Structural heterogeneity of milk casein micelles: a SANS contrast variation study† Antoine Bouchoux,‡ * ab Jorge Ventureira, ab Genevi ` eve G ´ esan-Guiziou, ab Fabienne Garnier-Lambrouin, ab Peng Qu, ab Coralie Pasquier, ab St ´ ephane P ´ ezennec, ab Ralf Schweins c and Bernard Cabane d We examine the internal structure of milk casein micelles using the contrast variation method in Small- Angle Neutron Scattering (SANS). Experiments were performed with casein dispersions of different origins (i.e., milk powder or fresh milk) and extended to very low q-values (9  10 4 ˚ A 1 ), thus making it possible to precisely determine the apparent gyration radius R g at each contrast. From the variation of I(q / 0) with contrast, we determine the distribution of composition of all the particles in the dispersions. As expected, most of these particles are micelles, made of casein and calcium phosphate, with a narrow distribution in compositions. These micelles always coexist with a very small fraction of fat droplets, with sizes in the range of 20–400 nm. For the dispersions prepared from fresh milk, which were purified under particularly stringent conditions, the number ratio of fat droplets to casein micelles is as low as 1 to 10 6 . In that case, we are able to subtract from the total intensity the contribution of the fat droplets and in this way obtain the contribution of the micelles only. We then analyze the variation of this contribution with contrast using the approach pioneered by H. B. Stuhrmann. We model the casein micelle as a core–shell spherical object, in which the local scattering length density is determined by the ratio of calcium phosphate nanoclusters to proteins. We find that models in which the shell has a lower concentration of calcium phosphate than the core give a better agreement than models in which the shell has a higher density than the core. Introduction The milk casein micelle is one of those natural and ordinary colloids that have always been part of our everyday life. But despite this apparent familiarity, the casein micelle still remains a mysterious and fascinating object for (bio)physicists. The recurrent question of its internal structure, which has been the subject of a myriad of papers and reviews in the last 50 years, 1–9 is surely the best illustration of this. In the present paper, we aim at giving novel information about that very question. The approach followed is essentially based on the 1974 inuential work of H. B. Stuhrmann 10 and involves precise experiments of Small-Angle Neutron Scattering (SANS) at varying contrasts. Casein micelles are globular particles of sizes mostly comprised between 50–200 nm. 11,12 They result from the asso- ciation of four types of casein (denoted as a s1 -, a s2 -, b-, and k-) together with 7–8% in dry mass of phosphate and calcium ions; 13 the latter being in the form of amorphous CaP nano- clusters. 2,6,14 Besides proteins and minerals, the casein micelle also contains a large amount of water (3–4 g per g of caseins), 15 a constituent that presumably plays a central role in micellar stability. 1 How these constituents are arranged within the micelle is a crucial question for at least two reasons: (i) it is obviously important to gain new fundamental knowledge about this biological object, thus contributing to some other fasci- nating questions such as milk secretory process and micelle assembly in lactating cells. 16,17 (ii) The performance of many dairy processes, as well as the quality of many dairy products, is intimately linked to the structural properties of the casein micelle. 18–21 Also it is decisive to identify and understand these properties if one wants to develop new applications, such as the promising use of casein micelles for drug delivery. 22 Various methods of investigation have been used for studying the structure of the casein micelle in the past decades, among which three were clearly privileged: the biological route, a INRA, UMR1253 Science et Technologie du Lait et de l'Œuf, F-35042 Rennes, France. E-mail: Antoine.Bouchoux@insa-toulouse.fr b Agrocampus Ouest, UMR1253 Science et Technologie du Lait et de l’Œuf, F-35042 Rennes, France c Institut Laue-Langevin, DS/LSS group, F-38042 Grenoble Cedex 9, France d Laboratoire CBI, CNRS UMR8231, ESPCI, 10 rue Vauquelin, F-75231 Paris Cedex 05, France † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sm01705f ‡ Present address: Laboratoire d'Ing´ enierie des Syst` emes Biologiques et des Proc´ ed´ es/LISBP, UMR5504/792 INRA-CNRS-INSA, 135 avenue de Rangueil, F-31077 Toulouse Cedex 04, France. Cite this: Soft Matter, 2015, 11, 389 Received 1st August 2014 Accepted 29th September 2014 DOI: 10.1039/c4sm01705f www.rsc.org/softmatter This journal is © The Royal Society of Chemistry 2015 Soft Matter, 2015, 11, 389–399 | 389 Soft Matter PAPER