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Plasmin plays an important role in casein hydrolysis during cheese ripening, which may influence the properties of the casein network and the texture of the final product. In view of this, the relation between plasmin-induced casein hydrolysis and textural changes of cheese during ripening was investigated in this study. Four batches of model cheese with different concentrations of added plasmin (0, 0.4, 0.6 and 1.0 μL/g milk) were prepared, and were stored for 12 weeks at 16 °C. During this period, plasmin activity, casein hydrolysis, textural properties and other compositional characteristics (pH, dry matter) were determined. Our results show that the addition of plasmin had significant effect on both the degree and the pattern of proteolysis. As a result, cheeses with different plasmin content showed different textural properties. With increased plasmin concentration, Young's modulus, hardness, resilience and cohesion decreased, while brittleness increased. All textural properties showed linear relations with the degree of casein hydrolysis, and logarithmic relations with the percentage of intact casein fractions. At the beginning of ripening, only slight changes in textural properties were found, although a substantial part (40–60 %) of the casein fractions was already been broken down. When ripening progressed, ongoing proteolysis significantly weakened the protein network and consequently led to noticeable textural changes. Model cheeses became softer, more brittle and less elastic. The knowledge gained from this study provide new insights in the changes of different textural parameters of model cheese. This will help to optimize the existing products and create new ones.

The consumption of animal proteins in general, and dairy proteins in particular, is associated with sustainability and animal welfare issues. Recombinant synthesis of milk proteins is therefore receiving increasing interest, with several studies showing synthesis of milk proteins using a wide range of expression systems. Achieving a high yield and purity is essential for economic production. Besides the synthesis, also the construction of the specific structure in which milk proteins are present in animal milks, casein micelles, is needed. Looking at the current state-of-the-art, the steps to produce recombinant dairy products are technically feasible, but whether it can be implemented at low cost, with the process being environmentally friendly, remains to be seen in the coming years.

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

In this study, protein and salt distributions in the milk of 48 Holstein-Friesian cows were studied. Gross composition of milk samples and variation between individual cows was in line with expectation. However, the mineralisation of the casein fraction, expressed in mmol protein-associated calcium per 10 g casein, differed >2-fold between the milk samples. Reasons for this variation are unknown and could not be correlated to casein composition and/or genetic variants of caseins. Furthermore, notable differences (>5-fold) in non-sedimentable (100,000 × g for 60 min) casein were observed. Percentages of non-sedimentable caseins differed for κ-casein genetic variants (BB > AB > AA) and increased with increasing degree of glycosylation. Higher levels of non-sedimentable caseins with increasing degree of glycosylated κ-casein could be attributable to increased repulsion between the micelles, leading to the formation of a less cohesive sediment on centrifugation.

In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.

The white appearance of skim milk is due to strong light scattering by colloidal particles called casein micelles. Bovine casein micelles comprise expressed proteins from four casein genes together with significant fractions of the total calcium, inorganic phosphate, magnesium and citrate ions in the milk. Thus, the milk salts are partitioned between the casein micelles, where they are mostly in the form of nanoclusters of an amorphous calcium phosphate sequestered by caseins through their phosphorylated residues, with the remainder in the continuous phase. Previously, a salt partition calculation was made assuming that the nanoclusters are sequestered only by short, highly phosphorylated casein sequences, sometimes called phosphate centres. Three of the four caseins have a proportion of their phosphorylated residues in either one or two phosphate centres and these were proposed to react with the nanoclusters equally and independently. An improved model of the partition of caseins and salts in milk is described in which all the phosphorylated residues in competent caseins act together to bind to and sequester the nanoclusters. The new model has been applied to results from a recent study of variation in salt and casein composition in the milk of individual cows. Compared to the previous model, it provides better agreement with experiment of the partition of caseins between free and bound states and equally good results for the partition of milk salts. In addition, new calculations are presented for the charge on individual caseins in their bound and free states.

Destabilization of UHT milk during its shelf life is mainly promoted by the residual proteolytic activity attributed to the psychrotrophic bacterial proteases and native milk proteases. In this study, we built skim UHT milk-based model systems to which either the major bacterial protease (AprX from Pseudomonas fluorescens), or the major native milk protease (plasmin) was added, to allow a direct comparison between the destabilization of skim UHT milk by both categories of enzymes. The physical and chemical properties were studied during 6 weeks. Our results showed AprX induced compact gels when almost all the κ-casein was hydrolyzed and the degree of hydrolysis (DH) exceeded 1.3%. Plasmin induced soft gels when around 60% of both β- and αs1-casein were hydrolyzed and the DH reached 2.1%. The knowledge gained from this study may be used for developing diagnostic tests for determining the protease responsible for UHT milk destabilisation.

The caseins exhibit a high degree of heterogeneity as a result of posttranslational modifications (PTMs). Phosphorylation of the αs1-, αs2-, and β-caseins and glycosylation of κ-casein are the best-known modifications and are critical for the formation and stability of casein micelles. κ-Casein, in particular, has long been known to exhibit a high degree of variability in glycosylation, specifically in the caseinomacropeptide region, which is responsible for the stabilization of the micelle. It is somewhat surprising to see so much variability in such important structural features. In recent years, the adoption of proteomic techniques has greatly enhanced our ability to unravel heterogeneity in proteins arising from complex and variable patterns of PTMs. In this chapter, a summary of our knowledge of the PTMs of caseins in bovine milk is attempted, with a particular emphasis on κ-casein and the implications that variations in PTMs have for dairy processors. As increasing attention is also being paid to PTMs in milks from other species, either to learn more on the stability of the casein in the milk from these species or for nutritional purposes, a section on PTMs of the caseins in the milks from other species is added at the end of the chapter.

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra-high-temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat-resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf-life.

The objective of this study was to investigate the impact of natural variations in αS1-casein and β-casein composition of milk on chymosin-induced hydrolysis of these caseins in milk gels and in sodium caseinate solutions. At 50% casein degradation, 15% more of αS1-casein with eight phosphate groups was hydrolysed compared with αS1-casein with nine phosphate groups in chymosin-induced milk gels. Furthermore, in sodium caseinate solutions, >10% more β-casein A2 was degraded compared with β-casein A1 and B at 50% casein degradation. Proteolysis by chymosin was not impacted by natural variation in the αS1-casein/β-casein ratio. Natural variation in αS1-casein/β-casein ratio did not impact upon firmness and gel strength in milk gels. Overall, comparison of sodium caseinate solutions to milk gels showed that differences in either phosphorylation of αS1-casein or amino acid composition of β-casein caused significant differences in degradation by chymosin, possibly due to changes in physical conformation of caseins.

Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the internal structure of the casein micelles. α(S1)-Casein (α(s1)-CN) is of special interest because it constitutes up to 40% of the total casein fraction in milk, and it has 2 common phosphorylation states, with 8 (α(S1)-CN-8P) and 9 (α(S1)-CN-9P) phosphorylated serine residues. Factors affecting this variation in the degree of phosphorylation are not currently known. The objective of this research was to determine the genetic background of α(S1)-CN-8P and α(S1)-CN-9P. The genetic and phenotypic correlation between α(S1)-CN-8P and α(S1)-CN-9P was low (0.18 and 0.19, respectively). This low genetic correlation suggests a different genetic background. These differences were further investigated by means of a genome-wide association study, which showed that both α(S1)-CN-8P and α(S1)-CN-9P were affected by a region on Bos taurus autosome (BTA) 6, but only α(S1)-CN-8P was affected by a region on BTA11 that contains the gene that encodes for β-lactoglobulin (β-LG), and only α(S1)-CN-9P was affected by a region on BTA14 that contains the diacylglycerol acyltransferase 1 (DGAT1) gene. Estimated effects of β-LG protein genotypes showed that only α(S1)-CN-8P was associated with the β-LG A/B polymorphism (g.1772G>A and g.3054C>T); the AA genotype of β-LG was associated with a lower concentration of α(S1)-CN-8P (-0.32% wt/wt) than the BB genotype (+0.41% wt/wt). Estimated effects of DGAT1 K232A genotypes showed that only α(S1)-CN-9P was associated with the DGAT1 gene polymorphism; DGAT1 AA genotype was associated with a higher α(S1)-CN-9P concentration (+0.53% wt/wt) than the DGAT1 KK genotype (-0.44% wt/wt). The results give insight in phosphorylation of α(S1)-CN-8P and α(S1)-CN-9P, which seem to be regulated by a different set of genes.

Caseins are phosphoproteins and constitute about 80% of the protein in milk. Caseins exhibit a high degree of heterogeneity as a result of post-translational modifications (PTMs). Phosphorylation of the α- and β-caseins and glycosylation of κ-casein are the best-known modifications and are critical for the formation and stability of casein micelles. κ-Casein has long been known to exhibit a high degree of variability in glycosylation. This chapter summarizes current understanding on PTMs of the αs1-, αs2-, β-, and κ-caseins. Caseins are assembled in a colloidal complex with calcium phosphate and small amounts of other minerals. The casein micelle structure is critical in determining the physical properties of milk and for the provision of amino acids, calcium, and phosphorus for infant nutrition. A critical factor in micelle formation and stability is the presence PTMs such as phosphorylation, glycosylation, and perhaps disulfide bond formation that occur in the endoplasmic reticulum and/or Golgi complex after synthesis of the polypeptide chain. In recent years, the development of proteomic technologies has greatly enhanced our ability to analyze milk proteins, particularly with respect to PTMs. Two-dimensional electrophoresis (2-DE) provides a high-resolution methodology for displaying the heterogeneity of the major milk proteins. Advances in mass spectrometry (MS) have enhanced our ability to analyze the proteins arrayed on 2-D gels. Therefore, not only is it possible to resolve many proteins and their isoforms but it is also possible to characterize them, particularly with respect to the many PTMs that affect their electrophoretic mobility. Furthermore, the chapter includes an extended discussion on the functional significance of κ-casein heterogeneity.

The average casein micelle size varies widely between milk samples of individual cows. The factors that cause this variation in size are not known but could provide more insight into casein micelle structure and into the physiology of casein micelle formation. The objective of this research was therefore to determine factors that influence average casein micelle size in milk from individual cows. Average casein micelle size of milk samples was associated with the A and B genetic variants of k-casein, and differences in concentration of glycosylated k-casein as a fraction of total milk protein. Milk samples with a low average casein micelle size were associated with the B variant of k-casein and a higher relative concentration of glycosylated k-casein, compared with milk samples with a high average casein micelle size. Differences observed may be attributed to the effect of glycosylated k-casein groups on casein micelle formation in the mammary gland.

The protein and fat content of Dutch bulk milk has been monitored since the 1950s and has increased considerably, by 11 and 20%, respectively, whereas milk yield has more than doubled. The change in protein and fat content of milk is advantageous for the dairy industry, as these are the 2 most economically valuable constituents of milk. Increases in protein and fat content of milk have allowed increases in the yield of various products such as cheese and butter. However, for cheese and other applications where casein micelles play a crucial role in structure and stability, it is not only casein content, but also the properties of the casein micelles that determine processability. Of particular importance herein is the salt partition in milk, but it is unknown whether increased protein content has affected the milk salts and their distribution between casein micelles and milk serum. It was, therefore, the objective of this research to determine the salt composition and protein content for individual cow milk and bulk milk over a period of 1 yr and to compare these data to results obtained during the 1930s, 1950s, and 1960s in the last century. Calcium, magnesium, sodium, potassium, and phosphorus content were determined by inductively coupled plasma atomic emission spectrometry and inorganic phosphate, citrate, chloride, and sulfate content by anion-exchange chromatography in bulk milk and milk ultracentrifugate. In addition, ionic calcium and ionic magnesium concentration were determined by the Donnan membrane technique. We concluded that historical increase in milk yield and protein content in milk have resulted in correlated changes in casein content and the micellar salt fraction of milk. In addition, the essential nutrients, calcium, magnesium, and phosphorus in milk have increased the past 75 yr; therefore, the nutritional value of milk has improved. Key words: milk protein , casein , calcium phosphate , magnesium

The European Physical Journal Special Topics, 222 (1), ISSN:1951-6355, ISSN:1951-6401