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.     

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.     

Factors affecting the acid gelation of sodium caseinate

Acid gelation, turbidity and particle size development of dispersions of sodium caseinate and other protein fractions were studied. Sodium caseinate dispersions became particulate prior to the onset of gelation. Casein particles had enhanced stability to gelation in the presence of sodium chloride. Removal of the hydrophilic part of the κ-casein molecule through renneting and acidification of the soluble sodium para-caseinate resulted in increased gelation pH. Removal of most of the κ-casein through ethanol fractionation of sodium caseinate resulted in an α_s1-β-fraction, which was markedly destabilised at higher pH values during acidification in the presence of sodium chloride. Preheated β-lactoglobulin/sodium caseinate dispersions had similar acid gelation profiles in the presence of sodium chloride with or without N-ethylmaleimide, suggesting that secondary thiol-disulphide interchange reactions between κ-casein and pre-heated β-lactoglobulin aggregates did not effect the gel point and final storage modulus in the short time-frame (120 min) of acidification. It was found that κ-casein and sodium chloride played a significant role in both particle development and subsequent stability of sodium caseinate dispersions on acidification.     

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.     

Modification to the renneting functionality of casein micelles caused by nonionic surfactants

Nonionic emulsifiers of small molecular weight such as polysorbates are widely used in dairy products. Nevertheless, the mechanism of interaction between these surfactants and milk proteins is not yet fully understood. This work investigated the effect of Tween 20 on casein micelles by studying the renneting behavior of skim milk in the presence of different amounts of surfactant. The presence of Tween accelerated both the first and second phase of renneting in skim milk. The gel obtained showed a higher elastic modulus than that of a skim milk gel, but also showed similar brittleness. By varying the size of the surfactant (Tween 20 or Tween 80) as well as the colloidal state of the proteins in solution, it was possible to demonstrate that the surfactant did not have a direct effect on the activity of the enzyme, but rather had a direct effect on the casein micelles. The effect of surfactant on the gelation point was reduced by increasing surfactant size. The presence of Tween caused an increase in the size of the micelles without affecting their stability. In addition, Tween did not alter the amount of caseins free in the serum phase. These findings can contribute to improving our ability to custom design final structures in rennet-induced gels, though further studies are needed to fully understand the mechanism at play when casein micelles are enzymatically cleaved in the presence of nonionic surfactants of small molecular weight.     

Casein–whey protein interactions in heated milk: the influence of pH

Heat-treatment of milk causes denaturation of whey proteins, leading to a complex mixture of whey protein aggregates and whey protein coated casein micelles. In this paper we studied the effect of pH-adjustment of milk (6.9–6.35) prior to heat-treatment on the distribution of denatured whey proteins in aggregates and coating of casein micelles. Proteins were fractionated using an alternative fractionation technique based on renneting. Acid- and rennet-induced gelation of these milks were used to obtain more information on the characteristics of the milk. Acid-induced gelation appeared to be mainly influenced by the presence of whey protein aggregates. Rennet-induced gelation was determined by the whey protein coating of the casein micelles. Both the quantity of whey proteins present on the surface of the casein micelles and the homogeneity of the coating were determining the renneting properties. These results extend the current knowledge on pH dependent casein–whey protein interactions. In order to present a clear picture of the changes occuring during heat treatment of milk at various pH, the results are summarized in a model. In this model we propose that heating at a pH>6.6 lead to a partial coverage of the casein micelles and the formation of separate whey protein aggregates. Heating at a pH<6.6 lead to an attachment of all whey proteins to the casein micelles. At pH 6.55 the coverage is rather homogeneous but lowering the pH further lead to an inhomogeneus coverage of the casein micelles. Surprisingly small changes of the pH at which the milk was heated had considerable effects on the gelation behaviour both in renneting and in acid gelation.     

Gelation of casein micelles in ��-casein reduced milk prepared using membrane filtration

Pasteurized skim milk was subjected to membrane filtration using a molecular weight cut-off of 80 kDa and a plate and frame pilot scale system at temperatures below 10 °C. Via this process, transmission of whey proteins and ??-casein through the membrane was achieved. The milk was concentrated to two times (based on volume reduction), and whey protein-free permeate was added to return to the original volume fraction of casein micelles in milk. This diafiltration process was carried out four times, and the retentate obtained was nearly free of whey proteins and with approximately 20% of ??-casein removed. The same membrane filtration was also carried out at 25 °C to achieve transmission of whey protein but not of ??-casein, and to obtain whey protein-depleted milk without depletion of ??-casein.The gelling behaviour of these samples, reconstituted to the original casein volume fraction, was examined using rheology and diffusing wave spectroscopy. When compared to the original skim milk it was found that there were no statistically significant differences in gelation behaviour during acidification, but differences were noted in gelation time and final stiffness modulus for samples undergoing renneting. These differences were attributed mostly to the changes in ionic composition, as when the serum composition of the retentates was re-equilibrated against the original skim milk by dialysis; the gelation behaviour of the samples was comparable to that of skim milk. The results clearly indicate the importance of the milk's overall ionic balance in the early stages of aggregation of rennet-induced gelation of milk.     

Fortification of milk protein content with different dairy protein powders alters its compositional,rennet gelation,heat stability and ethanol stability characteristics

Skim milk (SM) was fortified from 3.3 to 4.1% protein using different milk protein powders: skim milk powder (SMP), native phosphocasein (NPC), calcium-reduced phosphocasein (CaRPC), sodium caseinate (NaCas) or calcium caseinate (CaCas). Compared with SMP or NPC, fortification with NaCas and CaRPC, and to a lesser extent CaCas, resulted in milk samples having higher proportions of non-sedimentable casein and calcium, and lower- and higher-levels of κ- and α_S1-casein, respectively, as a proportion of non-sedimentable casein. These changes coincided in milk samples fortified with NaCas, CaRPC or CaCas failing to undergo rennet-induced gelation, and having higher heat stability in the region 6.7–7.2 and ethanol stability at pH 6.4. The study demonstrates that the aggregation behaviour of protein-fortified milk samples is strongly influenced by the degree of mineralisation of the protein powder used in fortification, which affects the partitioning of casein and calcium in the sedimentable and non-sedimentable phases.     

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.     

Pulse NMR of Casein Dispersions

The spin-spin relaxation time, T₂, of skimmilk, sodium caseinate dispersions and milk ultrafiltrate was measured as a function of pH and temperature using pulse proton NMR. The T₂ shows a maximum around pH = 5.2 for skimmilk, decreases with decreasing pH for sodium caseinate dispersions and is independent of pH for milk ultrafiltrate. For all systems studied T₂ increases with temperature, but, the extent of increase depends on the system. It is concluded that the T₂ of casein dispersions is mainly determined by factors determining the state of aggregation of the caseins. Apart from affecting the extent of aggregation of the casein particles, micellar calcium phosphate probably contributes also directly to the measured T₂.     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

Stability of casein micelles cross-linked by transglutaminase

In this study, caseins micelles were internally cross-linked using the enzyme transglutaminase (TGase). The integrity of the micelles was examined on solubilization of micellar calcium phosphate (MCP) or on disruption of hydrophobic interactions and breakage of hydrogen bonds. The level of monomeric caseins, determined electrophoretically, decreased with increasing time of incubation with TGase at 30°C; after incubation for 24 h, no monomeric β- or κ-caseins were detected, whereas only a small level of monomeric α_S1-casein remained, suggesting near complete intramicellar cross-linking. The ability of casein micelles to maintain structural integrity on disruption of hydrophobic interactions (using urea, sodium dodecyl sulfate, or heating in the presence of ethanol), solubilization of MCP (using the calcium-chelating agent trisodium citrate) or high-pressure treatment was estimated by measurement of the L*-value of milk; i.e., the amount of back-scattered light. The amount of light scattered by casein micelles in noncross-linked milk was reduced by >95% on complete disruption of hydrophobic interactions or complete solubilization of MCP; treatment of milk with TGase increased the stability of casein micelles against disruption by all methods studied and stability increased progressively with incubation time. After 24 h of cross-linking, reductions in the extent of light scattering were still apparent in the presence of high levels of dissociating agents, possibly through citrate-induced removal of MCP nanoclusters from the micelles, or urea- or sodium dodecyl sulfate-induced increases in solvent refractive index, which reduce the extent of light-scattering.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Effect of micellar structure of casein and its modification on plasmin-induced hydrolysis

Plasmin-induced hydrolysis of casein in milk can lead to many defects including proteolysis, age gelation, and bitterness. The susceptibility of casein to plasmin can be affected by micellar structure and modification of the lysine residues on caseins. Different levels of casein modification and dissociation of the casein micelle structure were achieved through succinylation. Succinylation occurred at residues Lys7, Lys34, Lys36, Lys42, Lys83 and Lys124 in α_S1-casein; Lys80, Lys150, Lys152, Lys158 and Lys165 in α_S2-casein; Lys28, Lys29, Lys32, Lys99, Lys105, Lys107 and Lys113 in β-casein, as identified using liquid chromatography–tandem mass spectrometry. The dissociation of caseins from the casein micelle reduced steric hindrance and made the protein more readily susceptible to hydrolysis by plasmin. However, the formation of succinyl-lysine rendered β-casein unrecognisable to the substrate-binding pocket of plasmin, resulting in a non-linear decrease in level of hydrolysis because of the competitive effect of micelle dissociation.     

A method for the large-scale isolation of β-casein

A method for the large-scale isolation of β-casein from renneted skim milk was developed. The curd from renneted skim milk was dispersed in hot (?70 °C) water to inactivate residual chymosin. The heated curd was subsequently recovered by centrifugation, resuspended in water and incubated at 5 °C, during which β-casein dissociated from the curd; the suspension was centrifuged and the aqueous phase lyophilised. The isolated protein consisted mainly of β-casein, containing a minor amount of γ-caseins and traces of other caseins. Unless chymosin was fully inactivated by heating, some β-casein was hydrolysed at the Leu₁₉₂–Tyr₁₉₃ bond. The yield of β-casein increased with incubation time, up to ∼20% of the β-casein present in the milk after 24 h at 5 °C. Reducing milk pH to 5.5 or 6.0, prior to renneting, caused a high level of contamination with α_s-caseins. This isolation procedure can be easily scaled-up to an industrial process and the β-casein-depleted curd may be used for the manufacture of rennet casein or processed cheese.     

The effect of hexametaphosphate addition during milk powder manufacture on the properties of reconstituted skim milk

Skim milk powders with various levels of sodium hexametaphosphate (NaHMP) were prepared. Reconstituted skim milk samples were prepared from these powders. NaHMP slightly reduced the pH, markedly reduced the serum and ionic calcium and markedly increased the serum phase orthophosphate levels of the milks. This shift in the mineral equilibrium resulted in a drastic reduction in casein micelle integrity, with a marked dissociation of casein from the micelles. κ-Casein was the predominant casein dissociated, although significant levels of α_S-casein and β-casein were also transferred to the serum phase. This dissociation of the casein micelles caused a marked decrease in size and scattering properties of the casein micelles. In addition, a small decrease in the zeta potential of the casein micelles in the milk was observed. Heat treatment of the milks with added NaHMP induced further dissociation of κ-casein, although much of the α_S-casein and β-casein re-associated with the micelles.     

Effect of temperature and pH on the solubility of caseins: environmental influences on the dissociation of α(S)- and β-casein

Selective precipitation is a common method for the isolation of β-casein, using the different calcium sensitivities of the individual caseins and the selective solubility of β-casein at a low temperature. In previous studies, it has been indicated that the β-casein yield depends on the physicochemical characteristics of the casein raw material used for fractionation. The objective of this study was to evaluate and compare the solubility of α(S)- and β-casein in solutions of micellar casein, sodium caseinate, and calcium caseinate as a function of pH and temperature. Additionally, the solubility of isolated α(S)- and β-casein fractions in demineralized water, ultrafiltration permeate, and a calcium-depleted milk salt solution was investigated depending on the pH and temperature. Furthermore, micellar casein, sodium caseinate, and calcium caseinate were subjected to a calcium chloride-precipitation process to determine the solubility of α(S)- and β-casein in calcium chloride precipitate, which is produced during selective precipitation, as a function of temperature and pH. Generally, the temperature had only a marginal influence on the α(S)-casein solubility compared with the β-casein solubility, whereas the solubility was shown to be strongly influenced by the pH. Our results suggest that the yield of β-casein obtained during isolation by means of selective precipitation may be a result of the solubility characteristics of α(S)- and β-casein in calcium chloride precipitate. Manufacturers may consider a simple solubility experiment before the β-casein isolation process by means of selective precipitation to predict β-casein yield.     

Impaired or accelerated aggregation of proteins in sterilised milk by adding surfactants

The effect of small-molecule surfactants on the stability of sterilised milk (with added sterol esters) was determined by measurement of the sensitivity towards renneting, and of particle size upon freeze-thaw treatment. Water-soluble surfactants like diacetyl tartaric esters of monoglycerides and sodium stearoyl lactylates reduced freeze-thaw stability and the rate of renneting. Oil-soluble surfactants, like monoglycerides and citric acid esters of monoglycerides had an opposite effect. Water-soluble surfactants could coat the proteins and render casein micelles more dense; hence they could reduce age gelation through reduced access by enzymes. The same surfactants, however, also coated the outside of the micelles and rendered them more hydrophobic, which resulted in reduced colloidal stability. The oil-soluble surfactants had an opposite effect: they accelerated renneting, and improved colloidal stability. Because of the latter they are alternatives to polyphosphates or carrageenan. We also found indications that polyphosphates increased protein-enzyme repulsion.     

Effect of transglutaminase and acidification temperature on the gelation of reconstituted skim milk

Effects of transglutaminase (TG), acidification temperature and total milk solids level on the acid gelation of skim milk were investigated. Despite similar acidification kinetics, TG-treated milk acidified at ≥35 °C showed differences in elastic modulus (Gʹ) with acidification time, particularly by inhibiting the formation of the peculiar shoulder (Gʹ_shoulder) observed at an early stage of gelation in the control milk. Regardless of the milk solids content, the G'_shoulder was absent in both types of milk at 30 °C. However, control milk above 2.5% (w/w) milk solids showed the G'_shoulder at 45 °C. G'_shoulder is proposed as the transition from the first increase in Gʹ, due to aggregation of soluble protein complexes at the early stage of acidification, to the second, due to further aggregation of casein micelles (and aggregated soluble complexes) as acidification progresses. The G'_shoulder was absent in acidified TG-treated milk due to the lack of soluble protein complexes containing caseins.     

Composition and Structure of Fat Globule Surface Layers in Recombined Milk

Protein coverage, composition and structure of surface layers of fat globules in recombined milk were determined. Average protein load was ～6 mg/m² fat surface. Both casein and whey proteins were present in the fat globule surface layer, with casein adsorbed in preference to whey proteins and α_s (α_sl+α_s2)-casein adsorbed in preference to β-casein. Transmission electron microscopy showed that the surface layer of fat globule was made up of casein micelles, fragments of casein micelles and a thin layer of protein, possibly whey proteins. Experiments with surface layers that had been dispersed in EDTA showed that the extent of dissociation of caseins followed the order: β-casein > α_s-casein ≦ K-casein, suggesting that most of the K-casein was probably associated directly with the fat surface.