Characterisation of milk protein adsorption onto hydroxyapatite

Adsorption behaviour of α_S-casein, β-casein, κ-casein, β-lactoglobulin and α-lactalbumin on hydroxyapatite (HA) was characterised by determination of adsorbed protein levels and surface charge of HA. Individually, the proteins were able to bind onto HA causing a decrease in the zeta-potential magnitude of the HA particles. The maximum amount of protein that could bind onto HA and the affinity of the proteins for HA were quantified using a Langmuir model, and were different between the different proteins. α_S-Casein and β-casein could bind to higher levels onto HA and had a higher affinity for HA, probably because of the presence of clusters of phosphoserine residues in their primary structures. β-Casein was also able to displace adsorbed β-lactoglobulin from the HA surface when added in a suspension of β-lactoglobulin-covered particles, probably because the affinity of the casein phosphoserine residues for HA was stronger than that of the carboxyl groups of the whey protein.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.     

Effect of pressure treatment of recombined whole milk on fat globule membrane composition and acid gelation functionality

Recombined whole milk was prepared by pressure treating skim milk (200 to 600 MPa/30 min) then homogenizing with milkfat (HPHO) or by homogenizing milkfat with skim milk then pressure treating the recombined whole milk (HOHP). β-Lactoglobulin denaturation increased at higher pressures. Low levels of α-lactalbumin were denatured at 600 MPa only. Denaturation was similar in the HPHO and HOHP milk. The HPHO milk had statistically similar levels of total protein, higher levels of whey protein and κ-casein and lower levels of α_s-casein adsorbed to the fat globules compared with the HOHP milk. The HPHO milk had a higher proportion of β-casein directly at the interface at all pressures and a higher proportion of κ-casein and a lower proportion of denatured whey proteins at pressure up to 400 MPa than the HOHP milk. Acid gels prepared from the HOHP milk had higher final G′ and yield stresses than those from the HPHO milk. These differences are discussed in relation to the compositions of the proteins adsorbed to the fat globules and how these interact during acidification.     

Effects of Environmental Factors and Milk Protein Polymorphism on Composition of Casein Fraction in Bovine Milk

Test-day samples were collected from individual Holstein cows in 62 herds enrolled in the Quebec Dairy Herd Analysis Service. Samples were analyzed for protein, fat, casein, and serum protein content, somatic cell count, and relative percentages of α-, β-, and κ-casein and a-lactalbumin. Cows included in the study were phenotyped for the genetic variants of α_s1-, β-, and κ-casein. Unadjusted means for relative percentages of α_s-, β-, and κ-casein were 59.85, 31.23, and 8.93%, respectively. Least-squares analyses showed that month of test, stage of lactation, age of the cow, somatic cell count, and phenotype of the cow for β-casein contributed to variations in the relative percentages of α_s- and β-casein. Month of test, somatic cell count, and phenotype of the cow for κ-casein also had a significant effect on the relative percentage of κ-casein. When test-day milk yield; percentages of fat, protein, casein, and serum protein; casein to protein ratio; and relative percentage of α-lactalbumin were included in the model as covariates, only casein percentage did not have a significant effect on the relative percentages of α_s- and β- casein. For κ-casein, only fat percent was significant.     

Effects of pressurized thermal processing on native proteins of raw skim milk and its concentrate

Heating, pressurization, and shearing can modify native milk proteins. The effects of pressurized heating (0.5 vs. 10 MPa at 75 or 95°C) with shearing (1,000 s^?1) on proteins of raw bovine skim milk (SM, ～9% total solids) and concentrated raw skim milk (CSM, ～22% total solids) was investigated. The effects of evaporative concentration at 55°C and pressurized shearing (10 MPa, 1,000 s^?1) at 20°C were also examined. Evaporative concentration of SM resulted in destabilization of casein micelles and dissociation of α_S1- and β-casein, rendering CSM prone to further reactions. Treatment at 10 MPa and 1,000 s^?1 at 20°C caused substantial dissociation of α_S1- and β-casein in SM and CSM, with some dissociated caseins forming shear-induced soluble aggregates in CSM. The pressure applied at 10 MPa induced compression of the micelles and their dissociation in SM and CSM at 75 or 95°C, resulting in reduction of the micelle size. However, 10 MPa did not alter the mineral balance or whey proteins denaturation largely, except by reduction of some β-sheets and α-helices, due to heat-induced conformational changes at 75 and 95°C.     

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

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

Changes in the surface protein of the fat globules during ultra-high pressure homogenisation and conventional treatments of milk

Disruption of fat globules upon homogenisation provokes a reduction of their size and a concomitant increase in their specific surface area. In order to overcome this phenomenon, the milk fat globule membrane (MFGM) adsorbs non-native MFGM proteins. The aim of the present study was to examine the effects of UHPH conditions (temperature and pressure) on the milk fat globule and the surface proteins by comparison with conventional treatments applied in the dairy industry. Transmission electron microscopy and SDS-PAGE revealed major differences. In UHPH-treated milk, casein micelles were found to be adsorbed on the MFGM in a lesser extent than in conventional homogenisation–pasteurisation. However, greater adsorption of directly bonded casein molecules, released by UHPH through the partial disruption of casein micelles, was observed especially at high UHPH pressures. Adsorption of whey proteins on the MFGM of conventionally homogenised–pasteurised milk was mainly through intermolecular disulfide bonds with MFGM material, whereas in UHPH-treated milk, disulfide bonding with both indirectly and directly adsorbed caseins was also involved.     

The effect of hexametaphosphate addition during milk powder manufacture on the properties of reconstituted skim milk

Skim milk powders with various levels of sodium hexametaphosphate (NaHMP) were prepared. Reconstituted skim milk samples were prepared from these powders. NaHMP slightly reduced the pH, markedly reduced the serum and ionic calcium and markedly increased the serum phase orthophosphate levels of the milks. This shift in the mineral equilibrium resulted in a drastic reduction in casein micelle integrity, with a marked dissociation of casein from the micelles. κ-Casein was the predominant casein dissociated, although significant levels of α_S-casein and β-casein were also transferred to the serum phase. This dissociation of the casein micelles caused a marked decrease in size and scattering properties of the casein micelles. In addition, a small decrease in the zeta potential of the casein micelles in the milk was observed. Heat treatment of the milks with added NaHMP induced further dissociation of κ-casein, although much of the α_S-casein and β-casein re-associated with the micelles.     

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk

Casein proteins (α_S1-, α_S2-, β- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 10¹⁵ micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pK_a) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pK_a = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pK_a = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ～5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ～65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine 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.     

Interaction of artificial casein micelles with β-lactoglobulin

By means of spectroscopy in visible light, the interaction was followed between artificial casein micelles and β-lactoglobulin at 20 °C and after heating to 80 °C for IO min. The micelles consisted of either α_s1-casein-χ-casein or β-casein-χ-casein. In the presence of β-lactoglobulin, the micelles of the α_s1-casein-χ-casein type were found to be of spherical shape. On heating, they dissociated to particles of a smaller size and this dissociation was followed by reassociation to aggregates of a larger size than that of micelles in the absence of β-lactoglobulin. The aggregates formed in the system of β-casein-χ-casein in the presence of β-lactoglobulin were also of spherical shape but of a smaller size.     

Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of β-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of β-, α_s1-, and α_s2-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

Effects of Packaging Materials and Filling Methods on Selected Characteristics of Otlu (Herby) Cheese

In this research, two kinds of filling methods (block and pieced) in two container types (pot and plastic) were used for the preparation of Otlu cheeses. The uses of different containers influenced (P < 0.01) acidity, pH, dry matter, fat contents and water soluble nitrogen. Degradation of α?s₁- and β-casein were higher in pieced cheeses in pot (P1F2). Also, breakdown products of casein were higher in P1F2 cheeses compared to others. The amounts of α?s₁- and β-caseins of all treatments decreased during ripening, while the amounts of α?s₁-I casein and breakdown products increased. The acceptability of cheeses in block form was higher.     

TWO-DIMENSIONAL ANALYSIS OF SKIM MILK PROTEINS USING PREPARATIVE ISOELECTRIC FOCUSING FOLLOWED BY POLYACRYLAMIDE GEL ELECTROPHORESIS

ABSTRACT A preparative isoelectric focusing (IEF) method was applied to separate skim milk proteins using the Rotofor device in a pH 3–10 gradient containing 4 M urea/1% triton X-100. Each of the 20 fractions obtained from the Rotofor device was then analyzed by polyacrylamide gel electrophoresis (PAGE). Both urea-PAGE and SDS-PAGE were used to separate purified caseins and skim milk resulting in comparable two-dimensional patterns. The major bovine caseins (αs1, αs2, β, K-casein) were resolved better on urea-PAGE. The αs1-and β-casein were focused at pH ～ 4.5 and 4.8, respectively, whereas αs2-caseins focused as several bands at pH 6.2–6.8. The A variant of K-casein focuses at Fractions 6–9 which is slightly more acidic than the B variant that focuses at Fractions 7–13. No sample pretreatment was necessary to analyze skim milk proteins and urea-PAGE clearly resolved bands of all major caseins and whey proteins. Preparative isoelectric focusing followed by PAGE was found to be a useful and powerful method to analyze milk proteins in two-dimensions. This technique facilitates the analysis of the relative amounts of proteins in milk, as well as simplifies the detection of changes and foreign proteins in milk.     

Functional characterization of protein stabilized emulsions: Emulsifying behaviour of proteins in a valve homogenizer

Protein stabilised emulsions have been prepared in a valve homogeniser incorporated into a recirculating emulsification system, where the power input and number of passes have been varied. The food proteins studied were a soy-bean protein isolate, a whey protein concentrate (WPC) and a sodium caseinate. The emulsions obtained were characterized in terms of particle size distribution and amount of protein adsorbed on to the fat surface (protein load). Generally, the final fat surface area of the emulsions obtained increases more as a function of power input than as a function of number of passes. Distribution width, c_s, decreases mostly with increasing power supply and number of passes, but at the highest power input c_s increases. The protein load on the fat globules is largely determined by the fat surface area and by the type of protein adsorbed. The soy proteins give a high protein load and the caseinates give a low protein adsorption at small fat surface areas created. This relation is reversed at large surface areas of the fat globules. The relation between percentage protein adsorbed from bulk as a function of surface area suggests that the caseinates mainly cover the newly created interface by adsorption from the bulk, whereas the soy proteins fulfil this task mostly by spreading at the interface. Salt addition to 0.2M-NaCl enhances protein adsorption at the fat globule interface in the case of soy protein and caseinate, but for the whey proteins protein load is higher in distilled water.     

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

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

Behaviour of homogenized fat globules during the spray drying of whole milk

The changes in milk fat globule size and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure prior to spray drying using a disc atomization drier, were examined. The average fat globule size (d₃₂) of the spray-dried milk powders was smaller than that of the corresponding concentrates, but a small proportion of very large globules (4–80 μm) was also formed during spray drying. As a consequence, total surface protein (mg protein g⁻¹ fat) increased due to the adsorption of casein micelles at the fat globule surface during spray drying. Confocal micrographs of the powders showed some apparent spreading of the fat on the surface of the powder particles, particularly when the concentrates were homogenized at low pressure. These results indicate disruption of the milk fat globules during spray drying, which consequently causes changes in the fat globule surface protein layer.     

Mozzarella-Type Curd Made from Buffalo, Cows’ and Ultrafiltered Cows’ Milk

Buffalo milk contains (40–60 %) more protein, fat and calcium than cows’ milk. These constituents were enhanced by ultrafiltration (UF) of cows’ milk to give a product with similar levels to those found in the buffalo milk. Mozzarella-type curd was made from buffalo, cows’ and UF cows’ milk to compare the overall curd yield and quality. The curd yield on both dry and wet weight basis, curd moisture content and overall curd fat retention were found to be higher in the UF cows’ milk than for either the buffalo or the cows’ milk preparations. The minimum whey fat losses occurred in the UF cows’ curd when compared to the cows’ and the buffalo curd. The whey protein losses were found to be higher in the UF cows’ curd than those for the buffalo and the cows’ curds. The total mineral content of the curd was also higher in the UF cows’ milk than that found in either the buffalo or the cows’ milk. SEM micrographs showed that casein micelles sizes were different in the two different types of milk. Casein micelles were also observed to be deformed in the UF cows’ milk samples. UF cows’ milk contained higher amounts of both the α_s1- and α_s2-casein moieties than either the buffalo or the cows’ milk. Buffalo milk was found to contain a higher concentration of β-casein than either the UF cows’ or untreated cows’ milk samples. Gel strength was found to be higher in the resultant buffalo curd than for curds made from either native cows’ milk or those made from UF cows’ milk. The mineral distribution was also different in the three different types of bovine milk, measured by energy-dispersive X-ray (EDX) analysis. Differences in the curd quality observed between the buffalo and the cows’ milk appear to result from the differences in casein composition and overall micelle structure, rather than casein concentration alone.     

Enzymatic dephosphorylation of caseins and creaming behaviour of o/w emulsions stabilized with dephosphorylated casein fractions

α_s- and β-casein were treated with acid or alkaline phosphatase. The dependence of the dephosphorylation on incubation time was investigated by electrophoresis and by determination of the residual phosphate content. The degree of dephosphorylation is correlated with the stability of emulsions prepared with the modified proteins. Incubation of α_s- and β-casein with alkaline phosphatase increased the creaming stability 6- and 10-fold respectively.