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.     

Influence of pH and NaCl on rheological properties of rennet-induced casein gels made from UF concentrated skim milk

Milk concentrates are used in the production of cast cheese. The effects of pH (5.19–6.21) and NaCl concentration (0, 1.75% and 3.50%, w/w) on the rheological and microstructural properties of rennet-induced casein gels made from ultra filtered skim milk (19.8%, w/w casein) were investigated. Low pH and high NaCl concentration reduced the development rate of the gel elasticity after coagulation of the ultra filtrated skim milk. Strain at fracture and stress at fracture from uniaxial compression of casein gels 48 h after coagulation showed maximum and minimum values at pH ∼5.8 and 5.29, respectively. Young's modulus from uniaxial compression of the same gels was almost constant between pH 5.52 and 6.21 but much lower at pH 5.28. Addition of NaCl resulted in increased Young's modulus in the interval pH 6.21–5.52. As pH decreased, the level of colloidal calcium phosphate decreased concomitantly, giving less cross-links in the casein network and partly explaining the changes in the rheological properties. Increased ionic strength by adjusting pH and addition of NaCl also influenced rheological results. The microstructure examined with confocal laser-scanning microscopy was unaffected by the changes in pH and concentrations of NaCl in the range studied as revealed by image analysis and calculations of two- and three-dimensional data from micrographs.     

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.     

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).     

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.     

Micellar Transition State in Casein Between pH 5.5 and 5.0

pH-induced changes in casein micelles during direct acidification and bacterial fermentation of reconstituted skim milk at 20°C were monitored by scanning electron microscopy (SEM) in combination with biochemical and rheological methods. For SEM casein micelle observations, an original method of milk sample preparation with porous inorganic membranes was developed. Micrographs suggested that different stages of micellar association were related to pH and that between pH 5.5 and 5.0 casein micelles coalesced. Correlations between microstructural and biochemical changes in casein micelles, and rheological behavior of milk or gel, help to explain the different steps leading to the final protein network of the acid milk gel.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

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

Effect of Serine Proteolytic Enzymes (Trypsin and Plasmin), Trypsin Inhibitor,and Plasminogen Activator Addition to Ultra-High Temperature Processed Milk

Proteolysis and gelation were investigated in single strength, 2% fat, UHT-processed milk following aseptic addition of combinations of plasmin, plasminogen, trypsin, trypsin inhibitor (Kunitz), and urokinase (plasminogen activator). Individual 250-ml milk containers processed by direct or indirect methods were examined for the following attributes over 10 mo: growth on slants, appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples in the study did not gel. Addition of trypsin at 1.5 or 7.5 mg protein/L of milk or addition of plasmin at .3 or 1.5 mg protein/L did not result in gelation. However, containers with plasminogen at .3 mg protein/ L began forming a gel at 5.5 mo. Enzyme activity in plasminogen-treated samples was not detected spectrophotometrically using an L-lysine-p-nitroanilide substrate, but extensive casein breakdown was apparent by SDS-PAGE. The evidence suggests plasminogen-derived activity promotes UHT milk gelation.     

Iron fortification of skim milk: Minerals and 57Fe Mössbauer study

Reconstituted skim milk was fortified at 2, 5, 10 and 20 mmol Fe kg^?1 with ferric and ferrous chloride iron with or without reversible acidification by injection of CO₂ under pressure. Carbonation improved transfer of iron from the soluble to the colloidal phase and accelerated ferrous iron oxidation. ⁵⁷Fe Mössbauer spectra of the freeze-dried casein micelles collected by centrifugation demonstrated that iron is present in a distorted octahedral coordination and is chelated by phosphate rather than citrate. Milk iron fortification induced several changes in the mineral component of the casein micelles, which are explained by the formation of a ternary complex: inorganic phosphate–iron–organic phosphate.     

Effects of reverse CO2 acidification cycles,calcium supplementation,pH adjustment and chilled storage on physico-chemical and rennet coagulation properties of reconstituted low- and medium-heat skim milk powders

The reversibility extent of one and two reverse CO₂ acidification cycles on the physico-chemical and rennet coagulation properties of milks reconstituted from low- (LH) or medium- (MH) heat skim powder, enriched or not with calcium and pH adjusted or not was investigated. The ionized calcium concentration, buffering properties and average casein micelle size of untreated and CO₂-treated milks were evaluated before and after a chilled storage for 2 days. The ionized calcium concentration and buffering properties have been modified by the CO₂-treatment, particularly after a second CO₂-cycle. These modifications were highly dependent on the initial milk properties and chilled storage. Inversely, the average casein micelle size was not significantly changed. In addition, the rennet-clotting behaviour checked by near infrared spectroscopy (NIR-S) and rheology (SAOR) indicated the main factors responsible for changes in the casein micelles environment and dynamic casein micellar calcium phosphate reorganization, especially after two CO₂-cycles. A single CO₂-cycle induced a better rennetability for non Ca-enriched milk reconstituted from MH-powder. A second CO₂-cycle was particularly efficient to improve Ca-enriched pH-adjusted milks.     

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.     

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 a CO2-acidification cycle on physicochemical and acid gelation properties of skim milk reconstituted with added pectin

The influence of a CO₂-acidification cycle on the acid (glucono-δ-lactone, GDL) gelation properties of skim milk with and without added low-methoxyl pectin (LM-pectin) was assessed. Ionic calcium level, zeta potential, particle size, buffering properties and small amplitude oscillatory rheology moduli were monitored. The presence of LM-pectin in milk had an impact on the average size of the casein micelles and a large and dominant influence on its rheological behaviour during GDL acidification. The application of a CO₂–pH cycle (pH_target 4.9) as a milk pretreatment induced during GDL acidification a stabilization of the colloidal system in wide pH range (pH 6.0–5.1) with modifications of the structure of the casein micelles before the onset of gelation. These modifications induced a significant improvement on its acid gelation behaviour. The measurements of Ca²⁺ level during GDL acidification showed that an important and significant part of the Ca²⁺ released during the CO₂–pH cycling was electrostatistically trapped by pectin molecules in the serum.     

Casein Composition of Cow's Milk of Different Chymosin Coagulation Properties

Five good and four poor chymosincoagulating individual cow milk samples were analyzed for casein composition using hydroxyapatite chromatography and polyacrylamide gel electrophoresis to establish possible relationships between casein fractions and differences in coagulation properties.Samples exhibited wide variation in casein composition. Poor chymosincoagulating milk had higher content of γ- and degraded caseins and lower κ- and β-caseins than the good-coagulating milk. One milk sample that did not coagulate 30 min after chymosin addition had low α_S-casein concentration and an additional major casein fragment (not identified). A substantial peak representing unidentified minor caseins was apparent in a poorcoagulating milk sample, which coagulated early but whose coagulum did not become firm in 30 min. Excluding the nonclotting sample, less variability was observed in α_S-casein concentrations than in the other casein components.     

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R² = 0.95).     

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.     

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 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.     

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 chemical phosphorylation on solubility of buffalo milk proteins

Buffalo milk proteins (casein, co-precipitate or whey protein concentrate) were phosphorylated with phosphorus oxychloride (POCl₃) at three different pH values (5.0, 7.0 and 9.0). The solubilities of phosphorylated milk proteins were examined over the pH range 3.0–9.0 in water and ionic (0.1 m NaCl or 10–70 mm Ca²⁺) systems. The solubilities of buffalo milk proteins decreased at pH 3.0, while there was an increase in the solubilities of casein and co-precipitate near their isoelectric points upon phosphorylation. Solubilities of these phosphorylated milk proteins were pH dependent in 0.1 m NaCl but there was a decrease in their solubilities with increase in calcium ion concentration. This alteration could be due to the shifting of isoionic points of phosphorylated buffalo milk proteins towards acidic pH.