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.     

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.     

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.     

Effects of milk pH alteration on casein micelle size and gelation properties of milk

The effects of changes in pH above and below the natural pH of milk (ca 6.6) on the casein micelle size and the gelation properties of the pH adjusted and restored samples were investigated. The size of casein micelles increased at pH 7.0 and 7.5, then started to decrease above pH 8.5. It is postulated that at pH below 8.5 the casein micelles swell, while elevated pH causes their dissociation. Conversely, the size of casein micelles decreased on acidification to pH 5.5 and increased when the pH dropped below 5.5, due to the shrinkage of casein micelles at lower pH before their aggregation at pH below 5.5. In response to neutralising treated milk back to normal milk pH of 6.6, it was found that the casein micelle size of treated milk with a narrow range of pH change between 6.0 and 7.0 was reversible, while beyond this range the structure of casein micelles became irreversible. The restoration of casein micelle size was followed by the restoration of some parameters such as soluble calcium, ethanol stability, and milk whiteness. Acid-induced gelation did not change the elastic modulus, while rennet-induced gelation was affected by initial milk pH. In reference to the size of reversible range elastic modulus (G’) of acid or rennet gels made from restored milk, the sizes were similar to control milk after 6 h of gelation.     

Effect of heat treatment of rennet skim milk induced coagulation on the rheological properties and molecular structure determined by synchronous fluorescence spectroscopy and turbiscan

Heat treatment applied to milk induces denaturation of whey proteins, leading to a complex mixture of whey protein and whey protein coated casein micelles. The present paper investigates the effects of heat treatment (60 and 80°C during 20min) and rennet-induced coagulation temperature (30 and 40°C) determined by rheology, synchronous fluorescence spectroscopy (SFS) and turbiscan measurements. The gelation times determined by rheology and SFS increased with the increase of heat treatment applied to milk. The rise in temperature induced a decrease in the maximum curd firming rate and an increase in the viscosity of the investigated milk samples. The principal component analysis (PCA) applied, separately, to the SF and turbiscan spectra showed a clear discrimination between: (i) raw milks and heated milks; and (ii) milks renneted at 30°C from those renneted at 40°C. The results showed the ability of SFS as a rapid and non-destructive technique for the: (i) monitoring network structure and molecular interaction during the coagulation process; and (ii) determination of gelation time of rennet-induced coagulation of studied milk samples.     

Effects of added plasmin on the formation and rheological properties of rennet-induced skim milk gels

Elevated plasmin enzyme activity has been suggested as a likely cause of impaired functional properties that occur in milk from cows either in their late-lactational period or that are experiencing mastitis. However, there are conflicting reports on the impact of plasmin on rennet coagulation properties of milk. The effects of added plasmin on the rheological properties, at small and large deformation, of rennet-induced gels were investigated. The microstructure of rennet-induced gels was studied, using confocal scanning laser microscopy. Porcine plasmin was added to reconstituted milk, and samples were incubated at 37 degrees C for between 0.5 to 8 h. The hydrolysis reaction was terminated using soybean trypsin inhibitor. The extent of degradation of caseins was determined with SDS-PAGE. The extent of breakdown of alpha(s)- and beta-caseins increased with incubation time with plasmin. Storage modulus of rennet gels decreased linearly with increasing degradation of caseins. There was an increase in the loss tangent parameter of the gels with increasing casein degradation, indicating a more liquid-like gel character. Gelation time decreased until approximately 3 h of incubation with plasmin (when the amounts of intact alpha(s)- and beta-caseins were approximately 46 and 50%, respectively); thereafter, gelation time increased considerably. Yield stress of rennet-induced gels decreased with increasing casein breakdown. When the level of casein hydrolysis was high (<40% of intact caseins), the microstructure of rennet-induced gels was drastically altered. Even when there were low levels of casein hydrolysis, the rheological properties of rennet gels were altered, which could have negative impacts on cheese yield and texture.     

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.     

Effect of heat treatment on the rennet coagulation properties of recombined high total solids milk made from milk protein concentrate powder

The effects of heat treatment on the small and large deformation rheological properties of rennet gels made from recombined high total solids milk made from milk protein concentrate were studied. Heat treatment of recombined milk resulted in a slower rate of increase in the storage modulus (G') of rennet induced gels, a reduction in the gelation time and a decrease in the yield force required to fracture gels. The extent of whey protein denaturation (as a result of heat treatment) was related to the decrease in the G' value of gels as well as the yield force.     

The relationship between rheological parameters and whey separation in milk gels

The relation between whey separation of rennet-induced gels and rheological properties of those gels is reasonably well understood. A low fracture stress and a high value for the loss tangent at low frequencies have been correlated with a tendency to exhibit syneresis in rennet gels. In contrast, little is known about the relationship between mechanical properties of gels and whey separation in acid-induced milk gels, such as yoghurt, although this continues to be a major defect. In recent work, it has been found that conditions such as high milk heat treatment, fast rates of acidification and high incubation temperatures all gave high levels of whey separation compared with gels made from unheated milk that were incubated at low temperatures and where the rate of acidification was slow (i.e. when bacterial cultures were used instead of the acidogen, glucono-δ-lactone). The tendency to exhibit whey separation in acid gels made from heated milk was related to a low fracture strain and an increase in the loss tangent (observed even at high frequencies) during the gelation process (a high value indicates conditions favouring relaxation of bonds). Excessive rearrangements of particles in the gel network before and during gelation were implicated as being responsible for whey separation and rheological conditions that appeared to indicate this defect are described. It was also concluded that techniques that measure the spontaneous formation of surface whey should be distinguished from those that measure the expression of whey from networks under pressure as the latter tests only measure gel rigidity.     

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.     

RHEOLOGICAL PROPERTIES OF RENNET-INDUCED GELS DURING THE COAGULATION AND CUTTING PROCESS: IMPACT OF PROCESSING CONDITIONS

The effects of gelation temperature (GT), pH, milk solids nonfat (MSNF) content and aging time on the small and large deformation rheological properties of rennet‐induced skim milk gels were studied. Small amplitude oscillatory rheometry (SAOR) was used to study gel formation. A constant shear rate was applied to gels of various ages to try to simulate the cutting process used in cheese vats. Second‐order polynomial models successfully predicted (R² ≥ 0.83) the relationship between processing parameters and rheological properties of gels. The processing parameters – gelation pH, GT and MSNF – had a significant effect on the rheological properties of rennet‐induced gels. The type and the nature of bonds in these networks and the time scale of applied deformation affected the rheological properties of rennet gels. As time after rennet addition increased, storage modulus, loss modulus and yield stress values increased. This resulted from an increase in the number and strength of bonds with time. The yield strain decreased with time probably because of rearrangements in the network making the gel shorter/brittle in texture. When the impact of the time scale of the applied deformation was compared between the small (storage modulus as a function of frequency) and large (yield stress as a function of constant shear rate) deformation properties of rennet‐induced gels, similar power law exponents were obtained. This similarity presumably reflects the type and relaxation behavior of bonds in this casein network. These results identify the impact of several important processing variables on both the small and the large deformation rheological properties of rennet‐induced gels, which could be useful in identifying gelation properties that improve cheese yield.     

Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk

The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was approximately 130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171-177, 1998) which postulates that, following cleavage of the stabilizing K-casein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles.     

RHEOLOGICAL ASPECTS OF THE RENNETING OF MILK CONCENTRATED BY ULTRAFILTRATION

The kinetics of clotting by rennet of ultrafiltrated milk were studied by determining induction and clotting times, and development of the rigidity modulus as a function of rennet concentration and protein content in the sample. Creep cmpliance-time studies were conducted on the resultant gels at three different stresses. Clotting time increased with protein content and decreased with rennet concentration. The protein effect on clotting time decreased with increasing rennet concentration and was practically nil at 200 mg enzyme/kg sample. The ultimate rigidity and the viscoelastic parameters of gels depended on the protein concentration in a double logarithmic relationship. The calculated values of the constants indicated that the viscosities were influenced to a greater degree by the protein concentration than were the elastic moduli. The renneted milk gels exhibited creep compliance-time response at low shear stresses indicative of the viscoelastic behaviour of an interlinked three-dimensional network. Gelation followed a second order reaction in its initial stages and an approximately third order reaction in the final stage. The results are discussed in terms of potential mechanism and type of bonds involved.     

Rheological and structural properties of differently acidified and renneted milk gels

In this study we assessed the rheological and structural properties of differently acidified and renneted milk gels by controlling pH value and renneting extent. Skim milk were exactly renneted to 4 extents (20, 35, 55, and 74%) and then direct acidified to the desired pH (4.8, 5.0, 5.2, 5.5, 5.8, and 6.2), respectively. Rheological properties were assessed by dynamic rheological measurements, structural properties were studied by spontaneous whey separation and confocal laser scanning micrograph, and protein interactions were studied by dissociation test. Results showed that minimally renneted milk samples (20 and 35%) formed weak gels with low storage modulus, and the acidification range within which gels could form was narrow (pH ≤5.2). Highly renneted milk samples formed more gels with high storage modulus. The results of this study revealed that acidification determined the structural properties of highly renneted milk gels. As pH increased from 5.0 to 6.2, highly renneted milk gels had lower loss tangent, decreased spontaneous syneresis, and smaller pores. For both the low and high rennetings, divalent calcium bonds contributed less at low pH than at high pH. In conclusion, renneting increased the pH range suitable for gel formation; acidification determined the spontaneous syneresis and microstructure of highly renneted milk gels.     

Gelation and Water Binding Properties of Transglutaminase-treated Skim Milk Powder

Transglutaminase (TGase)-treated skim milk powder (TG-SMP) was prepared by freeze-drying skim milk after TGase treatment (10 U/g milk protein, 40°C for 3 h), followed by TGase inactivation at 85°C for 5 min. TGase modification resulted in significant increases in hardness and water holding capacity (WHC) of heat-induced gels (10% protein, w/v). A marked increase in storage modulus (G') of TG-SMP upon heating suggests that TG-SMP has a greater gelling ability than control-SMP (C-SMP) prepared with predenatured TGase. Acid gels prepared from TG-SMP had a significantly higher WHC at all solid levels (12%, 14%, and 16%) tested and formed a more elastic network than C-SMP.     

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.     

酶凝干酪素凝胶性研究进展及其在干酪中的应用

介绍酶凝干酪素凝胶形成机理和冷却机制研究进展,概述乳化盐、蛋白和脂肪、pH 值、温度、钙离子对凝胶形成的影响：乳化盐能促进蛋白质溶解形成凝胶,蛋白和脂肪含量可对凝胶的复合模量和黏弹性产生影响,适当的温度和pH 值条件能形成良好的凝胶结构,钙离子增加对凝胶形成不利,以及酶凝干酪素在加工干酪中的应用。     

Rheological Properties and Microstructure of Acid Milk Gels as Affected by Fat Content and Heat Treatment

Gels were made from recombined milks containing 0, 1.5 or 3.5% fat that were heated at 75, 80 or 90°C for 30 min, followed by acidification with glucono-8-lactone at 30°C. The rheological and microstructural properties of acid gels were investigated using dynamic low-amplitude oscillatory rheology and confocal scanning laser microscopy (CSLM). Heating milks, at temperatures ≥80°C, increased the storage moduli (G′) and decreased the gelation time. Recombined milks containing high fat (3.5%) had higher G′ than gels made from low-fat or skim milk. Milk heat treatment resulted in gels with a cross-linked microstructure. Recombined fat globules appeared to be embedded in the protein matrix.     

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.     

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.