Properties of casein micelles cross-linked by transglutaminase

Factors affecting the cross-linking of milk proteins by transglutaminase (TGase) were studied. Cross-linking of caseins in bovine skim milk was optimal over a very wide pH range. The role of micellar calcium phosphate (MCP) in maintaining the integrity of TGase-treated casein micelles was studied by incubating skim milk with 0.01% (w/v) TGase at 30°C for 1–24 h, followed by removal of MCP from untreated or TGase-treated milk by acidification and dialysis. The protein content and profile of the samples were determined by Kjeldahl and SDS-PAGE, respectively. Whey proteins in unheated milk were not susceptible to TGase-induced cross-linking. The higher level of sedimentable protein in MCP-free TGase-treated milk than in MCP-free control milk indicated that TGase treatment partially prevented disintegration of the micelle on removal of MCP, probably due to extensive intramicellar TGase-induced cross-linking of casein molecules which led to the formation of sedimentable covalently bonded casein aggregates.     

Supramolecular structure of the casein micelle

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of α_s1-, α_s2-, or β-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

Effect of transglutaminase on the heat stability of milk: a possible mechanism

Treatment of milk with transglutaminase (TGase) affects its heat stability, but the manner in which it does so depends on whether or not the milk had been preheated before incubation and on the temperature of preheating. In raw milk, it appears that cross-link formation between the individual caseins is responsible for preventing the dissociation of kappa-casein from the micelles at pH values in the region of minimum stability. In milks preheated before incubation with TGase, denaturation of whey protein may have allowed the formation of cross-links by TGase between denatured whey proteins and the individual caseins which, in combination with cross-linking of the caseins, contributed to greatly improved heat stability at pH > 6.5. It appears from the results of this study that TGase has potential commercial applications as a food-grade additive capable of improving the heat stability of milk.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

P NMR spectroscopic investigations of caseins treated with microbial transglutaminase

Caseins - the main constituents of bovine milk proteins - self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state ³¹P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state ³¹P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state ³¹P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state ³¹P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state ³¹P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state ³¹P HR NMR in combination with dynamic light scattering (DLS). Solid-state ³¹P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.     

Heat stability of transglutaminase-treated milk

Heat-induced coagulation of unconcentrated (9%, w/w) and concentrated (18%, w/w) reconstituted skim milk was determined after incubation with transglutaminase (TGase). Cross-linking ∼20% of κ-casein strongly increased the heat stability of unconcentrated milk at pH >6.9, presumably by preventing heat-induced dissociation of κ-casein, whereas increased heat stability of unconcentrated milk at pH 6.6–6.8 was only observed when >80% of casein was cross-linked. Treatment with TGase reduced heat stability of unconcentrated milk at pH <6.6, presumably due to the increased susceptibility of partially cross-linked casein micelles to coagulation arising from heat-induced acidification. A low degree of cross-linking increased the heat stability of concentrated milk at pH >6.8, but more extensive cross-linking progressively reduced heat stability. The degree of cross-linking studied did not increase the heat-stability of concentrated milk at its natural pH. The outcomes of this study substantiate the crucial roles of heat-induced acidification and casein dissociation in heat stability of milk.     

Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk

The objective of this work was to study the effect of different salts and salt concentration on the isolation of casein micelles from bovine raw skim milk by tangential flow microfiltration. Tangential flow microfiltration (0.22 μm) was conducted in a continuous process adding a modified buffer to maintain a constant initial sample volume. This buffer contained calcium chloride (CaCl₂), sodium phosphate (Na₂HPO₄), or potassium citrate (K₃C₆H₅O₇) in concentrations ranging from 0 to 100 mM. The concentrations of caseins and whey proteins retained were determined by sodium dodecyl sulfate-PAGE and analyzed using the Scion Image software (Scion Corporation, Frederick, MD). A complete isolation of caseins from whey proteins was achieved using sodium phosphate in the range of 10 to 50 mM and 20 times the initial volume of buffer added. No whey proteins were detected at 50 mM but this was at the expense of low caseins being retained. When lower sodium phosphate concentrations were used, the amount of caseins retained was higher but a small amount of whey proteins were still detected by sodium dodecyl sulfate-PAGE. Among the salts tested, calcium chloride at 50 mM and all volumes of buffer showed the higher retention of casein proteins. The highest casein:whey protein ratio was found at 30 mM CaCl₂, but no complete casein micelle isolation was achieved. Potassium citrate was the most ineffective salt because a rapid loss of caseins and whey proteins was observed at all concentrations and with low quantities of buffer added during the filtration process. Our results show the potential of altering the mineral balance in milk for isolation of casein micelles from whey proteins in a continuous tangential flow microfiltration system.     

Interactions of caseins with phenolic acids found in chocolate

To investigate the interactions between caseins and phenolic acids, such as the ones present in chocolate, casein was incubated with protocatechuic acid or p-coumaric acid at 55 °C. In addition, casein was isolated from chocolate and the phenolic compounds within these caseins were quantified. Electrophoresis results revealed that casein–phenolic interactions were induced by incubation; minor aggregation of casein subunits was observed after incubation of casein with protocatechuic acid. Minor aggregation of casein isolated from milk chocolate was also observed. In vitro hydrolysis of casein control, casein–protocatechuic acid, casein–p-coumaric acid, caseins isolated from milk chocolate and white chocolate using trypsin showed degree of hydrolysis of 19.3, 18.6, 17.7, 10.4 and 17.8% respectively. The presence of protocatechuic acid and p-coumaric acid in the model system and the presence of phenolic compounds in milk chocolate, in addition to the structural changes occurring during processing, affected the peptide profiles of casein hydrolysates.     

Influence of enzymatic cross-linking on milk fat globules and emulsifying properties of milk proteins

The enzyme transglutaminase (TGase) can modify dairy protein functionality through cross-linking of proteins. This study examined the effects of TGase treatment on milk fat globules and the emulsifying properties of milk proteins. The extent of TGase-induced cross-linking of caseins increased with incubation time, with no differences between whole and skim milk. Extensive clustering of fat globules in extensively cross-linked raw whole milk occurred on homogenisation at 400 or 800 bar. Considerably less clustering of fat globules was observed when recombined milk (90 g fat L^–1) was prepared from TGase-treated skim milk and homogenised at 400 or 800 bar. TGase treatment did not affect fat globule size in cream, but prevented coalescence of fat globules therein, possibly through cross-linking of milk fat globule membrane components. TGase-induced cross-linking of milk proteins affected their emulsifying properties and may increase the stability of natural milk fat globules against coalescence.     

The stabilizing behaviour of soybean soluble polysaccharide and pectin in acidified milk beverages

The mechanisms of stabilization of soybean soluble polysaccharide (SSPS) and high methoxyl pectin (HMP) in acidified milk drinks were studied focusing on the differences in behaviour between the two polysaccharides. The changes in casein micelles size during acidification with glucono-δ-lactone or by direct acidification were measured using light scattering. When HMP was added to skim milk before acidification, pectin adsorbed on the surface of the casein micelles via electrostatic interactions and prevented casein aggregation. Results suggested that adsorption of pectin occurred from the beginning of acidification and somewhat affected the rearrangement of casein micelles in the pH range between 5.8 and 5.0. On the other hand, SSPS, at concentrations up to 2% (w/w), did not interact with caseins at pH >4.6. At pH <4.2 SSPS showed better stabilizing properties than HMP. In addition, between pH 4.2 and 3.2, SSPS-stabilized acid dispersions were not affected by pH, while dispersions homogenized with pectin showed a size distribution that depended on pH. The differences in structure between SSPS and HMP account for the unique functionalities of the two polysaccharides in acid milk systems.     

High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy

Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300 MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400 MPa favoured formation of smaller casein assemblies, with sizes between 30 nm and 100 nm. Measurements of free calcium concentration [Ca²⁺] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk.     

Influence of transglutaminase treatment on some physico-chemical properties of milk

Transglutaminase (TGase) is an enzyme that cross-links many proteins, including milk proteins. In this study, the effects of TGase on some physico-chemical properties of milk were studied. TGase-treated milk was not coagulable by rennet, which was due to failure of the primary (enzymic) stage of rennet action rather than the non-enzymic secondary phase. Dissociation of TGase-treated casein micelles by urea or sodium citrate or removal of colloidal calcium phosphate by acidification and dialysis was reduced, presumably due to the formation of cross-links between the caseins. Casein micelles in TGase-treated milks were also resistant to high pressure treatment and to hydrolysis by plasmin. Results of the present study show that milk proteins are fundamentally modified by the action of TGase, which may have applications in the manufacture of functional proteins for use as novel food ingredients.     

Physicochemical Properties of Acylated Casein Micelles in Milk

ABSTRACT: The objective of this study was to determine the effects of acetylation or succinylation on the physicochemical properties of casein micelles in milk. Analysis of untreated and modified milk samples revealed solubilization of caseins and calcium phosphate from the micelle under mild acylation treatments. Solvation of micelle was affected, whereas no influence in size dia and potential zeta arose from the acylation treatments. Physicochemical characteristics of acetylated milk sample were intermediate between untreated and succinylated ones. Changes were also observed after calcium addition to reconstituted milk. Calcium supplementation counteracted the effect of chemical modification of caseins by reducing the susceptibility of casein micelle to disassembly.     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Effect of SDS on Acid Milk Coagulability

ABSTRACT To study the importance of hydrophobic interaction on the mechanism of acid milk gel formation, milk coagulation process was monitored at 30 °C in presence of various levels of sodium dodecyl sulphate (SDS). As a function of the SDS concentration, acid milk coagulability was either enhanced or reduced. Main pH‐induced biochemical changes were preserved despite the presence of SDS (such as pH‐induced demineralization and pH‐induced protein solubilization). It could be assumed that SDS‐modified casein micelles ability to coagulate by lowering of pH might seem to be governed essentially by the level of SDS‐induced k‐casein micellar dissociation, at natural milk pH.     

Sodium caseinate-stabilized fat globules inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave Spectroscopy

Sodium caseinate (NaCas)-stabilized oil-in-water emulsions were added to skim milk and the rennet-induced aggregation was observed in situ using light scattering and dynamic oscillatory rheology. The gelation of the recombined milk was greatly inhibited by the addition of the oil droplets, at volume fractions >0.025. The development of the turbidity parameter, 1/l*, and the apparent hydrodynamic radius during renneting were determined using diffusing wave spectroscopy. Although the recombined milk samples contained two scattering particles, namely, casein micelles and fat globules, the latter overwhelmingly contributed to the overall light-scattering signal. This made possible to follow the behaviour of NaCas-stabilized fat globules during the gelation process. The enzymatic reaction associated with the hydrolysis of micellar κ-casein was not significantly affected by the presence of the NaCas-stabilized fat globules. However, the emulsion droplets impeded the aggregation of rennet-altered casein micelles preventing the formation of a gel network. The inability of renneted casein micelles to develop a gel network can be attributed in part to an altered equilibrium between soluble and micellar calcium phosphate, caused by the association of soluble Ca²⁺ with casein molecules, but mostly can be attributed to the effect of non-adsorbed caseins on the surface of the casein micelles.     

Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature

The effect of pH (from 5.5 to 7.5) and temperature (from 5 to 40°C) on the turbidity of reconstituted skim milk powder was investigated at ambient pressure and in situ under pressure (up to 500 MPa) by measurement of light scattering. High-pressure treatment reduced the turbidity of milk for all combinations of pH and temperature due to micelle dissociation. The turbidity profiles had a characteristic sigmoidal shape in which almost no effect on turbidity was observed at low pressures (100 MPa), followed by a stronger pressure dependency over a pressure range of 150 MPa during which turbidity decreased extremely. From the turbidity profiles, the threshold pressure for disruption of micelle integrity was determined and ranged from 150 MPa at low pH to 350-400 MPa at high pH. The threshold pressure diagram clearly showed a relationship between the barostability of casein micelles and pH, whereas almost no effect of temperature was shown. This remarkable pH effect was a consequence of pressure-induced changes in the electrostatic interactions between colloidal calcium phosphate and the caseins responsible for maintaining micellar structure and was explained by a shift in the calcium phosphate balance in the micelle-serum system. Accordingly, a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium plays a crucial role in the micelle dissociation process.     

CPG-chromatography studies of the stability of casein micelles.

Casein micelles fractionated on controlled pore glass (CPG-10/3000) were shown to be stable by recycling experiments. Only minor effects on the size distribution of the casein micelles were found after heating skim-milk to 100 degrees C for 10 min, or freeze-drying skim-milk at -70 degrees C followed by resuspension in the synthetic milk serum of Jenness & Koops (1962). The heating caused some whey proteins (beta-lactoglobulin) to enter the micelle fractions while the freeze-drying caused some of the largest micelles to disrupt. In colloidal clacium phosphate-free skim-milk prepared according to Pyne & McGann (1960) all the micelles appeared to dissociate into monomeric caseins.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.