Casein micelle dissociation in skim milk during high-pressure treatment: Effects of pressure, pH, and temperature

The effect of pH (from 5.5 to 7.5) and temperature (from 5 to 40°C) on the turbidity of reconstituted skim milk powder was investigated at ambient pressure and in situ under pressure (up to 500 MPa) by measurement of light scattering. High-pressure treatment reduced the turbidity of milk for all combinations of pH and temperature due to micelle dissociation. The turbidity profiles had a characteristic sigmoidal shape in which almost no effect on turbidity was observed at low pressures (100 MPa), followed by a stronger pressure dependency over a pressure range of 150 MPa during which turbidity decreased extremely. From the turbidity profiles, the threshold pressure for disruption of micelle integrity was determined and ranged from 150 MPa at low pH to 350-400 MPa at high pH. The threshold pressure diagram clearly showed a relationship between the barostability of casein micelles and pH, whereas almost no effect of temperature was shown. This remarkable pH effect was a consequence of pressure-induced changes in the electrostatic interactions between colloidal calcium phosphate and the caseins responsible for maintaining micellar structure and was explained by a shift in the calcium phosphate balance in the micelle-serum system. Accordingly, a mechanism for high pressure-induced disruption of micelle integrity is suggested in which the state of calcium plays a crucial role in the micelle dissociation process.     

Temperature-dependent dynamics of bovine casein micelles in the range 10–40 °C

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10–40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10–40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.     

High pressure effects on the structure of casein micelles in milk as studied by cryo-transmission electron microscopy

Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300 MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400 MPa favoured formation of smaller casein assemblies, with sizes between 30 nm and 100 nm. Measurements of free calcium concentration [Ca²⁺] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk.     

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50°C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4 ± 5.3 nm at 6°C, 177.4 ± 1.9 nm at 20°C, and 137.3 ± 2.7 nm at 50°C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20°C. Simulated milk ultrafiltrate seemed to give accurate results only at 20°C. Results obtained in simulated milk ultrafiltrate at 6°C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50°C, precipitation of amorphous calcium phosphate affected the DLS measurement.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.     

Processing of phosphocasein dispersions by dynamic high pressure: Effects on the dispersion physico-chemical characteristics and the binding of α-tocopherol acetate to casein micelles

Dispersions of phosphocaseins (PCs) containing (w/w) 2.5% proteins at pH 6.7 were processed using a ~ 15 L/h homogeniser with a high-pressure valve immediately followed by cooling devices. The effect of dynamic high-pressure (or ultra-high pressure homogenisation, UHPH) at 100–300 MPa and two initial temperatures (T_in = 14 °C or 34 °C) was investigated on (i) casein micelle size distributions, (ii) the turbidity and viscosity of PC dispersions, and (iii) the binding efficiency of α-tocopherol acetate (α-TA) to casein micelles (α-TA:PC molar ratio ~ 1:1). A significant and gradual decrease of casein micelle sizes was observed after UHPH up to 300 MPa at T_in = 14 °C. The decrease in micelle sizes was less extensive after UHPH at T_in = 34 °C. Notably, the binding efficiency of α-TA significantly (p < 0.001) increased after processing, suggesting combined effects of temperature and dynamic high-pressure.

Industrial relevance

Industrial operators in food, cosmetic and pharmaceutical areas are currently interested in developing encapsulating systems to delivery bioactive compounds, generally hydrophobic, unstable and sensitive to light, temperature or/and oxygen. The present study suggests that processing of casein micelle dispersions by dynamic high pressure at ≥ 200 MPa could modify casein micelle organisation with an enhancement of the binding of hydrophobic ligand (α-tocopherol acetate) to the newly UHPH-formed neo-micelles. Here is a potential application for UHPH, a physical technology that offers the added advantage of notably reducing the microbial load of processed samples.     

Interaction of curcumin with phosphocasein micelles processed or not by dynamic high-pressure

The binding of curcumin to native-like phosphocaseins (PC) dispersed in simulated milk ultrafiltrate at pH 6.6 was assessed by fluorescence spectrophotometry. Curcumin binds to native-like PC micelles with ∼1 binding site per casein molecule, and a binding constant of 0.6–5.6 × 10⁴ M⁻¹. Dynamic high pressure (or ultra-high pressure homogenisation, UHPH) at 200 MPa did not affect the binding parameters of curcumin to processed PC. UHPH-processing of PC dispersions at 300 MPa was followed by a slight but significant (p = 0.05) increase in the binding constant of curcumin to processed PC, which may result from the significant UHPH-induced dissociation of initial PC micelles into neo-micelles of smaller sizes, and from the corresponding 1.5–2-fold increase in micelle surface area. PC–curcumin complexes were resistant to pepsin but were degraded by pancreatin, providing the possibility of a spatiotemporally controlled release and protection of bound biomolecules. UHPH-processed PC did not induce TC7-cell damage or major inflammation as assessed by LDH release or IL-8 secretion, respectively, compared with native-like PC. PC micelles could provide a valuable submicron system to vectorise drugs and nutrients.     

Disruption and sedimentation of casein micelles and casein micelle isolates under high-pressure homogenization

Native casein micelles were isolated from raw skim milk by ultrafiltration (< 30 kDa) or microfiltration (< 0.2 μm) and subjected to high-pressure homogenization (HPH) at 100, 200, 250, 300, and 350 MPa. Of particular interest was the effect of HPH on casein micelle size in solutions varying in ionic strength (0, 5, 10, and 15 mM CaCl₂) and micelle size populations. Particle size distribution reflected an initial decrease in micelle diameter in all samples at 100 MPa. In samples containing 10 and 15 mM CaCl₂, there was an abrupt increase in particle size and subsequent casein precipitation followed by sedimentation upon centrifugation at elevated pressures (300 and 350 MPa). The amount of sedimentable casein protein increased as CaCl₂ concentration (10 and 15 mM) and pressure (300 and 350 MPa) increased as determined by UV absorbance of sample supernatant. SDS-PAGE indicated extensive micellar disruption at elevated pressures (300 and 350 MPa) and confirmed that the sedimented portion of the samples contained casein proteins and minimal amounts of whey proteins. Results indicated that through HPH treatment casein micelle size can be modified based on CaCl₂ concentration and pressure applied. Based on these findings, HPH in combination with an appropriate suspending medium has the ability to modify micelles to a desired size for a number of potential applications.Industrial relevanceThe modification of structure-function properties of the casein micelle from bovine milk by using high-pressure homogenization is relevant in (1) the development of new ingredients to change rheological/textural properties of dairy based foods, and (2) the discovery of new and/or improved functionalities for protein quaternary structures.     

Association of Triclosan to Casein Proteins Through Solvent-Mediated High-Pressure Homogenization

ABSTRACT:  The association of triclosan (TCS), a widely used hydrophobic compound, to the bovine casein micelle is investigated in this study. The use of high-pressure homogenization (HPH) at 0, 100, 200, and 300 MPa was introduced as a method for the dissociation of casein micelles in a skim milk/ethanol solution (1: 1, v/v) in the presence of TCS at 20, 80, and 160 mg/L where ethanol evaporation served as the final step for TCS association to caseins. The majority of TCS (over 80%) was associated with the caseins regardless of initial TCS concentration or applied pressure. TCS association to caseins was enhanced by 30% with continued pressurization to 300 MPa. Micellar dissociation and reassociation was found to be an irreversible process as evidenced by microscopy images. Pressurization to 300 MPa resulted in the formation of an integrated protein network of casein proteins and noncovalently linked whey proteins where the solubility of TCS was enhanced up to 40 times its reported water solubility at the highest initial TCS level of 160 mg/L. Reformed micelles exhibited Newtonian flow behavior at all pressure levels. This study provides evidence for the solubility enhancing quality of TCS through the solvent-mediated pressure/shear-induced dissociation of casein proteins.     

Effect of high hydrostatic pressure and whey proteins on the disruption of casein micelle isolates

High hydrostatic pressure disruption of casein micelle isolates was studied by analytical ultracentrifugation and transmission electron microscopy. Casein micelles were isolated from skim milk and subjected to combinations of thermal treatment (85 degrees C, 20 min) and high hydrostatic pressure (up to 676 MPa) with and without whey protein added. High hydrostatic pressure promoted extensive disruption of the casein micelles in the 250 to 310 MPa pressure range. At pressures greater than 310 MPa no further disruption was observed. The addition of whey protein to casein micelle isolates protected the micelles from high hydrostatic pressure induced disruption only when the mix was thermally processed before pressure treatment. The more whey protein was added (up to 5 g/l) the more the protection against high hydrostatic pressure induced micelle disruption was observed in thermally treated samples subjected to 310 MPa.     

Effects of high pressure on some constituents and properties of buffalo milk

In this study, effects of high pressure (HP) on some constituents and properties of buffalo milk were examined. HP treatment at 100-600MPa for 30 min affected casein micelle size only slightly, whereas treatment at 800 MPa increased it by approximately 35%. Levels of non-micellar alpha(S1)and beta-caseins were increased by treatment > or = 250MPa, and were highest after treatment at 400-800MPa. The level of non-micellar calcium increased with increasing pressure up to 600 MPa. The L*-value of the milk decreased gradually with increasing pressure, from approximately 82 for untreated milk to approximately 65 for milk treated at 800 MPa. Milk pH was increased by approximately 0.07 units after treatment at 100-800 MPa, with no significant difference between treatment pressures. Denaturation of alpha-lactalbumin occurred at pressures > or = 400 MPa, and reached >90% after treatment at 800 MPa, whereas beta-lactoglobulin (beta-Ig) was denatured > 100 MPa, reaching approximately 100% after treatment at 400MPa; after treatment > or = 400MPa, all beta-Ig was associated with the casein micelles. The rennet coagulation time of buffalo milk increased with increasing pressure, whereas the strength of the coagulum formed decreased after treatment at 250-800 MPa. Overall, HP treatment affected many constituents and properties of buffalo milk; some of these effects have also been observed in the milk from other species, but the extent of the effects, and the pressure at which they occurred, differed considerably.     

Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study

Curcumin, a polyphenolic compound present in turmeric, is a hydrophobic molecule that has been shown to bind to casein micelles. The present work tested the hypothesis that surface changes in the casein micelles caused by heat-induced interactions with the whey proteins would affect the binding of curcumin. Binding was quantified by direct and tryptophan quenching fluorescence spectroscopy. Curcumin binds to the hydrophobic moieties of the casein proteins, with a 10 nm blue shift in its fluorescence emission peak, and causes quenching of the intrinsic fluorescence spectra of the proteins. The fluorescence intensity of curcumin increased after heating of milk at 80 °C for 10 min; a similar trend in the binding constants was also observed with casein micelles separated from the soluble proteins by centrifugation. There was an increase in the non-specific interactions with heating milk at 80 °C for 10 min, both in milk as well as in casein micelles separated from the serum proteins. The increased capacity of milk proteins to bind curcumin after heat treatment can be attributed to whey protein denaturation, as whey proteins bind to the surface of casein micelles with heating.     

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.     

Effect of skim milk treated with high hydrostatic pressure on permeate flux and fouling during ultrafiltration

Ultrafiltration (UF) is largely used in the dairy industry to generate milk and whey protein concentrate for standardization of milk or production of dairy ingredients. Recently, it was demonstrated that high hydrostatic pressure (HHP) extended the shelf life of milk and improved rennet coagulation and cheese yield. Pressurization also modified casein micelle size distribution and promoted aggregation of whey proteins. These changes are likely to affect UF performance. Consequently, this study determined the effect of skim milk pressurization (300 and 600 MPa, 5 min) on UF performance in terms of permeate flux decline and fouling. The effect of HHP on milk proteins was first studied and UF was performed in total recycle mode at different transmembrane pressures to determine optimal UF operational parameters and to evaluate the effect of pressurization on critical and limiting fluxes. Ultrafiltration was also performed in concentration mode at a transmembrane pressure of 345 kPa for 130 or 140 min to evaluate the decline of permeate flux and to determine fouling resistances. It was observed that average casein micelle size decreased by 32 and 38%, whereas β-lactoglobulin denaturation reached 30 and 70% at 300 and 600 MPa, respectively. These results were directly related to UF performance because initial permeate fluxes in total recycle mode decreased by 25% at 300 and 600 MPa compared with nonpressurized milk, critical flux, and limiting flux, which were lower during UF of milk treated with HHP. During UF in concentration mode, initial permeate fluxes were 30% lower at 300 and 600 MPa compared with the control, but the total flux decline was higher for nonpressurized milk (62%) compared with pressure-treated milk (30%). Fouling resistances were similar, whatever the treatment, except at 600 MPa where irreversible fouling was higher. Characterization of the fouling layer showed that caseins and β-lactoglobulin were mainly involved in membrane fouling after UF of pressure-treated milk. Our results demonstrate that HHP treatment of skim milk drastically decreased UF performance.     

High-pressure homogenization of raw and pasteurized milk modifies the yield, composition, and texture of queso fresco cheese

High-pressure homogenization (HPH) of milk was studied as an alternative processing operation in the manufacturing of queso fresco cheese. Raw and pasteurized (65°C for 30 min) milks were subjected to HPH at 0, 100, 200, and 300 MPa and then used to manufacture queso fresco. The cheeses were evaluated for yield, moisture content, titratable acidity, nitrogen content, whey protein content, yield force, yield strain, and tactile texture by instrumental or trained panel analyses. The combination of HPH and thermal processing of milk resulted in cheeses with increased yield and moisture content. The net amount of protein transferred to the cheese per kilogram of milk remained constant for all treatments except raw milk processed at 300 MPa. The highest cheese yield, moisture content, and crumbliness were obtained for thermally processed milk subjected to HPH at 300 MPa. The principal component analysis of all measured variables showed that the variables yield, moisture content, and crumbliness were strongly correlated to each other and negatively correlated to the variables yield strain, protein content (wet basis), and sensory cohesiveness. It is suggested that the combination of thermal processing and HPH promotes thermally induced denaturation of whey protein, together with homogenization-induced dissociation of casein micelles. The combined effect results in queso fresco containing a thin casein-whey matrix that is able to better retain sweet whey. These results indicate that HPH has a strong potential for the manufacture of queso fresco with excellent yield and textural properties.     

Effect of moderate inlet temperatures in ultra-high-pressure homogenization treatments on physicochemical and sensory characteristics of milk

The effect of ultra-high-pressure homogenization (UHPH) on raw whole milk (3.5% fat) was evaluated to obtain processing conditions for the sterilization of milk. Ultra-high-pressure homogenization treatments of 200 and 300 MPa at inlet temperatures (Ti) of 55, 65, 75, and 85°C were compared with a UHT treatment (138°C for 4 s) in terms of microbial inactivation, particle size and microstructure, viscosity, color, buffering capacity, ethanol stability, propensity to proteolysis, and sensory evaluation. The UHPH-treated milks presented a high level of microbial reduction, under the detection limit, for treatments at 300 MPa with Ti of 55, 65, 75, and 85°C, and at 200 MPa with Ti = 85°C, and few survivors in milks treated at 200 MPa with Ti of 55, 65, and 75°C. Furthermore, UHPH treatments performed at 300 MPa with Ti = 75 and 85°C produced sterile milk after sample incubation (30 and 45°C), obtaining similar or better characteristics than UHT milk in color, particle size, viscosity, buffer capacity, ethanol stability, propensity to protein hydrolysis, and lower scores in sensory evaluation for cooked flavor.     

Yield, composition and rheological characteristics of cheddar cheese made with high pressure processed milk

High Pressure (HP) treatment of milk prior to cheese-making was shown to increase the yield of cheese due to increased protein and moisture retention in cheese. Cheeses were made with raw milk or milk treated with high temperature short-time (HTST) pasteurization, and HP treatments at two levels (483 and 676 MPa) at 10 °C, 483 MPa HP at 30 °C, and 483 MPa HP at 40 °C. Cheese yield, total solids, protein, fat and salt contents were evaluated, and fat and protein recovery indices were calculated. Cheeses from HP treatments of 676 MPa at 10 °C and 483 MPa at 30 °C exhibited wet yields of 11.40% and 11.54%, respectively. Protein recovery was 79.9% for HP treatment of 676 MPa at 10 °C. The use of slightly higher pressurization temperatures increased moisture retention in cheese. Visco-elasticity of cheeses was determined by dynamic oscillatory testing and a creep-recovery test. Rheological parameters such as loss (G″) and storage (G′) moduli were dependent on oscillation frequency. At high (173 rad/s) and low (2.75 rad/s) angular frequencies, cheeses made from milk treated at 483 MPa at 10 °C behaved more solid-like than other treatments. Creep tests indicated that cheeses from milk treated with 483 MPa HP at 10 °C showed the smallest instantaneous compliance (J_o), confirming the more solid-like behavior of cheese from the 483 MPa at 10 °C treatment compared to the behavior of cheeses from other treatments. Cheeses made with pasteurized milk were more deformable, exhibited less solid-like behavior than cheeses made with HP treated milk, as shown by the J_o value. With more research into bacteriological implications, HP treatment of raw milk can augment Cheddar cheese yield with better curd formation properties.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

The two-stage coagulation of milk proteins in the minimum of the heat coagulation time-pH profile of milk: effect of casein micelle size

Milks with casein micelles larger or smaller than control milk were prepared by differential centrifugation. The heat stability of these modified milks increased markedly throughout the pH range 6.4 to 7.1 with decreasing casein micelle size. Within the region of the minimum in the heat coagulation time-pH profile, the control milk coagulated by a two-stage process, but the modified milks, because of their narrower casein micelle size distribution, coagulated by a single-stage process at the pH of minimum stability. The content of kappa-CN and protein hydration increased as the size of the casein micelles decreased, and the level of glycosylation of kappa-CN and protein surface hydrophobicity increased as a function of micelle size. The effect of casein micelle size on the heat stability of milk is likely to be related to changes in the above physico-chemical properties.