Composition and Structure of Fat Globule Surface Layers in Recombined Milk

Protein coverage, composition and structure of surface layers of fat globules in recombined milk were determined. Average protein load was ～6 mg/m² fat surface. Both casein and whey proteins were present in the fat globule surface layer, with casein adsorbed in preference to whey proteins and α_s (α_sl+α_s2)-casein adsorbed in preference to β-casein. Transmission electron microscopy showed that the surface layer of fat globule was made up of casein micelles, fragments of casein micelles and a thin layer of protein, possibly whey proteins. Experiments with surface layers that had been dispersed in EDTA showed that the extent of dissociation of caseins followed the order: β-casein > α_s-casein ≦ K-casein, suggesting that most of the K-casein was probably associated directly with the fat surface.     

The effect of dephosphorylation and succinylation on the properties of casein proteins adsorbed to the surface of latex particles

Dephosphorylation decreased the absolute zeta potential and diameters of α_S1-casein-coated latex particles. Similar effects were observed for α_S1-casein dephosphorylated prior to or after adsorption. If α_S1-casein adopts a loop arrangement at hydrophobic surfaces, then it appears that dephosphorylation causes the loop to move closer to the surface. Dephosphorylation caused a larger change for particles coated with α_S1-casein than β-casein, whereas small changes were observed for κ-casein, consistent with the phosphorylation levels of these caseins. Succinylation increased the absolute value of zeta potential and the diameters of α_S1-casein-coated latex particles. A higher level of succinylated α_S1-casein was required to saturate the particle surface. It is proposed that succinylated α_S1-casein has the C-terminal region adsorbed to the particle surface and N-terminal region extending from the surface as a hair. Results of this study help explain the effect of dephosphorylation and succinylation on the functional properties of the caseins, particularly in stabilising emulsions.     

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.     

Short communication: Bovine α-casein is a ferritin-binding protein and inhibitory factor of milk ferritin immunoassay

Commercial bovine milk α-casein, but not β- and κ-caseins, bound to bovine spleen ferritin, as determined by an immunoassay for ferritin. In contrast, α-casein did not bind to apoferritin. The binding of α-casein to bovine spleen ferritin was strongly inhibited by increasing ionic strength by the addition of 0.5 M (NH₄)₂SO₄. The addition of α-casein to a known amount of bovine spleen ferritin resulted in significantly lower recovery (78-80%) of added ferritin, although β- and κ-caseins showed little inhibitory effect in the ferritin immunoassay. These results indicate that bovine α-casein is a specific ferritin-binding protein that may inhibit milk ferritin immunoassay.     

Rheological Evaluation of Maturing Cheddar Cheese

A number of Cheddar cheese samples of different age, pH and moisture content have been examined rheologically and electrophoretically to determine whether the progressive changes in cheese texture were related to casein proteolysis. The force-compression curves obtained by crushing cubes of cheese between small flat plates at constant speed were different for the different cheese samples and were affected by the moisture content, pH and extent of α_sl-casein proteolysis that had taken place in the cheese. These results support a model of cheese micro structure in which an extensive network involving α_sl- casein molecules traverses the cheese and as the cheese ripens, chymosin cleavage of α_sl-casein weakens the protein network. Such a model explains the rapid decrease in Cheddar cheese yield-force that occurs during the early stages of ripening.     

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.     

Variation in caseinolytic properties of six cheese related Lactobacillus helveticus strains

A large degree of strain variation was observed in caseinolytic properties of six cheese related Lactobacillus helveticus strains. Activity on intact α_s1- and β-casein was observed only after growth in milk and not in MRS. Totally 27 peptides from α_s1- and 22 from β-casein were identified from MS/MS fragmentation patterns. All six strains released peptides from the amino end of α_s1-casein, and the bonds Ile₆-Lys₇ and Gln₉-Gly₁₀ were identified as primary cleavage sites. Strain variation in the activity on intact β-casein was observed and five of the six strains released peptides from the C-terminal region. The strains had very different activities and some strains had only trace activities. L. helveticus CNRZ 32 had the highest activity towards α_s1-casein while L. helveticus LHC2 had the highest activity towards β-casein, and these two strains also produced unique peptides from both α_s1- and β-casein.     

On the activity and specificity of cardosin B,a plant proteinase,on ovine caseins

The proteolytic activity of cardosin B, an aspartic proteinase from the thistle, Cynara cardunculus, on ovine α_s-caseins and β-caseins (independently or present together in sodium caseinate) was followed by urea polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography. This enzyme degraded both types of caseins, but not to the same degree. In sodium-caseinate, by 10 h at 30°C, α_s-caseins were more susceptible to proteolysis by cardosin B than β-casein whereas, in isolated form, the reverse was observed. Sequencing of the peptides produced by hydrolysis of Na-caseinate showed that the major cleavage sites in α_s1-casein were Leu156-Asp157 and Trp164-Tyr165 whereas, in β-casein, they were Leu127-Thr128, Leu165-Ser166 and Leu90-Tyr191. The bonds Trpl64-Tyr165 and Leu165-Ser166 were the most susceptible to cardosin B when this enzyme acted upon isolated α_s1- and β-casein, respectively.     

Interactions Between Lipids and Milk Proteins in Emulsion

The interactions between nitroxide homologs of fatty acids and purified components of milk proteins in a model food emulsion were studied using electron spin resonance. The importance of the interactions in emulsions decreased in the order α_s1-casein > β-lacto-globulin > β-casein β whole casein. α_s1-Casein resulted in a better organization of the lipid monolayer. Neither the flexibility nor the tertiary structure of the proteins explained this result. It appeared that the interactions occurred through lipid polar heads and protein polar side chains due to hydrogen bonds and/or electrostatic interactions.     

Short communication: Effect of subclinical mastitis on proteolysis in ovine milk

The aim of this work was to evaluate the effect of intramammary infection (IMI) on the endogenous proteolysis of milk. Four control checks were carried out in the half-udder milk of 10 ewes that acquired unilateral subclinical mastitis. Two of these checks were conducted before the infection was established and 2 after. Ten healthy ewes were tested as a control group. The presence of a subclinical IMI involved an increase of the products of casein hydrolysis, the proteose-peptone (p-p) fraction and minor (m) caseins, and a decrease of β-casein. As a result, a significant increase in the proteolysis index (PI), calculated as the ratio of m-casein to the sum of caseins (α + β + κ), took place. α-Casein and κ-casein were not significantly affected by IMI. Correlations confirmed the scenario: log₁₀ of somatic cell count (SCC) was positively correlated with p-p content and negatively with β-casein, whereas log₁₀ SCC was not correlated with α-casein or κ-casein. On the other hand, p-p content was positively correlated with m-casein and PI and negatively with β-casein, but no correlation was detected between p-p content and α- or κ-casein. Furthermore, between casein fractions, m-casein was only significantly correlated with β-casein. These results suggest that use of indices of proteolysis of caseins such as p-p, m-casein, and PI, could be applied together with SCC to evaluate the cheese-making quality of milk.     

Relative Proteolytic Action of Milk-Clotting Enzyme Preparations on Bovine α-, β- and β-casein

Concentrations of seven milk-clotting enzyme preparations were standardized to equal clot times. Portions of bovine α_s-, β- and κ-casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6-trinitrobenzene-sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on α_s- and β-caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β-casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on α_s- and β-caseins but was the least proteolytic on κ-casein.     

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde,Normande, and Holstein dairy cattle breeds

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h²) were generally higher for protein fractions than for protein contents. They were moderate to high for α_S1-casein, α_S2-casein, β-casein, κ-casein, and α-lactalbumin (0.25 < h² < 0.72). In each breed, β-lactoglobulin was the most heritable trait (0.61 < h² < 0.86). Genetic correlations (r_g) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < r_g < 0.46) and κ-casein and α_S2-casein (0.36 < r_g < 0.49). Between protein contents, r_g estimates were positive, with highest values found between caseins (0.83 < r_g < 0.98). In the 3 breeds, β-lactoglobulin was negatively correlated with caseins (?0.75 < r_g < ?0.08), in particular with κ-casein (?0.75 < r_g < ?0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection.     

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.     

Effect of β-casein concentration in cheese milk on rennet coagulation properties,cheese composition and cheese ripening

Effects of the use of a β-casein powder to enrich cheese milk on rennet coagulation properties of milk, cheese composition and cheese ripening were investigated. Casein content of control milk was 2.5%, whereas that for the three enriched milks was adjusted with β-casein powder at 2.7%, 2.9% and 3.1%. The β-casein to α-casein ratio of these cheese milks was, respectively, 0.70, 0.79, 0.89 and 0.99. Rennet coagulation properties were related not only to casein concentration but also to the proportion of β-casein and α_s-casein presents in milks. Milk with higher concentration of β-casein had poorer coagulation properties. Cheeses could be produced by using a miniature cheese making process. Moisture, ash and calcium contents decreased, while protein content and β-casein increased in cheese as casein and β-casein concentration increased in milk. As a result, hardness was higher in enriched cheeses than in control cheese. During cheese ripening, α-casein was hydrolyzed, but the rate of degradation of α-casein decreased as protein and β-casein concentration increased in cheese. β-Casein seemed to be not hydrolyzed. The rate of decrease of hardness was also slower for enriched cheeses.     

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.     

Plasmin digestion of photooxidized milk proteins

Plasmin-mediated hydrolysis of 6 different milk protein preparations [α_S-casein (α_S1 + α_S2), β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of α_S-casein, consisting of a mixture of α_S1- and α_S2-casein and a preparation of β-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.     

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.     

Time-dependent competitive adsorption of milk proteins and surfactants in oil-in-water emulsions

Competitive adsorption of pure milk proteins and non-ionic surfactants has been studied in model oil-in-water emulsions (4 g kg^?1 β-lactoglobulin or β-casein, 200 g kg^?1 n-hexadecane) as a function of the age of the adsorbed protein layer at the oil-water interface. With β-lactoglobulin-stabilised emulsions containing oil-soluble surfactant C₁₂ E₂ (diethylene glycol n-dodecyl ether), there is found to be a steadily increasing amount of protein associated with the emulsion droplets over a few hours following emulsification. Addition of water-soluble surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) to a β-lactoglobulin-stabilised emulsion (with or without C₁₂E₂) leads to less protein displacement if the emulsion is aged prior to addition of Tween 20. Moderate additions of C₁₂E₂ or Tween 20 produce no time dependence in the competitive adsorption in β-casein-stabilised emulsions, although some time dependence is observed when C₁₂E₂ and a high concentration of Tween 20 are present together. Crystallisation of the oil phase in β-casein-stabilised emulsions at pH 7 leads to a lowering of the measured protein surface concentration, especially in the presence of C₁₂E₂ and a reduction in the surfactant to protein molar ratio required for complete protein displacement by water-soluble surfactant (Tween 20 or octaethylene glycol n-dodecyl ether). Under more acidic conditions of pH 5 or pH 3, the surface coverage and ease of displacement of β-lactoglobulin at the surface of liquid emulsion droplets is substantially different from that under neutral pH conditions.     

Transglutaminase cross-linking kinetics of sodium caseinate is changed after emulsification

Enzymatic cross-linking is an important method of modifying the structure of food products to control their texture and stability. In this paper we look at the effect that adsorption to the oil–water interface of triglyceride oil-in-water emulsion has on rates of cross-linking of sodium caseinate by microbial transglutaminase. The kinetics of cross-linking has also been assessed for the individual casein proteins within the caseinate. In solution the rates were α_s2-casein > β-casein > α_s1-casein > κ-casein. This order is not as expected given the rheomorphic nature of the proteins and the number of glutamine and lysine residues in each protein. In particular, the α_s1-casein was cross-linked much more slowly than expected. When sodium caseinate was adsorbed to an emulsion the rates for all constituent caseins were decreased but the cross-linking rate for α_s1-casein was markedly reduced, indicating the most significant change in accessibility following adsorption. This knowledge will facilitate optimal production of cross-linked emulsions for use in future studies aimed at engineering emulsions with improved nutritional quality.