Effect of Ultra-high Temperature Treatment on the Enzymatic Cross-linking of Micellar Casein and Sodium Caseinate by Transglutaminase

ABSTRACT: It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide ε-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.     

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.     

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.     

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.     

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.     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

Casein molecular assembly affects the properties of milk fat emulsions encapsulated in lactose or trehalose matrices

Properties of spray-dried anhydrous milk fat emulsions stabilized by micellar casein (milk protein isolate—MPI) or non-micellar casein (sodium caseinate—Na-caseinate) with trehalose or lactose as encapsulants were studied. A lower concentration of Na-caseinate (0.33%) compared with MPI (1.26%) was sufficient to stabilize a 10% fat emulsion. Reconstituted emulsions showed larger droplet size than fresh emulsions, especially for MPI systems (from<1 μm to around 14 μm), which was attributed to lower shear resistance during atomization. Creaming behavior reflected changes in particle size. Powder surface free fat was affected by protein type and concentration. Trehalose systems (regardless of protein type) released significantly lower amounts of encapsulated fat upon crystallization compared with those containing lactose. Individual and hence, more mobile and flexible casein molecules, as opposed to aggregated and less mobile casein micelles, appear to result in superior co-encapsulation properties of Na-caseinate compared with MPI.     

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.     

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.     

Impact of cryoconcentration on casein micelle size distribution,micelles inter-distance,and flow behavior of skim milk during refrigerated storage

Cryoconcentration combined with a cascade effect was used to concentrate skim milk up to 25.12% total dry matter. Size, shape, and inter-micellar distance of casein micelles were characterized by ZetasizerNano-ZS, transmission electron microscopy, and ImageJ analyses. Flow properties of the cryoconcentrated skim milk were evaluated during 5 weeks of storage under refrigerated condition at 4 °C. Milk color was also evaluated according to the L*, a*, and b* system. The cryoconcentrated skim milk obtained after three cryoconcentration cycles was characterized by a monomodal distribution of its micelles with a tendency to smaller casein micelles. Approximately 60% of the total micellar volume was occupied by the casein micelles with a size of 100–200 nm, less than 18% of the volume with a size of 50–100 nm and only less than 1% was occupied by micelles with a size > 350 nm. This result shows that cryoconcentration changed the distribution of the mean size of the casein micelles to smaller units. No significant difference was observed on the inter-micellar distance. Cryoconcentration significantly improved the color of skim milk by increasing the L* value up to 67 which was similar to that of whole milk. Transition from a Newtonian to a non-Newtonian behavior was observed from the fourth week storage with a slight increase of casein micelle size.Industrial relevanceA concentration procedure of skim milk based on a complete block cryoconcentration technique was proposed. Application of this sub-zero technology permitted the concentration of skim milk total dry matter up to 25%. The casein micelle size was positively affected by moving the major part of the micelles toward the smaller size, whereas the inter-micellar distance was not affected. This new knowledge can be exploited in milk-based products to enhance the product stability. The cryoconcentrated skim milk color was positively affected since its L* value, which represents the milk whiteness, was significantly improved. The flow behavior of the cryoconcentrated milk was of Newtonian type up to 4 weeks of storage at 4 °C. The generated knowledge in this study can be easily used by the milk processing industry in order to make stable milk product with high dry matter content without adding milk powder, which negatively affects the product sensory properties (floury consistency).     

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900 MPa for 5 min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450 MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250 MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900 MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥ 700 MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550 MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800 MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700 MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900 MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550 MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.     

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.     

Comparison of rheological properties of acid gels made from heated casein combined with β-lactoglobulin or egg ovalbumin

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β-lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ-potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β-lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.     

Transglutaminase cross-linking of milk proteins and impact on yoghurt gel properties

A novel yoghurt process was investigated in which milk proteins were covalently cross-linked by a microbial transglutaminase (TG) preparation containing glutathione (TG+GSH). As unheated milk is normally less reactive towards TG, TG+GSH was applied to enable non-inhibited cross-linking without requiring a pre-heat treatment beyond pasteurisation conditions. After the TG+GSH incubation phase, the enzyme was inactivated by heat treatment of the yoghurt milk prior to fermentation. During yoghurt fermentation, no negative effect of TG+GSH on fermentation time was found. Protein cross-linking by TG+GSH was enhanced, resulting in higher apparent viscosity and a higher degree of protein polymerisation than that given by TG without GSH. Furthermore, different ratios of casein to whey proteins (CWP ratios) were used to investigate the effect of both protein fractions on covalently cross-linked yoghurt gel structures. The results showed a relationship between extent of cross-linking, apparent viscosity and CWP ratio of the yoghurt gels. During storage for up to 6 weeks at 4 °C, no changes in rheological properties and degree of protein polymerisation were measurable for stirred yoghurt gels prepared from cross-linked milk proteins.     

Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua

The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. High-pressure (HP) treatment of L. innocua 4202 (1×10⁹ cfu mL⁻¹) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a ∼5.0 log reduction in phosphate-buffered saline and a ∼2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 mm), magnesium (5 mm), citrate (10 mm) and phosphate (20 mm), suspended in SMUF increased survival by ∼3.3, ∼1.7, ∼3.3 and ∼3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP.     

High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

High hydrostatic pressure processing on microstructure of probiotic low-fat yogurt

The effect of milk processing on the microstructure of probiotic low-fat yogurt was studied. Skim milk fortified with skim milk powder was subjected to three treatments prior to innoculation: thermal treatment at 85 °C for 30 min, high hydrostatic pressure at 676 MPa for 5 min, and combined treatments of high hydrostatic pressure (HHP) and heat. The processed milk was then fermented by using two different starter cultures containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum. The microstructure of heat-treated milk yogurt had fewer interconnected chains of irregularly shaped casein micelles, forming a network that enclosed the void spaces. On the other hand, microstructure of HHP yogurt had more interconnected clusters of densely aggregated protein of reduced particle size, with an appearance more spherical in shape, exhibiting a smoother more regular surface and presenting more uniform size distribution. The combined HHP and heat milk treatments led to compact yogurt gels with increasingly larger casein micelle clusters interspaced by void spaces, and exhibited a high degree of cross-linking. The rounded micelles tended to fuse and form small irregular aggregates in association with clumps of dense amorphous material, which resulted in improved gel texture and viscosity.     

Effect of soy protein substitution for sodium caseinate on the transglutaminate-induced cold and thermal gelation of myofibrillar protein

The influence of soy protein isolate (SPI) substitution for sodium caseinate (SC) on the properties of cold-set (4 °C) and heat-induced gels of pork myofibrillar protein (MP) incubated with microbial transglutaminase (TG) was investigated. The strength of cold-set MP–SC gels (formed in 0.45 M, NaCl, 50 mM phosphate buffer, pH 6.25) increased with time of TG incubation, but those gels with more than 66% SPI substituted for SC had a >26% reduced strength (P < 0.05). Upon cooking, both incubated and non-incubated protein sols were quickly transformed into highly elastic gels, showing up to 6000 Pa in storage modulus (G′) at the final temperature (72 °C). However, no differences (P < 0.05) in G′ were observed between heated samples with SPI and SC. Myosin heavy chain, casein and soy proteins gradually disappeared with TG incubation, contributing to MP gel network formation. Both cold-set and heat-induced gels had a compact protein matrix, attributable to protein cross-linking by TG.     

Adsorption of Positively-Charged β-Lactoglobulin Derivatives to Casein Micelles

Positively-charged β-lactoglobulin derivatives prepared by amidation or esterification of available carboxyl groups of the native protein were bound strongly by casein micelles. Increasing amounts of these additives progressively decreased rennet coagulation time of casein micelles. Higher concentrations of positively-charged proteins coagulated casein micelles without addition of rennet extract. Of the modified proteins tested, approximately 1.0 g amidated β-lactoglobulin, 2.0 g ethyl-esterified β-lactoglobulin, or 1.0 g methyl-esterified β-lactoglobulin would be required to coagulate 100 ml of casein micelles at concentrations in milk.     

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.