Factors influencing casein micelle size in milk of individual cows: Genetic variants and glycosylation of k-casein Etske Bijl a , Ruben de Vries a , Hein van Valenberg a, * , Thom Huppertz b , Toon van Hooijdonk a a Dairy Science and Technology Group, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands b NIZO Food Research, P.O. Box 20, 6710 BA Ede, The Netherlands article info Article history: Received 30 May 2013 Received in revised form 28 July 2013 Accepted 1 August 2013 abstract 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 con- centration 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. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The average hydrodynamic diameter of casein micelles in bulk milk is rather constant, at around 200 nm (De Kruif & Holt, 2003); however, casein micelle size of individual milk samples, although constant during lactation, can vary markedly between the milk from cows, between 154 and 230 nm (De Kruif & Huppertz, 2012). This variation in casein micelle size can influence several techno- logical aspects of milk, such as the rennet-induced gel formation (Ekstrand, Larsson-Raznikiewicz, & Perlmann, 1980; Glantz et al., 2010). In addition, understanding the nature of differences in casein micelle size can provide more insight into casein micelle structure and the physiology of micelle formation in the mammary gland. To exploit the possibilities of differences in casein micelle size be- tween cows, it is important to understand the factors that influence micelle size. Besides the variation in average casein micelle size between cows, a large variation also exists in protein composition of the milk of individual cows (Heck et al., 2008). The sources of these varia- tions in the major milk proteins include genetic polymorphism (Bobe, Beitz, Freeman, & Lindberg, 1999; Hallen, Wedholm, Andren, & Lunden, 2008; Heck et al., 2009) and post-translational modifi- cation (PTM) (Holland, 2009). Genetic polymorphism is mainly caused by the substitution or deletion of amino acids within the polypeptide chains, which result in genetic variants of milk caseins (Caroli, Chessa, & Erhardt, 2009; Farrell et al., 2004). The complexity of caseins is further increased by PTM. PTMs of caseins include the glycosylation of k-casein (k-CN), the phosphorylation of all caseins and the disulphide bridging of a S2 -casein (a S2 -CN) and k-CN (Holland, 2009; Huppertz, 2013). The microheterogeneity of k-CN is particularly high, since it can be present as the A, B and E genetic variant in Holstein-Friesian cows, with 0e6 glycans and 1e3 phosphate groups attached to specific threonine and serine resi- dues, respectively, in its C-terminus (Holland, Deeth, & Alewood, 2006). In addition, the N-terminus of k-CN contains two cysteine residues, which engage in intermolecular disulphide-bridging (Bouguyon, Beauvallet, Huet, & Chanat, 2006; Holland, Deeth, & Alewood, 2008; Rasmussen, Hojrup, & Petersen, 1992). Glycosyla- tion of k-CN starts in the Golgi apparatus of milk epithelial cells (Vilotte, Whitelaw, Ollivier-Bousquet, & Shennan, 2003; Witsell, Casey, & Neville, 1990). These glycans can be present as mono-, di-, tri- and tetrasaccharides. The most common form is the tetra- sacharide (56%), which is composed of galactose, N-acetylga- lactosamine and two neuraminic acid groups (Saito & Itoh, 1992). To determine correlations between size and milk composition, most studies have used bulk milk (Dalgleish, Horne, & Law, 1989; * Corresponding author. Tel.: þ31 317 482 284. E-mail address: Hein.vanValenberg@wur.nl (H. van Valenberg). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.08.001 International Dairy Journal 34 (2014) 135e141