulation, respectively. Overall, the higher the renneting temperature and the lower the TGase level, the lower were the yields of the rennet gels. The coagulation times (tc) of the gels decreased with decreased initial milk pH and increased coagulation temperature. The optimum initial milk pH, coagulation temperature and TGase concentration were determined to be 6.3, 30 °C and 1.8 U g−1 protein.

The rennet coagulation mechanisms of a concentrated casein suspension as observed by PFG-NMR diffusion measurements

Pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was used to monitor the diffusion of caseins throughout the rennet coagulation of a highly concentrated casein suspension. Two types of casein species were distinguished according to their diffusing properties and attributed to the dissolved casein fraction, i.e. dissociated caseins and caseinomacropeptides (CMP), and casein particles, respectively. The NMR signal intensity coming from the dissolved casein fraction, which is related to the number of protons it contains, increased all along the experiment whereas the opposite tendency was observed for casein particles. This was explained by the increasing amount of CMP in the whey and the reciprocal loss of protons contained by casein particles, and used to quantitatively estimate the kinetics of the chymosin action. The diffusion of the dissolved casein fraction remained nearly constant during the coagulation process whereas, as revealed by rheometry measurements, the diffusion of casein particles was very sensitive to the structural reorganization of the sample. It decreased by about 35% during the sol–gel transition and increased in similar proportions during the succeeding rise in gel porosity. Our results also provide different types of information on the respective behaviors of dissociated caseins and casein particles during the course of the process, the most remarkable one being that all the casein particles did not aggregate during the sol–gel transition of our sample. This strongly suggests that the rennet gelation of a concentrated dairy solution may be better visualized by the formation of a network backbone during the sol–gel transition which is thereafter reinforced upon further incorporation of casein particles.

Modification of the renneting process in Berridge substrate by transglutaminase

The influence of enzymatic cross‐linking on the rennet coagulation of Berridge substrate was investigated in the rennet gel samples prepared by three different methods using a microbial Tgase. Protein cross‐linking was carried out at each of three stages: before the addition of rennet, simultaneously with rennet and after the addition of rennet. The results showed that the cross‐linking of milk proteins before the addition of rennet inhibited the primary enzymatic phase of rennet coagulation. It was observed that enzymatic cross‐linking affected both the primary and the secondary phases of rennet coagulation. The most suitable application method was the addition of transglutaminase at the 5th minute after the addition of rennet.

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.

Protein interactions in heat-treated milk and effect on rennet coagulation

Skim milk was heated at different pH values to cause differential association of whey proteins (WP) with the casein micelles. All of these milk treatments coagulated poorly with rennet. To understand this in more detail, the casein micelles from heated milk were redispersed in unheated serum or unheated micelles were suspended in the sera from heat-treated milk. Systems containing micelles from milk heated at pH 6.7 and 7.1 were marginally better than the heated milk, but that from milk heated at pH 6.3 was not. The sera from milk heated at pH 6.7 and 7.1 impaired the clotting of native micelles but that from the pH 6.3 milk did not. Native casein micelles were suspended in permeates or dialyzed (against unheated milk) sera from heat-treated milk. Permeate systems free of WP/κ-casein complexes produced significantly stronger rennet gels; as did dialyzed systems. The impaired rennet clotting of heat-treated milk was attributed to a synergistic effect of the casein micelles with their heat-modified surfaces, the soluble serum WP/κ-casein complexes, and other dialyzable serum components.

Adjustment of vat milk treatment to optimize whey protein transfer into semi-hard cheese: A case study

The influence of heat treatment combined with microfiltration on the protein transfer from milk to cheese was investigated. Small-scale laboratory cheese-making experiments (10 L vat) showed that, upon adjusting coagulant and CaCl 2 addition, curd-making was possible as long as β-lactoglobulin denaturation was below 40%. It was also possible to compensate the negative effect of heating on rennet gel formation by protein enrichment through microfiltration. Depending on heat treatment intensity, the protein yield in curd increased to approximately 6%. Using Gouda cheese technology, pilot-scale experiments (500 L vat) showed that, depending on heat treatment intensity (up to 85 °C/60 s) and concentration (up to a concentration factor 2.0), it was possible to reduce the amount of protein which is lost in permeate and whey by 15–30%. Dry matter and crude protein of cheeses were significantly affected by heat treatment and microfiltration of milk, and proteolysis during maturation was delayed.

Short communication: The effect of dry period duration and dietary energy density in early lactation on the rennet gelation properties of milk

This study was carried out to examine the effects of decreasing the dry period (DP) duration and altering the energy density of the diet during early lactation on the rheological characteristics of milk. Forty mature Holstein-Friesian cows were used in a completely randomized design with a 2 × 2 factorial arrangement of treatments. Cows were randomly assigned to 1 of 2 dry period treatments and 1 of 2 nutritional treatments. The DP treatments were continuous milking (CM) or an 8-wk standard dry period (SDP), and the nutritional treatments were a standard energy diet (SE) or a high energy diet (HE). Actual DP lengths were 6.3±1.7 d and 62.1±1.9 d for cows on the CM and SDP treatments, respectively. Milk samples were collected at 2, 6, and 10 wk postpartum. The concentrations of fat, protein, and lactose were determined in each sample. The rennet gelation properties were measured at 31°C using dynamic low-amplitude strain oscillatory rheometry. The following parameters were obtained from the resultant elastic shear modulus (G′): gelation time, maximum curd firming rate, and gel strength. Reducing the DP duration from 62 to 6 d resulted in increases in milk protein concentration (31.8 vs. 34.7 g/kg), maximum curd firming rate (2.58 vs. 3.60 Pa/min), and gel strength (69.4 vs. 90.5Pa). Increasing the dietary energy density decreased percentage milk fat (43.1 vs. 37.7 g/kg) but otherwise had no effect. Gel strength was correlated with maximum curd firming rate (r=0.99), and both variables were correlated with milk protein concentration (r=0.71 and r=0.73, respectively). The results indicate that decreasing the duration of the DP increased milk protein concentration and improved the rennet gelation properties of milk, but that dietary energy density had little effect.

Effect of manufacture and reconstitution of milk protein concentrate powder on the size and rennet gelation behaviour of casein micelles

Samples of raw skim milk, ultrafiltration/diafiltration retentate, concentrated retentate and milk protein concentrate powder (MPC80) from a single commercial production run were analysed using photon correlation spectroscopy. Measurements revealed insignificant differences in casein micelle size between the samples. In addition, there was no discernable difference between raw skim milk and MPC powder dissolved at 60 °C in the amount of casein remaining in supernatants from centrifugation at either 25,000 × g or 174,200 × g. Casein micelles did not appear to be altered during manufacture of MPC. The rennet gelation behaviour of reconstituted MPC was compared with raw skim milk. Reconstituted MPC did not coagulate unless supplemented with approximately 2 m m calcium chloride, which was attributed to the mineral removal during ultrafiltration/diafiltration. Addition of sufficient calcium could restore rennet coagulation kinetics and gel strength of reconstituted MPC to approximately that of raw skim milk.

Influence of the emulsion droplet type on the rheological characteristics and microstructure of rennet gels from reconstituted milk

Rheological and microstructural properties of rennet-induced milk gels containing different fat globules were studied. Recombined milks were prepared by mixing reconstituted low-heat skim milk powder and anhydrous milk fat emulsified with reconstituted skim milk powder (SMP), sodium caseinate (NaCas), whey protein isolate (WPI) or Tween 20. Final elastic modulus of the rennet gels containing WPI- or Tween 20-stabilized fat globules showed significantly lower values compared with those prepared with SMP-emulsified fat globules. SMP-stabilized fat globules interacted with the continuous casein network reinforcing the gel structure. Confocal micrographs supported the rheological data revealing that gels containing SMP-stabilized fat globules formed a tighter network relative to other treatments. Microscopy images also showed some degree of droplet flocculation in the case of gels containing WPI- or Tween 20-stabilized fat globules, and this was most likely the cause of the increase of elastic modulus of these systems. Contrary to reports for acid-induced casein gels, NaCas-stabilized fat globules hindered the formation of rennet gels. These results illustrate that rennet gel structure is affected by droplet-droplet and droplet-casein interactions, which in turn are determined by the composition of the oil-water interface as well as the ionic equilibrium in the reconstituted milk gels.

THE IMPACT OF HOMOGENIZATION AND MICROFILTRATION ON RENNET‐INDUCED GEL FORMATION

ABSTRACT The effects of the independent variables, milk homogenization pressure (p1), concentration factor of milk microfiltration (i) and pH on the rheological properties of rennet milk gels were studied. Nondestructive oscillatory rheometry was used to determine rennet coagulation time (RCT), curd firming rate (CFR) and cutting time (CT). A central composite design, comprising two levels of i (1 and 2), pH (6.4 and 6.6) and p1 (0 and 8 MPa), was applied. Second‐order polynomial models successfully described (R2 > 0.92) the relationship between processing parameters and rheological properties of the gels. pH had the most important influence on RCT, while CFR and CT were strongly influenced by i, pH and the interaction of i and pH. In contradiction to studies on active filler interactions for acid milk gels, a discrepancy was observed between results obtained by compression test and rheometry. Rennet gel firmness strongly decreased with a rise in p1 when measured using the compression test, whereby CFR increased with an increase in p1 when measured using rheometry. The latter result corresponds to higher storage modulus values after a certain time indicating higher gel stiffness. This effect was stronger for concentrated milk than for unconcentrated milk.PRACTICAL APPLICATIONSThe use of microfiltration (MF) and homogenization may reduce raw material and processing time in conventional cheese manufacture. However, MF markedly influences milk composition, and homogenization alters the particle size distribution of fat globules. Hence, both technologies may influence rennet‐induced gel formation, syneresis, cheese composition and quality. Curd firmness of homogenized milk is often too weak to resist the extensive curd treatment applied in semi‐hard cheese manufacture which causes loss of curd fines during the syneresis process and finally decreases cheese yield. MF leads to high curd firmness if cutting is not performed at the appropriate time, which unnecessarily extends processing time. The study of the effect of the individual treatments, as well as of the combination of both on rennet‐induced gel formation, is the first important step to evaluate their impact on further processing steps in cheese making. The combination of both technologies may overcome the antagonistic effect of the individual technology as low curd firmness due to homogenization can be compensated by MF that increases curd firmness and vice versa.

Comparison of rennet gelation using raw and reconstituted skim milk

The gelation properties of raw skim milk were compared with milk reconstituted from low-heat skim milk powder. Coagulation of reconstituted milk, as monitored by capillary viscometry, was slower than that of raw milk, but increased markedly with increased reconstitution time. This was attributed to re-equilibration of calcium from the micelles to the serum. Freshly reconstituted milk supplemented with calcium confirmed this suggestion. The results emphasise the importance of calcium being present in the serum for coagulation of renneted micelles. Gel strength was also improved by increasing reconstitution time. However, gels made from raw milk were significantly stronger than those made from reconstituted milk, even when supplemented with 3 m m calcium. The decreased gel strength from reconstituted milk was attributed to a general increase in casein micelle size that occurs during powder manufacture and is not reversed upon reconstitution.

Effect of Buttermilk Made from Creams with Different Heat Treatment Histories on Properties of Rennet Gels and Model Cheeses

Although many studies have reported negative effects on cheese properties resulting from the use of buttermilk in cheese milk, the cause of these effects has not been determined. In this study, buttermilk was manufactured from raw cream and pasteurized cream, as well as from a cream derived from pasteurized whole milk. Skim milks with the same heat treatments were also manufactured to be used as controls. Compositional analysis of the buttermilks revealed a pH 4.6-insoluble protein content approximately 10% lower than that of the skim milk counterparts. Milk fat globule membrane (MFGM) proteins remained soluble at pH 4.6 in raw cream buttermilk; however, when heat was applied to cream or whole milk before butter making, MFGM proteins precipitated with the caseins. Rennet gel characterization showed that MFGM material in the buttermilks decreased the firmness and increased the set-to-cut time of rennet gels, but this effect was amplified when pasteurized cream buttermilk was added to cheese milk. The microstructure of gels was studied, and it was observed that gel appearance was very different when pasteurized cream buttermilk was used, as opposed to raw cream buttermilk. Model cheeses manufactured with buttermilks tended to have a higher moisture content than cheeses made with skim milks, explaining the higher yields obtained with buttermilk. Superior retention of MFGM particles was observed in model cheeses made from pasteurized cream buttermilk compared with raw cream buttermilk. The results from this study show that pasteurization of cream and of whole milk modifies the surface of MFGM particles, and this may explain why buttermilk has poor coagulation properties and therefore yields rennet gels with texture defects.

Rheological Properties of Rennet Gels Containing Milk Protein Concentrates

Different milk protein concentrates (MPC), with protein concentrations of 56, 70, and 90%, were dispersed in water under different treatments (hydration, shear, heat, and overnight storage at 4°C), as well as in a combination of all the treatments in a factorial design. The particle size distribution of the dispersions was then measured to determine the optimal conditions for the dispersion. Heating at 60°C for 30min with 5min of shear was chosen as the best condition to dissolve MPC powders. The samples were also characterized for composition, presence of protein aggregates, and ratio of calcium to protein. The total calcium present in MPC increased with increasing concentration of protein; however, the total calcium-to-protein ratio was lower in MPC90 than in MPC56 and MPC70. The level of whey protein denaturation, the presence of κ-casein-whey protein aggregates in the supernatant after centrifugation, and the amount of caseins dissociated from the micelle increased as the protein concentration in the powder increased. The total amount of casein macropeptide released was lower in samples from powders with a higher protein concentration than for MPC56 or the skim milk control. The gelation behavior of reconstituted MPC was tested in systems dispersed in water (5% protein) as well as in systems dispersed in skim milk (6% protein). The gelation time of MPC dispersions was considerably lower and the gel modulus was higher than those of reconstituted skim milk with the same protein concentration. When MPC dispersions were dialyzed against skim milk, a significant decrease in the gelation time and modulus were shown, with a complete loss of gelling functionality in MPC90 dispersed in water. This demonstrated that the ionic equilibrium was key to the functionality of MPC.

Production and functional properties of micellar casein/ κ-carrageenan aggregates

When mixtures of reconstituted skim milk powder (SMP; 10.5% solids) and κ-carrageenan (0.025%) were cooled from 60 to 25 °C while shearing, casein micelles and κ-carrageenan formed 10–100 μm aggregates. Aggregation decreased with increasing total milk solids at constant casein: κ-carrageenan ratios. Reducing ionic concentration from reconstituted SMP by ultrafiltration prior to κ-carrageenan addition restored aggregate formation, thus enabling production of a spray-dried powder containing 10–100 μm aggregates. When reconstituted, the aggregate powder had a lower viscosity than an unaggregated mixture of κ-carrageenan and skim milk and was very close to the retentate at the same concentration. Acidification and rennet gelation of the aggregates were greatly modified compared with the unaggregated mixture. A process for the manufacture of aggregated casein micelles in skim milk powder has been demonstrated.

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 6mM) 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 10h 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.