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.     

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.     

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.     

Formation of whey protein/κ-casein complexes in heated milk: Preferential reaction of whey protein with κ-casein in the casein micelles

Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90 °C 10 min⁻¹) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles.     

Effect of NaCl on some physico-chemical properties of concentrated bovine milk

The influence of added NaCl (75–850 mmol L⁻¹) on some physicochemical properties of 2× or 3× concentrated milk (18 or 27%, w/v, total solids, respectively) was investigated. Adding NaCl did not influence average casein micelle size, but reduced the net-negative charge on the casein micelles and milk pH. The level of soluble and ionic calcium was increased by addition of NaCl, but the level of soluble inorganic phosphorus was not influenced. Addition of NaCl shifted the maximum in the pH–heat coagulation time (HCT) profile of 2× or 3× concentrated milk to a higher pH value and certain concentrations increased the maximum HCT, probably due to the fact that NaCl reduced the extent of heat-induced dissociation of κ-casein. Added NaCl reduced the ethanol stability, with the extent of this effect increasing with the concentration of NaCl. The key-mechanism though which added NaCl induces changes in the physico-chemical stability of casein micelles appears to be through changes in the charge on the casein micelles.     

Enhancement of transglutaminase-induced protein cross-linking by preheat treatment of cows’ milk: A statistical approach

Optimization of heat treated milk towards protein cross-linking induced by transglutaminase was carried out. Capillary electrophoresis was employed to study the extent of cross-linking under different preheating temperatures (70–90 °C) and times (15–60 min). The experiments were arranged according to a central composite statistical design (3²+centre points). Response surface methodology was used to assess factor interactions and empirical models regarding relative peak area (%) of individual protein (α_s2-casein, α_s1-casein, α_s0-casein, κ-casein, β-casein A¹, β-casein A², α-lactalbumin and β-lactoglobulin) and total α_s-caseins, total β-caseins and whey proteins (sum of α-lactalbumin and β-lactoglobulin). Multi-response optimization was also performed on the total α_s-caseins, total β-caseins, κ-casein and whey proteins data set of the factorial design. The desirability function was the statistical tool employed in this multi-optimization step. The optimum preheating conditions that maximized the cross-linking reactions catalyzed by transglutaminase were achieved within 60 min at 84.5 °C.     

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.     

Bovine milk procathepsin D: Presence and activity in heated milk and in extracts of rennet-free UF-Feta cheese

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α_s1-I and para-κ-caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.     

Rheological properties and microstructure during rennet induced coagulation of UF concentrated skim milk

Rennet induced coagulation of ultrafiltrated (UF) skim milk (19.8%, w/w casein) at pH 5.8 was studied and compared with coagulation of unconcentrated skim milk of the same pH. At the same rennet concentration (0.010 International Milk Clotting Units g⁻¹), coagulation occurred at a slower rate in UF skim milk but started at a lower degree of κ-casein hydrolysis compared with the unconcentrated skim milk. Confocal laser scanning micrographs revealed that large aggregates developed in the unconcentrated skim milk during renneting. Following extensive microsyneresis the protein strands were shorter and thinner in gels from UF skim milk. Moreover, during storage up to 60 days (13 °C), the microstructure and the size of the protein strands of the UF gel changed only slightly. Hoelter–Foltmann plots suggested that the coagulation rate was reduced in the UF skim milk due to a high zero shear viscosity of the concentrate compared with the unconcentrated skim milk.     

Interactions Leading to Formation of Casein Submicelles

Gel chromatography was employed in studies of interactions of soluble whole casein that was prepared by dissociation of casein micelles with ethylenedia-minetetraacetate. With increasing protein concentration at pH 6.6 and 37°C, components of whole casein associate to polymers that approach molecular radii with apparent upper limit of 9.4 ± .4 nm. With decreasing protein concentration, κ-casein dissociates from the other casein components. This was shown by analysis of the eluted protein boundary by gel electrophoresis and radial immunodiffusion. The peak maximum elution volume and the broad, skewed character of the separated κ-casein peak indicate that in whole casein κ-casein exists in a size distribution of disulfide bonded polymers. This apparently suggests that SH-κ-casein monomers aggregate independently of the other casein components in the growth of casein submicelles, and additional studies with the purified casein components support this concept. However, after disulfide bond reduction with dithiothreitol, chromatography of whole casein over the same concentration range did not result in separation of SH-κ-casein polymers, because all of the casein was eluted under one peak. These findings show that, in vivo, casein submicelles could be formed by interaction of SH-κ-casein monomers with those of α_s- and β-casein, followed by S-S-κ-casein polymer formation through oxidation after milking.     

Assessment of the rennet coagulation of skim milk: A comparison of methods

Monitoring of rennet-induced coagulation of milk reconstituted from low-heat or medium-heat skim milk powders with or without calcium enrichment (6.25 mmol kg⁻¹) was carried out by five analytical methods. The four types of milk samples allowed evaluation of these methods on substrates characterized by very different rennetability. Thirteen parameters describing the coagulation process were extracted from the various measurements for each sample and compared. The visual flocculation and gelation times evaluated by the Berridge test and dynamic small amplitude oscillatory rheometer (DSAOR), respectively, were not significantly different from specific parameters identified from near-infrared (NIR) spectra analysis. DSAOR, piezoelectric rheometer, and NIR spectrometer allowed and evaluation of the beginning of gel firming. It clearly appeared that NIR spectroscopy, an on-line sensor, could best assess rennet-induced coagulation of studied samples.     

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.     

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).     

Partial renneting of pasteurised bovine milk: Casein micelle size,heat and storage stability

The effects of partial renneting at low temperature on the casein micelle (CM) size and the storage stability of milk were investigated. Low chymosin concentrations (≤ 0.03 IMCU mL^− 1) was applied to pasteurised skim milk at 4 °C and enzyme activity was terminated by thermal application at 60 °C/3 min and 85 °C/30 min, referred to as low heat (LHT) and high heat (HHT) treatment milk, respectively. The addition of rennet with concentrations of 0.01, 0.02 and 0.03 IMCU mL^− 1 for 15 min resulted in κ-casein hydrolysis of 10, 20 and 25%, respectively. Moreover, mean CM size of milk was reduced by up to 10 nm. For LHT milk, the renneted micelles appeared to be stable for up to 17 days, especially in response to the application of 0.01 IMCU mL^− 1 and at a storage temperature of 4 °C. Severe heating at 85 °C/30 min to inactivate the enzyme caused an increase in CM size.     

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.     

Cheese made from instant infusion pasteurized milk: Rennet coagulation,cheese composition,texture and ripening

Milk subjected to instant infusion pasteurization (IIP) at 72 °C, 100 °C and 120 °C (holding time 0.2 s) exhibited increased rennet coagulation time and decreased curd firming rate for increasing heat treatment temperature, when compared with raw or high temperature short time pasteurized (HTST) milk. However, addition of 4.5 mm or 9.0 mm of calcium restored the impaired rennet coagulation ability. Open texture cheeses produced from IIP milk (100 °C and 120 °C) contained significantly more moisture, had lower pH and shorter texture than similar cheese from IIP at 72 °C and HTST pasteurized milk. Cheese ripening was also affected by heat treatment, and different patterns of casein breakdown and peptide formation resulted from cheeses made from milk treated to IIP at 100 °C and 120 °C compared with cheeses made using IIP at 72 °C or HTST.     

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.     

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.     

Edible films produced with gelatin and casein cross-linked with transglutaminase

In this study, transglutaminase was used to produce cross-linked casein, gelatin and casein–gelatin blend (100:0, 75:25, 50:50, 25:75 and 0:100) edible films. Cross-linking was investigated by SDS–PAGE. Mechanical and water vapor barrier properties of the films were characterized using ASTM procedures, and the film morphology was evaluated using scanning electron microscopy. The casein–gelatin film showed significant greater elongation values (P < 0.05) with or without transglutaminase treatment, as compared to films made from gelatin or casein alone. Mixtures of casein and gelatin produced a synergistic effect only observed in the film elongation, while no improvement was detected for tensile strength and water vapor barrier properties, except for the casein:gelatin (75:25) formulation with transglutaminase, which showed the lowest water vapor permeability value (5.06 ± 0.31 g mm/m² d kPa). Enzymatic cross-linking also induced a substantial increase in the high molecular weight protein components in the film forming solutions.