Linking variation in the casein fraction and salt composition to casein micelle size in milk of Dutch dairy goats.

The casein (CN) composition, salt composition and micelle size varies largely between milk samples of individual animals. In goats, the link between those casein characteristics are unknown and could provide useful insights into goat casein micelle structure. In this study, the casein- and salt composition of 42 individual Dutch goats from 17 farms was studied and linked to casein micelle size. Micelle size, proportions of individual caseins, and protein content were associated with each other. Milk with smaller casein micelles was higher in protein content, salt content, and proportion of αs1-CN, but lower in αs2-CN and ß-CN. The higher salt content in milk with small casein micelles was mainly attributed to a higher protein content, but changes in casein composition might additionally contribute to differences in mineralization. The non-sedimentable casein content in goat milk correlated with non-sedimentable fractions of ß-CN and κ-CN and was independent of micelle size. Between large and small casein micelles, goat casein micelles showed more differences in casein and salt composition than bovine micelles, indicating differences in internal structure. Nevertheless, the casein mineralization in goat milk was similar to casein mineralization in bovine milk, indicating that mineralization of casein micelles follows a general principle. These results can help to better understand how composition and micelle structure in goat milk are related to each other, which may be useful to improve processing and product properties of goat milk in the future.

Effect of plasmin on casein hydrolysis and textural properties of rennet-induced model cheeses.

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.

Can recombinant milk proteins replace those produced by animals?

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.

Variations in N-linked glycosylation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) whey protein: Intercow differences and dietary effects.

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.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin.

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.

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review.

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.

A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk.

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 by protease AprX from Pseudomonas fluorescens and plasmin.

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 ß- 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.