Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

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.     

Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition

The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1 mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

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.     

A non-invasive method for the characterisation of milk protein foams by image analysis

An apparatus for the investigation of milk protein foams was introduced based on three jacket columns and exclusively image analysis. The method had a repetition coefficient <10%, and offered a high sample throughput and an expandable design. Sodium caseinate, micellar casein concentrate, whey protein isolate and whey protein concentrate foams were analysed as an application. Foaming properties depended on the protein, the composition of the preparations and the foaming conditions, e.g., stable foams at 20 °C were observed for micellar casein, while sodium caseinate showed a half-life of 22 min. At 50 °C, the stability of sodium caseinate decreased by about 70%. Additionally, a direct link between the foaming properties of sodium caseinate and its degree of enzymatic hydrolysis was found. No changes in foaming properties using Alcalase^® 2.5L occurred up to a degree of hydrolysis of about 3%, while higher degrees of hydrolysis led to decreased foaming properties.     

贮藏期UHT奶中蛋白质组成结构及其糖基化的变化

通过测定UHT奶在贮藏过程中蛋白水解,蛋白在胶束相和乳清相的分布,乳蛋白糖基化位点变化,探究乳蛋白在贮藏过程中性质的变化。结果表明贮藏过程中UHT奶中菌落总数增大,纤溶酶含量增大。UHT奶在内外源酶的作用下乳蛋白发生不同程度的水解。热处理导致从胶束表面脱落的κ-CN和乳清蛋白以热诱导聚合物的形式,在贮藏过程中重新结合到胶束表面,从而使胶束粒径增大,牛乳表观黏度增大。贮藏过程中胶束Zeta电位逐渐降低,流动行为指数n逐渐降低,说明牛乳呈现出更明显的剪切变稀能力,暗示胶束结构更加松散。贮藏过程中UHT奶色泽加深,美拉德反应继续进行,其产物半乳糖基赖氨酸和Nε-羧甲基赖氨酸(CML)含量不断增加。     

Hydration of casein micelles and caseinates: Implications for casein micelle structure

By studying the hydration of casein micelles using a variety of techniques, a distinction could be made between water that appeared bound by the protein (∼0.5 g g⁻¹ protein), water associated with the κ-casein brush (∼1.0 g g⁻¹ protein) and water entrapped in the casein micelles (∼1.8 g g⁻¹ protein), yielding a total micellar hydration of ∼3.3 g g⁻¹ protein, in line with casein micelle voluminosity derived from intrinsic viscosity measurements. For caseinate particles, however, the main contributor to intrinsic viscosity was not protein hydration but the non-spherical particle shape. These non-spherical particles in caseinate are likely to be naturally present as primary casein particles (PCP) in casein micelles. PCP could be used to build casein micelles by controlled introduction of micellar salts. Based on the findings of this study, casein micelles could be described as a porous network of non-spherical PCP linked by calcium phosphate nanoclusters.     

Effects of varying time/temperature-conditions of pre-heating and enzymatic cross-linking on techno-functional properties of reconstituted dairy ingredients

The effects of varying time/temperature-conditions of pre-heating and cross-linking with transglutaminase (TG) on the functional properties of reconstituted products from skim milk, WPC and sodium caseinate was analyzed. The degree of cross-linking (DC) of skim milk proteins could be increased from 54.4% to 70.5% by varying process conditions. Thereby the water-holding capacity (WHC) increased from 10% to 20%, while the heat stability decreased. The burning-on was lower than that of the non-treated products at optimum pre-heating conditions (90 °C/30 s). Using sodium caseinate as substrate for TG the DC increased from 39.2% to 100% due to the improvement of the process. As a result the WHC increased by 30% and the heat stability up to 380%. However, the burning-on of casein increased as well. TG-treated sodium caseinate started to gel at 10% protein, whereas untreated sodium caseinate gelled not before 15% protein. The WHC of enzyme-treated whey proteins was lowered. The heat stability of WPC could be doubled by TG-treatment, and the burning-on of the products was, especially at optimum pre-heating conditions, less pronounced. The degree of denaturation of TG-treated whey proteins was 2–5% higher than that of untreated samples.     

Gastric digestion of milk protein ingredients: Study using an in vitro dynamic model

The coagulation behavior and the kinetics of protein hydrolysis of skim milk powder, milk protein concentrate (MPC), calcium-depleted MPC, sodium caseinate, whey protein isolate (WPI), and heated (90°C, 20 min) WPI under gastric conditions were examined using an advanced dynamic digestion model (i.e., a human gastric simulator). During gastric digestion, these protein ingredients exhibited various pH profiles as a function of the digestion time. Skim milk powder and MPC, which contained casein micelles, formed cohesive, ball-like curds with a dense structure after 10 min of digestion; these curds did not disintegrate over 220 min of digestion. Partly calcium-depleted MPC and sodium caseinate, which lacked an intact casein micellar structure, formed curds at approximately 40 min, and a loose, fragmented curd structure was observed after 220 min of digestion. In contrast, no curds were formed in either WPI or heated WPI after 220 min of digestion. In addition, the hydrolysis rates and the compositions of the digesta released from the human gastric simulator were different for the various protein ingredients, as detected by sodium dodecyl sulfate-PAGE. Skim milk powder and MPC exhibited slower hydrolysis rates than calcium-depleted MPC and sodium caseinate. The most rapid hydrolysis occurred in the WPI (with and without heating). This was attributed to the formation of different structured curds under gastric conditions. The results offer novel insights about the coagulation kinetics of proteins from different milk protein ingredients, highlighting the critical role of the food matrix in affecting the course of protein digestion.     

Effect of beta-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk

The effect of beta-lactoglobulin and heat-induced precipitation of calcium phosphate on the pH dependence and mechanism of thermal coagulation of milk throughout the pH range 6.3-7.3 was studied using serum protein-free milk and sodium caseinate as models for micellar and non-micellar milk protein systems respectively. It appears that the specific effect of beta-lactoglobulin at the pH of maximum stability may be related to its ability to chelate calcium. The effect of beta-lactoglobulin at the pH of minimum stability does not appear to be directly related to heat-induced dissociation of K-casein or micellar integrity but may be due to its ability to sensitize casein micelles to heat-induced precipitation of calcium phosphate, by increasing micellar hydrophobicity. The extent of heat-induced precipitation of calcium phosphate, as a function of pH, is an inverse reflection of the pH dependence of heat stability. Micellar integrity appears to play a critical role in the heat stability of milk but for reasons not previously appreciated.     

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.     

Effect of Trichoderma reesei tyrosinase on rheology and microstructure of acidified milk gels

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.     

Effect of transglutaminase on rheological properties and microstructure of chemically acidified sodium caseinate gels

The mechanical properties and microstructure of 2.7% and 4.5% sodium caseinate gels chemically acidified by glucono-δ-lactone (GDL) and cross-linked by microbial transglutaminase (TG) were studied. The acidification was performed at different temperatures. According to SDS–PAGE TG clearly caused polymerisation of caseinate irrespective of the treatment temperature (4–50 °C), The cross-linking of the proteins was more extensive at temperatures 22–50 °C. Low amplitude viscoelastic measurements showed that 4.5% caseinate gels acidified at 50 °C were formed much faster than gels acidified at 22 °C. TG only slightly increased the time of gelling. Control gels prepared without TG at temperatures of 4, 22, 37 and 50 °C were mechanically weak. Examination of the control gels with a confocal laser scanning microscope showed that gels formed at 37 and 50 °C were coarse and porous with large cavities between particle aggregates, whereas those formed at 22 °C were much more homogeneous. The TG-treated and acidified sodium caseinate dispersions formed firm gels, indicating cross-linking of casein proteins. Interestingly, the strongest gels were formed at 22 and 37 °C. TG treatment improved the homogeneity of the gel structure at temperatures of 37 and 50 °C. The hardness of TG-treated gels acidified at 4 °C increased during 1 week of storage.     

Behaviour of partially cross-linked casein micelles under high pressure

High pressure (HP) treatment of a casein micelle suspension at 250 and 300 MPa leads to an initial rapid increase of its light transmission, as measured in situ , indicating micellar disruption. Subsequently, a much slower, partial reversal of the HP-induced increase in light transmission is observed, indicating re-association of micellar fragments. Partial internal cross-linking of the casein micelles by the enzyme transglutaminase prior to pressure treatment slows down both the disruption and the reassociation process considerably. It is proposed that covalent cross-linking provides the micelle with extra stability against pressure-induced disruption and also prevents a molecular reorganization process required to induce reassociation of micellar fragments during prolonged pressure treatment.     

PFG-NMR self-diffusion in casein dispersions: Effects of probe size and protein aggregate size

The self-diffusion coefficients of different molecular weight PEGs (Polyethylene glycol) and casein particles were measured, using a pulsed-gradient nuclear magnetic resonance technique (PFG-NMR), in native phosphocaseinate (NPC) and sodium caseinate (SC) dispersions where caseins are not structured into micelles. The dependence of the PEG self-diffusion coefficient on the PEG size, casein concentration, the size and the mobility of casein obstacle particles are reported. Wide differences in the PEG diffusion coefficients were found according to the casein particle structure. The greatest reduction in diffusion coefficients was found in sodium caseinate suspensions. Moreover, sodium caseinate aggregates were found to diffuse more slowly than casein micelles for casein concentrations >9 g/100 g H₂O. Experimental PEG and casein diffusion findings were analyzed using two appropriate diffusion models: the Rouse model and the Speedy model, respectively. According to the Speedy model, caseins behave as hard spheres below the close packing limit (10 g/100 g H₂O for SC (Farrer & Lips, 1999) and 15 g/100 g H₂O for NPC (Bouchoux et al., 2009)) and as soft particles above this limit. Our results provided a consistent picture of the effects of diffusant mass, the dynamics of the host material and of the importance of the casein structure in determining the diffusion behavior of probes in these systems.     

Impact of processing conditions and protein concentration on the assembly of carrageenan milk protein weak gels

The entropy-driven need for a minimized number of solvent's molecules used for hydration shells determines the assembly of composite carrageenan milk protein structures. Mixed kappa-2 (kappa/iota hybrid) carrageenan systems with different milk protein content were produced in an ultra-high-temperature (UHT) pilot plant with variation of the UHT temperature between 120 and 139 °C. The influence of process conditions and protein content was determined by rheological analyses of the structure point of a stress ramp and the phase-angle delta obtained by small-angle oscillation. The relationship between rheological properties and dynamic light scattering experiments was used for interpretation of the results.The protein content shows only influence on the weak gel if the UHT temperature is 132 °C and above. This can be related to calcium release of casein micelles. On the other hand, the size of the protein clusters is limited by the carrageenan-to-protein ratio. To determine the influence of particles with non-specific interactions to the polysaccharide, a variation of the preheating step was done. Systems, where coiled carrageenan can compete with the whey proteins for the casein micelle surface, build a stronger composite network compared to systems with casein micelles already covered by whey protein complexes when carrageenan is hydrated. This indicates that the assembly of the composite weak gel is basically entropy driven but improved by calcium bridging and specific interactions between casein micelles and carrageenan.     

Effect of SDS on Casein Micelles: SDS-Induced Milk Gel Formation

ABSTRACT: The addition of sodium dodecyl sulfate (SDS) during skim-milk reconstitution contributed to a modification of hydrophobic interactions and, consequently, to change in the micellar structure. SDS-induced modifications in casein micelles were investigated by biochemical measurements (soluble mineral and protein analyses, granulometric and electrokinetic potential measurements, and casein micelle solvation). SDS induced micellar κ-casein dissociation and caused a decrease in steric, hydration, and electrostatic repulsive forces between casein micelles and as a result altered micellar stability. Consequently, SDS-modified micelle aggregation occurred. Mineral analysis indicated that Ca, PO₄^3- and Mg partitioning between aqueous phase and curd is similar, suggesting a possible bridging mechanism via minerals and SDS molecules.     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

Size of native and heated casein micelles,content of protein and minerals in milk from Norwegian Red Cattle—effect of milk protein polymorphism and different feeding regimes

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca²⁺, Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α_s1-casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α_s1-casein (native mean size only) and κ-casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.