Influence of ethanol on the rennet-induced coagulation of milk

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10-20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of kappa-casein, was progressively slowed with increasing ethanol content (5-20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of kappa-casein-depleted micelles, was enhanced in the presence of 5-15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.     

Rennet coagulation properties of UHT‐treated phosphocasein dispersions as a function of casein and NaCl concentrations

The renneting properties of whey protein‐free, UHT‐heated (140 °C/10 s) casein dispersions were investigated as a function of casein and NaCl concentration. It was found that the rennet coagulation time and gel firmness can be optimised when the whey protein‐free casein concentration is increased, while the added NaCl concentration is kept low. The strongest gel firmness occurs at 0.05 and 0.08 m NaCl addition and at a micellar casein concentration between 6.0 and 6.6 g/100 mL. Weak rennet gels were formed at 3.0–3.6 g/100 mL casein at all NaCl concentrations tested.     

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.     

Interactive Effects of Milk Fat Globule and Casein Micelle Size on the Renneting Properties of Milk

The size of the casein micelles (CM) and the milk fat globules (MFG) vary depending on farming factors, such as seasonal variation and stage of lactation, and cow genetics. The MFG and CM size of milk can influence the renneting behavior and texture of manufactured dairy products. In this work, we investigated the combined effects of MFG and CM size on the onset of gelation, the maximum rate of gelation, the value for G′_60 min (the final storage modulus) and G″_60 min (the final loss modulus), and tan δ upon renneting. Fractionation of MFG on the basis of size was carried out using laboratory-based centrifugation, whereas milk of predominantly large (184–218 nm) or small (147–159 nm) CM was selected naturally on-farm. Casein micelle size had the dominant effect on curd firmness and gelation rates of milk, where small CM milk formed rennet gels earlier and resulted in a firmer gel than milk with large CM. However, MFG size also influenced the renneting properties. The strongest rennet gels were obtained when large MFG (3.88–5.78 μm) was combined with small CM (153–159 nm). Selecting milk on the basis of the microstructure of key milk components could be achieved by natural selection of dairy cows or via fractionation technologies. Selection may provide a useful tool for efficient manufacturing of different dairy products based on the desirable characteristics specific to each.     

Interactive effects of casein micelle size and calcium and citrate content on rennet‐induced coagulation in bovine milk

Interactive effects of casein micelle size and milk calcium and citrate content on rennet‐induced coagulation were investigated. Milk samples containing small (SM) and large (LM) micelles, obtained from individual Holstein cows, were modified by addition of calcium and/or citrate and milk coagulation properties were evaluated in a full factorial design. The results showed that LM milk had a higher relative proportion of casein, coagulated faster, and resulted in a stronger gel than SM milk. Addition of calcium slightly decreased casein micelle size, while addition of citrate slightly increased micelle size. Calcium addition resulted in a shorter coagulation time and the strongest gels, while citrate addition increased the coagulation time and resulted in the weakest gels. Addition of calcium and citrate in combination resulted in intermediate coagulation properties. The interactive effect of micelle size and citrate was significant for gel strength. Microstructural differences between the milk gels were consistent with the rheological properties, for example, the micrographs revealed that a more homogeneous network was formed when calcium was added, resulting in a stronger gel. A more inhomogeneous network structure was formed when citrate was added, resulting in a weaker gel. Thus, variations in casein micelle size and in calcium and citrate content influence rennet‐induced coagulation in bovine milk. The calcium and citrate contents in Swedish milk have changed over time, whereby calcium content has increased and citrate content has decreased. In practical cheese making, calcium is added to cheese milk, most likely altering the role of inherent citrate and possibly influencing casein micelle size. The observed interaction effect between casein micelle size and citrate in this study, suggests that larger micelles with moderate citrate level will result in firmer gels, whereas a higher citrate content reduced gel strength more in case of large than SM. Since firmer gels are likely to retain more protein and fat than less firmer gels, this interaction effect could have implications in practical cheese production.     

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.     

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.     

The effect of ultra high-pressure homogenization (UHPH) on rennet coagulation properties of unheated and heated fresh skimmed milk

The effect of ultra-high pressure homogenization at a pressure of 179 MPa on the renneting of milk has been studied. The homogenization has a small effect on the diameters of casein micelles, because of the loss of some of their surface κ-casein. This modification of the structure leads to a slightly decreased rennet coagulation time. Interactions between the casein micelles in homogenized and unhomogenized milk samples started at a degree of proteolysis of the κ-casein of about 65–70%, although aggregation of the micelles did not start until over 90% proteolysis. Homogenization improved the coagulation properties of heated milk only slightly; however, it was shown that the removal of stabilizing repulsions between the casein micelles in the heated milk seemed to proceed in the same way as in unheated milk. The removal of the κ-casein has the same effect in heated and unheated milk samples, and the casein micelles are destabilized; it is only in the final aggregation step that the two milks differ.     

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.     

High pressure-induced changes in milk proteins and possible applications in dairy technology

This paper reviews the newest information on the effects of high pressure (HP) on whey proteins, caseins and milk enzymes, and discusses their influence on milk properties. HP treatments cause substantial modification to milk proteins and to the mineral balance of milk. Casein micelles disaggregate into smaller particles or aggregate, depending the intensity and the temperature of the HP treatment. Whey proteins are denatured, possibly interacting with the remnants of the casein micelles, and give aggregated forms different from those produced by heat treatment. These events influence rennet coagulation properties of milk, with micellar disintegration favoring coagulation and whey protein denaturation hindering the aggregation of renneted micelles and enhancing cheese yield. HP treatment of milk favors acid coagulation and produces acid gels whose structure is greatly determined by the different micellar sizes attainable and the degree of whey protein denaturation. Milk gels can also be formed from concentrated milk under HP, providing new structures inaccessible via conventional methods.     

Changes in the physico-chemical properties of casein micelles during ultrafiltration combined with diafiltration

The objective of this work was to determine if after membrane processing, the physical properties of casein micelles change. Milk was concentrated by ultrafiltration, and also subjected to various levels of diafiltration, by addition of water to the retentate. After the membrane concentration process, the retentates were diluted back to their original concentration, to study their physico-chemical properties. For better comparison, all the samples were dialyzed against the original milk, to obtain similar serum compositions. For the first time, the effect of different levels of diafiltration was studied. Diafiltration induced losses of colloidal calcium phosphate and caused changes in the turbidity parameter (1/l*) measured by light scattering, as well as in the ultrasonic properties (velocity and attenuation) of the casein micelles. When tested in a similar serum environment, the reconstituted micelles after diafiltration showed a lower susceptibility to aggregation and the rennet induced gels had a lower storage modulus than those formed with the original milk, at the same protein concentration. This work brings for the first time evidence of the differences in the physical properties of the casein micelles as a function of membrane processing history.     

Effect of soluble calcium on the renneting properties of casein micelles as measured by rheology and diffusing wave spectroscopy

Addition of calcium chloride to milk has positive effects on cheese-making because it decreases coagulation time, creates firmer gels, and increases curd yield. Although addition of calcium chloride is a widely used industrial practice, the effect of soluble calcium on the preliminary stages of gelation is not fully understood. In addition, it is not known whether the manner of addition and equilibration of the soluble calcium would affect the rennetability of the casein micelles. Therefore, the aim of this paper was to study the details of the coagulation behavior of casein micelles in the presence of additional calcium, and to elucidate whether the manner in which this cation is added (directly as calcium chloride or by gradual exchange through dialysis) affects the functionality of the micelles. Calcium was added as CaCl(2) (1 mM final added concentration) directly to skim milk or indirectly using dialysis against 50 volumes of milk. Additional soluble calcium did not affect the primary phase of the renneting reaction, as demonstrated by the analysis of the casein macropeptide (CMP) released in solution; however, it shortened the coagulation time of the micelles and increased the firmness of the gel. The turbidity parameter of samples with or without calcium showed that similar amounts of CMP were needed for particle interactions to commence. However, the amount of CMP released at the point of gelation, as indicated by rheology, was lesser for samples with added calcium, which can be attributed to a greater extent of calcium bridging on the surface or between micelles. The results also showed that the manner in which calcium was presented to the micelles did not influence the mechanism of gelation.     

Effect of Temperature of Milk Acidification on Rennet Gel Properties

Changes in mineral solubilization and rennet reaction rate were investigated after decreasing milk pH to 6.3 by lactic acid addition at a temperature of acidification (TAC) of 25 or 35 °C with a short period of equilibration. With increasing TAC, casein micelles retained higher amounts of Ca and P, and at a given temperature of coagulation, rennet clotting time was increased, and dG'/dt decreased. This effect was confirmed by the microstructure of casein micelles during the first stage of the enzymic coagulation indicating that the aggregation of para-k-casein was observed later at higher TAC. The effect of TAC on rennet milk gel formation could be attributed to the nature of the micellar mineral content and the conformational state of casein micelles before rennet action.     

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.     

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.     

Physicochemical characteristics and renneting properties of camels' milk: A comparison with goats', ewes' and cows' milks

A great variability in physicochemical and microscopic characteristics of individual milk has been observed leading to the presence of differences in kinetics of the renneting reaction and in drainage ability. Furthermore, this study shows that casein concentration, total calcium content and mean diameter of casein micelles are the principal variables explaining the major part of the variation in the renneting properties. Finally, several relationships between coagulation characteristics, drainage ability and some compositional parameters of these kinds of milk were highlighted.     

Effect of extracts of oak (Quercus petraea) bark,oak leaves,aloe vera (Curacao aloe), coconut shell and wine on the colloidal stability of milk and concentrated milk

Addition of aloe vera extract, non-dialyzable red wine residue or aqueous methanol extracts of oak bark, oak leaves or coconut shell increased the heat stability of skim milk (at 140°C) and concentrated milk (at 120°C) and retarded rennet coagulation, but had no effect on the alcohol stability of milk. The calcium ion concentration in milk was reduced by the addition of these extracts but calcium chelation does not appear to be the exclusive mechanism responsible for promoting micellar stability. The extracts contained a high concentration of polyphenols, which are highly reactive and may be the active agents in the extracts, responsible for the enhanced stability of casein micelles.     

Effect of sodium chloride and pH on the rennet coagulation and gel firmness

The effect of sodium chloride concentration and pH of milk at renneting on the rennet clotting time (RCT) and gel firmness was studied. The results showed that rennet clotting activity and gel firmness decreased with increasing NaCl concentration in milk. The RCT increased as the pH of the salted milk decreased below 6.4. Milk containing 5 and 10 g NaCl/100 g did not coagulate at pH 5.0. The gel firmness increased with decreasing milk pH to 6.2 for unsalted milk. However, the firmness decreased as the pH of milk decreased below 6.2. In case of using salted milk, the firmness decreased as the pH at renneting dropped below 6.4.     

ADSA Foundation Scholar Award. Formation and physical properties of milk protein gels

Gelation of milk proteins is the crucial first step in both cheese and yogurt manufacture. Several types of milk gels are discussed, with an emphasis on recent developments in our understanding of how these gels are formed and some of their key physical properties. Areas discussed include the latest dual-binding model for casein micelles; some recent developments in rennet-induced gelation; review of the methods that have been used to monitor milk coagulation; and a discussion of some of the possible causes for the wheying-off defect in yogurts. Casein micelles are the primary building blocks of casein-based gels; however, controversy about its structure continues. The latest model proposed for the formation of casein micelles is the dual-binding model proposed by Horne, 1998, which suggests that casein micelles are formed as a result of two binding mechanisms, namely hydrophobic attraction and colloidal calcium phosphate (CCP) bridging. Most previous models for the casein micelle have treated milk gelation from the viewpoint of simple particle destabilization and aggregation, but they have not been able to explain several unusual rheological properties of milk gels. Although there have been many techniques used to monitor the milk gelation process over the past few decades, only a few appear attractive as possible in-vat coagulation sensors. Another important aspect of milk gels is the defect in yogurts called wheying-off, which is the appearance of whey on the gel surface. The factors responsible for its occurrence are still unclear, but they have been investigated in model acid gel systems.     

Reformation of casein particles from alkaline-disrupted casein micelles

In this study, the properties of casein particles reformed from alkaline disrupted casein micelles were studied. For this purpose, micelles were disrupted completely by increasing milk pH to 10.0, and subsequently reformed by decreasing milk pH to 6.6. Reformed casein particles were smaller than native micelles and had a slightly lower zeta-potential. Levels of ionic and serum calcium, as well as rennet coagulation time did not differ between milk containing native micelles or reformed casein particles. Ethanol stability and heat stability, >pH 7.0, were lower for reformed casein particles than native micelles. Differences in heat stability, ethanol stability and zeta-potential can be explained in terms of the influence of increased concentrations of sodium and chloride ions in milk containing reformed casein particles. Hence, these results indicate that, if performed in a controlled manner, casein particles with properties closely similar to those of native micelles can be reformed from alkaline disrupted casein micelles.