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Food Hydrocolloids 34 (2014) 46e53Contents lists availableFood Hydrocolloids
journal homepage: www.elsevier .com/locate/ foodhydPhysico-chemical properties of casein micelles in unheated skimmilk concentrated by osmotic stressing: Interactions and changesin the composition of the serum phase
Pulari Krishnankutty Nair, Marcela Alexander, Douglas Dalgleish, Milena Corredig*
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1a r t i c l e i n f o
Article history:Received 10 August 2012Accepted 3 January 2013
Keywords:Casein micellesConcentrated milkOsmotic stressingRheology* Corresponding author. Tel.: 1 519 824 4120; faxE-mail addresses: milena.corredig@uoguelph
0268-005X/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2013.01.001a b s t r a c t
The changes in processing functionality of concentrated milk are caused by a number of factors, amongstthe most important, the ionic equilibrium and the increase in the interactions between the casein mi-celles because of their increased volume fraction. The objective of this work was to characterize thephysico-chemical properties of casein micelles as a function of their volume fraction, by using osmoticstressing as a non-invasive method to obtain concentrated milk, in the attempt to preserve the ionicbalance during concentration. Osmotic concentration was carried out for 18 h at 4 C, using differentconcentrations of polyethylene glycol dissolved in permeate as the stressing polymer. The viscosity of theconcentrated milk could be predicted using established rheological models, when the changes occurringto the viscosity of the serum phase were taken into account. Both Eilers and Mendoza equations pre-dicted a maximum packing volume fraction of 0.8 for the casein micelles. After concentration up to 20%protein, the casein micelles did not show a change in their size upon redilution. Light scattering mea-surements carried out using diffusing wave spectroscopy without dilution suggested that casein micellesbehave as hard spheres with the characteristic of free diffusing Brownian particles up to a volumefraction of 0.3, and restricted motion at higher concentrations. Results of total and soluble calciumsuggested release of colloidal calcium phosphate from the micelles at volume fractions >0.35. Thisresearch brings new insights on the changes occurring in skim milk during concentration.
2013 Elsevier Ltd. All rights reserved.1. Introduction
The caseins are a family of calcium binding phosphoproteinsthat comprise about 80% of the protein present in milk with overallconcentration of approximately 25 g L1and the majority (Fox,2003) exist as particles of colloidal dimensions generally referredto as casein micelles. The remainder of the milk protein is wheyproteins, which are composed mostly of b-lactoglobulin, a-lactal-bumin and bovine serum albumin (Fox, 2003). The casein micellesare of great interest for colloid chemists as they represent theresponse of nature to the need to deliver a high level of calcium tothe neonate. These micelles are highly hydrated colloids (>4 g ofwater per g of protein), composed of a core of highly phosphory-lated caseins (as and b caseins) interacting with calcium phosphate,with a stabilizing layer of k-casein on the surface (Dalgleish, 2011).: 1 519 824 6631..ca, firstname.lastname@example.org
All rights reserved.The casein micelles contain calcium phosphate nanoclusters boundto the phosphoserine groups of the aS1, aS2, and b-caseins. Thiscolloidal component is in equilibrium with the calcium and phos-phate present in the soluble phase (Holt, 2002). The casein micellesare polydisperse in size (between 60 and 500 nm in diameter). Inbovine milk, the content of k-casein decreases as the micellar sizeincreases, balanced by an increase in the content of b-casein, whilethe proportions of aS1 and aS2-casein are independent of micellesize (Dalgleish, Horne, & Law, 1989). The micellar calcium phos-phate is distributed uniformly inside the micellar particles, inclusters of about 2.5 nm in diameter (Marchin, Putaux, Pignon, &Lonil, 2007).
The understanding of the supramolecular structure of nativecasein micelles and their changes during processing still holdsmany challenges. This aggregated structure is very dynamic as itresponds in various ways to environmental changes as well as tothe presence of the other components present in milk (Horne,2009). In the past years there has been an increased interest inthe study of the effects of concentration on the structure andprocessing functionality of casein micelles. With concentration, the
Delta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_given nameDelta:1_surnameDelta:1_given namemailto:email@example.com:firstname.lastname@example.org://crossmark.crossref.org/dialog/?doi=10.1016/j.foodhyd.2013.01.001&domain=pdfwww.sciencedirect.com/science/journal/0268005Xhttp://www.elsevier.com/locate/foodhydhttp://dx.doi.org/10.1016/j.foodhyd.2013.01.001http://dx.doi.org/10.1016/j.foodhyd.2013.01.001http://dx.doi.org/10.1016/j.foodhyd.2013.01.001
P. Krishnankutty Nair et al. / Food Hydrocolloids 34 (2014) 46e53 47serum compositionmay change as well as the interactions betweenthe colloidal particles, perhaps affecting the structure and functionof the casein micelles.
The viscosity of milk changes with concentration in a non-linearfashion (Snoeren, Damman, & Klok, 1982); as concentration in-creases there is a change from Newtonian to non-Newtonianbehaviour (Walstra & Jenness, 1984). The rheological properties ofconcentrated milk prepared by either heat evaporation (Ruiz &Barbosa-Cnovas, 1997; 1998), ultrafiltration (Karlsson, Ipsen,Schrader, & Ard, 2005; Pignon et al., 2004) or powder reconsti-tution (Anema, 2009; Dahbi, Alexander, Trappe, Dhont, &Schurtenberger, 2010) have been investigated. In addition to this,information is available on age gelation or renneting of ultra-filtered, concentrated milk (Bienvenue, Jimenez-Flores, & Singh,2003; Karlsson, Ipsen, & Ard, 2007) or evaporated milk,(Harwalkar, Beckett, McKellar, Emmons, & Doyle, 1983; Hwang, Lee,Park, Min, & Kwak, 2007) because of the technological implicationsin dairy processing. However, little research has been carried out onthe fundamentals of the interactions of casein micelles in concen-trated milk. When membrane filtration is employed as a means toconcentrate milk, shear effects and membrane fouling may occur,while when concentrating by evaporation, heating is applied andthe serum composition may change. These differences in process-ing history make a study on the properties of the casein micellesduring concentration quite challenging, and for this reason,knowledge of the fundamental aspects of how the physico-chemical properties of caseins evolve during concentration ofmilk is very limited.
Osmotic stress is an alternative process for concentrating milkwhich may be considered non-invasive. This technique has alreadybeen successfully employed to characterize concentrated milksystems by other groups (Bouchoux, Cayemitte, Jardin, Gsan-Guiziou, & Cabane, 2009; Bouchoux, Debbou, et al., 2009). By us-ing milk permeate (the serum phase of milk) it is possible tomaintain the original serum environment for the casein micelles.This has important implications for the equilibrium between thecolloidal and the soluble calcium, which may otherwise change,with consequences to proteineprotein interactions within thecasein micelle. In short, this allows the investigation of the funda-mental aspects of concentrated systems as a function of volumefraction of the casein micelles, without applying shear (as inmembrane filtration) or affecting the serum composition. Adetailed study of the dependence of the osmotic pressure of sodiumcaseinate solutions as a function of concentration has been pub-lished (Farrer & Lips, 1999), encompassing the dilute, semi-diluteand highly concentrated regimes. The relative viscosity of sodiumcaseinatewas shown to increase gradually with concentration up toabout 10% (w/w) and then steeply after (Farrer & Lips, 1999). Recentwork (Bouchoux, Cayemitte, et al., 2009) explored model disper-sions of phosphocaseinate over a wide range of casein concentra-tions (from 20 to 500 g L1). The results were described in terms ofthree compression regimes: dilute, transition and concentratedregimes. The same authors also studied in detail the rheologicalbehaviour of the system (Bouchoux, Debbou, et al., 2009),concluding that when casein micelles are below close-packingconditions, these protein particles behave like polydisperse hard-spheres. At concentrations close to close-packing (178 g L1), theelastic modulus increases rapidly and the system progressivelyshows a frequency independent elastic modulus.
The objective of this work was to extend the knowledge of thecolloidal properties of casein micelles in untreated skim milk as afunction of volume fraction. In addition to the determination of therheological properties, a detailed composition analysis of the serumphase was carried out as well as a study of the colloidal propertiesof the casein micelles using diffusing wave spectroscopy, a lightscattering technique which does not require dilution of the sample.This work will allow for a better understanding of the effects ofconcentration on the physico-chemical properties of the caseinmicelles, and may strengthen our current understanding of thestructure of these colloidal particles.
2. Materials and methods
2.1. Skim milk and permeate preparation
Sodium azide (0.2 g L1) was added to fres