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Dairy Sci. Technol. 89 (2009) 3–29 Available online at:c© INRA, EDP Sciences, 2009 www.dairy-journal.orgDOI: 10.1051/dst:2008033

Review

Formation and properties of the wheyprotein/κ-casein complexes in heated skim milk –

A review

Laurence Donato1, Fanny Guyomarc’h2,3*

1 Nestlé Research Center, P.O. Box 44, 1000 Lausanne 26, Switzerland2 INRA, UMR 1253, Science et Technologie du Lait et de l’Œuf, 65 rue de St Brieuc,

35000 Rennes, France3 Agrocampus Ouest, UMR 1253, Science et Technologie du Lait et de l’Œuf, 65 rue de St Brieuc,

35000 Rennes, France

Received 26 May 2008 – Accepted 3rd November 2008

Abstract – The formation of complexes between whey proteins and κ-casein during heat treat-ment of milk dramatically affects the protein organisation in both the colloidal casein and the serumphases of milk and consequently, its technological applications. This paper reviews the compositionand building interactions of these complexes and their localisation between the casein micelle andlactoserum. The currently proposed mechanisms that lead to their formation are also presented. Thephysico-chemical properties of these complexes, in terms of structure, size and surface propertiesare described and the technological means by which these properties could be controlled are dis-cussed. Finally, the current hypotheses that explain the functional properties of these complexesin the heat-induced changes of dairy applications are reviewed, with emphasis on acid gelation ofmilk.

heat treatment / whey protein / κ-casein / complex

摘摘摘要要要 –热热热处处处理理理脱脱脱脂脂脂乳乳乳中中中乳乳乳清清清蛋蛋蛋白白白/κ-酪酪酪蛋蛋蛋白白白复复复合合合物物物的的的形形形成成成和和和特特特性性性——综综综述述述。。。由于热处理导致的乳清蛋白/κ-酪蛋白复合物的形成严重影响了酪蛋白胶体和乳清蛋白的组织状态及其应用特性。本文综述了乳成分、复合物交互作用产生的原因、以及复合物在酪蛋白胶束和乳清之间的定位。概述了乳清蛋白/κ-酪蛋白复合物形成机理;描述了复合物的结构、颗粒形状、尺寸和表面特性等物理化学性质;以及控制复合物特性的技术方法。最后,阐述了热诱导的复合物在乳品加工中的作用,特别是对酸凝胶形成的作用。

热热热处处处理理理 /乳乳乳清清清蛋蛋蛋白白白 / κ-酪酪酪蛋蛋蛋白白白 /复复复合合合物物物

Résumé – Formation et propriétés des complexes protéines sériques/caséine κ dans le laitécrémé traité thermiquement. Revue. La formation de complexes entre les protéines sériqueset la caséine κ au cours du traitement thermique du lait modifie profondément l’organisa-tion des protéines dans la phase caséine micellaire et dans le lactosérum, et par conséquentses aptitudes technologiques. Cet article fait l’état de l’art de la composition, des interactionsimpliquées dans les complexes et de leur localisation entre caséine micellaire et lactosérum.

* Corresponding author (通讯作者): [email protected]

Article published by EDP Sciences

http://www.dairy-journal.org

http://dx.doi.org/10.1051/dst:2008033

http://www.edpsciences.org

4 L. Donato, F. Guyomarc’h

Les mécanismes actuellement proposés pour décrire la formation de ces complexes sont présen-tés. Les propriétés physico-chimiques des complexes, telles que leur structure, leur taille et leurspropriétés de surface, sont décrites et les moyens technologiques permettant de moduler ces pro-priétés sont discutés. Enfin, les hypothèses actuellement proposées pour expliquer les propriétésfonctionnelles des complexes au cours des procédés de transformation du lait sont exposées, avecune attention particulière pour la gélification acide du lait.

traitement thermique / protéine sérique / caséine κ / complexe

1. INTRODUCTION

Milk proteins are commonly divided be-tween caseins and whey proteins, corre-sponding, respectively, to about 80 and20% of the total protein. Caseins are es-sentially composed of four different types,namely the κ, αs1, αs2 and β caseins.In milk biological conditions, the caseinmolecules associate to form supramolec-ular assemblies named casein micelles,which are in dynamic equilibrium with thesoluble phase of milk. Whey proteins havea globular structure and essentially includeβ-lactoglobulin, α-lactalbumin, blood pro-tein immunoglobulins, and Bovine SerumAlbumin [100, 193]. When milk is heat-treated at temperatures of ∼ 60 ◦C andabove, the whey proteins unfold, irre-versibly denature [80, 148, 149, 159] andeventually aggregate through hydrophobicbonding and thiol/disulphide exchangeswith themselves and with, essentially,κ-casein, leading to the so-called wheyprotein/κ-casein complexes [70, 95, 135,171, 176].

Heat treatment is applied in many dairyprocesses either to enhance desirable prop-erties of the products, such as texture andtaste, or to ensure its safety and shelf-life.Heat-treated milk proteins have interestingfunctional properties that are widely ap-plied in food, cosmetics or pharmacy. Inthe last few decades, extensive researchhas been dedicated to the understanding ofthe heat-induced aggregation of denaturedwhey proteins in milk or in its fractions

(whey, whey protein isolates) as well as inmodel systems of individual proteins, espe-cially β-lactoglobulin [15,20,25,38,39,43,56,61,65,85–87,92,108,109,140,141,156,165, 179, 191]. However, the simple com-parison of heat-treated whey and skim milkstrongly suggests that the presence of ca-seins, especially κ-casein, dramatically af-fects the characteristics of the heat-inducedprotein complexes in milk [21, 51, 151].

Since the publication of earlier reviewsby Hill [84] and Sawyer [167], significantadvances have been made in understand-ing the formation and properties of wheyprotein/κ-casein complexes in heated milk.The present state of the art therefore aimsat updating this knowledge. First, the loca-tion and composition of these complexeswill be described in milk, and the pro-posed pathways that may yield to theirformation will be discussed. The struc-tural and physico-chemical properties ofthe whey protein/κ-casein complexes willthen be described, taking their possible in-trinsic variations into account. The forma-tion of whey protein/κ-casein complexesaffects many dairy processes such as, e.g.,cheesemaking and recovery of the wheyprotein [69], yoghurt-making [41, 123],storage of UHT milks [129] and prepa-ration of functional ingredients. To im-prove these processes, technological strate-gies must therefore be grounded on anextensive knowledge of the properties ofthe whey protein/κ-casein complexes. Fi-nally, further possible prospects for re-search and application will be proposed.

Whey protein/κ-casein heat-induced complexes 5

2. FORMATION OF κ-CASEIN/WHEY PROTEIN COMPLEXESIN HEATED MILK

2.1. Composition and buildinginteractions of the complexes

2.1.1. Composition of the heat-induced protein complexesin milk

Early studies have long demon-strated that model mixtures of isolatedβ-lactoglobulin and κ-casein yielded co-valent complexes through thiol-disulphideexchanges on heat treatment [46, 128, 195]that may even gel at sufficient proteinconcentration [47]. In skim milk or re-constituted skim milk systems, covalentcomplex formation between κ-caseinand β-lactoglobulin has also been clearlydemonstrated [95, 134, 135, 171, 176] andevidence has also been presented thatα-lactalbumin is significantly involvedin these complexes [33, 105, 134, 135],most likely through similar mediationof β-lactoglobulin like that reported inmodel systems [48]. To a minor extent,BSA, lactoferrin [33, 50, 105, 161] andαs2-casein, containing two disulphidebridges [70, 133, 150], are also involvedin these complexes via thiol/disulphideexchanges. Immunoglobulins were sus-pected to partially associate with thecomplexes through hydrophobic interac-tions only [139, 141]. In serum complexesisolated from skim milk, cysteine-freeαs1- and β-caseins have sometimes beenreported in small proportions [96] andthought to be involved in a second type ofwhey protein/casein heat-induced complexas the pH of heat treatment is increased(see also below the effect of pH [50]).

A molar or mass ratio of 1–5 whey pro-teins to 1 κ-casein, or of 0.5–3.5 whey pro-teins to 1 κ-casein, was, respectively, foundfor the serum complexes [3,50,70,96,162]and for the micelle-bound complexes iso-lated from heated skim milk [3, 28–30,

70] (see also Sect. 2.2). Whey proteinsadded to skim milk are incorporated intothe complexes on heating [28, 29, 51, 176]while unreacted κ-casein has been foundin milk after heat treatment [51, 70, 152].These results indicate that the whey pro-teins, and especially β-lactoglobulin, arethe reaction-limiting proteins to the growthof the heat-induced complexes in milk.

2.1.2. Covalent binding throughthiol/disulphide interchanges

Only recently, mass spectroscopyhas been applied to skim milk andβ-lactoglobulin/κ-casein mixtures to tryto identify the intermolecular disulphidebonds that are formed on heat treatment.In all studies, Cysteine 160 (Cys 160)of β-lactoglobulin was implicated in theformation of intermolecular disulphidebridges, e.g. with κ-casein on the surfaceof goat’s milk casein micelles [83], withκ-casein in either the micelle or serumphase of heated skim milk [116] and withκ-casein in model protein mixtures [116],as well as with other β-lactoglobulinmolecules (i.e. involving other Cys thanintramolecular-bound 66) in heated modelprotein solutions [32, 114, 115, 178].Livney and Dalgleish [114] found thatCys 119/121 of β-lactoglobulin wasinvolved in intermolecular bonds withall the other possible cysteines of bothκ-casein and β-lactoglobulin. This re-sult was in agreement with Surrocaet al. [178] and with the role playedby β-lactoglobulin Cys 121 in initiatingcomplex formation, but was not confirmedby the other studies, which rather showedthat Cys 160 and 66 were implicatedin intermolecular disulphide bridges,possibly as a consequence of an earlyintramolecular thiol/disulphide exchangebetween Cys 121 and one of these twocysteine residues [32, 83, 115, 116]. Loweet al. [116] suggested that differences inthe disulphide-bonded products across


systems may be due to changes in theexperimental conditions and/or in thestructure of the κ-casein in isolatedor casein micelle forms. No formationof the intermolecular bond involvingβ-lactoglobulin Cys 106 could be reportedin heated milk [116], which suggestedthat it remained buried in the core ofthe protein on heating [114]. No obviouspreference between Cys 11 and 88 ofthe κ-casein was reported, suggestingthat both cysteines are randomly en-gaged in thiol/disulphide exchanges.Heat-induced disulphide bond formationin β-lactoglobulin/α-lactalbumin solu-tions have been shown to particularlyinvolve α-lactalbumin Cys 6, 61, 111and 120 [115]. Livney et al. [115] notedthat α-lactalbumin seemed a good chaincarrier for thiol/disulphide exchanges onceactivated through the initial opening ofone internal disulphide bond.

2.1.3. Hydrophobic interactions

Analysis of the heat-inducedκ-casein/whey protein complexes found inthe supernatant of heated milk in native,dissociative and reducing conditionsby chromatography or electrophoresishave shown that covalent disulphideintermolecular interactions are the ma-jor building bonds in the complexes,followed by hydrophobic interac-tions [96, 140, 141, 171]. Addition ofthiol-blocking N-ethylmaleimide (NEM)to model mixtures of β-lactoglobulin andκ-casein prior to heat treatment has, how-ever, shown that heat-induced complexformation could yet occur on the solebasis of hydrophobic interactions [128],which were proposed to come preliminaryto thiol/disulphide exchanges [76, 78].However, to the authors’ knowledge, nosuch demonstration has been made in milk;in particular, whether or not κ-casein isinvolved in such non-covalent complexesis still unclear. Blockage or absence of

β-lactoglobulin’s free thiol group in milksystems have shown that most of the heat-induced technological changes in milkthat depend on whey protein denaturationwere partly reduced when thiol/disulphideexchanges were inhibited [10, 62, 120].

2.1.4. Electrostatic and ionicinteractions

According to studies using eitherreconstituted lactose-free milk ormodel protein mixtures, the role ofionic interactions in the building ofβ-lactoglobulin/κ-casein complexes isminor and somewhat controversial, asionic screening by e.g. calcium, respec-tively promotes [176] or inhibits [45, 77]formation of complexes. Denaturedβ-lactoglobulin is able to chelate sig-nificant amounts of calcium on heattreatment, both in solution [75] and inskim milk [136]. The hypothesis that somecalcium could be incorporated in wheyprotein/κ-casein complexes of heated milkcan therefore be made. In their study,however, Parker et al. [145] observedthat the addition of sodium caseinate toskim milk, or to the serum phase of skimmilk, did not affect the formation of wheyprotein/κ-casein complexes, at least in theserum phase, although the concentrationsof soluble calcium and phosphate wereprobably decreased as a consequence oftheir uptake by the caseinate [35]. Thisresult indicates that calcium may not bea required component in the formation ofthe heat-induced (serum) complexes.

2.2. Location of the κ-casein/wheyprotein complexes in milk

Because of its consequences on therennet coagulation properties of milk,as detailed later, location of the heat-induced complexes as bound to the sur-face of the casein micelle has been ac-cepted from early studies on and largely


investigated (e.g. [152, 158, 195]). Ev-idence of heat-induced disulphide link-age between whey proteins and micel-lar κ-casein was presented by Jang andSwaisgood [95], and Dalgleish [33] usingcontrol pore glass separation. The bind-ing of heat-induced whey protein/κ-caseincomplexes further accounted for the roughsurface or appendages on the casein mi-celles of heated milk as observed by elec-tron microscopy [97]. Because of its gen-erally accepted predominant location onthe surface of the casein micelle [88],it has been proposed that κ-casein be-haved as a nucleation site for complexformation [34]. In comparison, the oc-currence of β-lactoglobulin/κ-casein com-plexes in the serum phase of heatedskim milk has received little attention,despite early reports by, e.g., Smits andvan Brouwershaven [176] or Creameret al. [31]. Separation of the serum ofheated milk using either centrifugation orrenneting, and its analysis using gel elec-trophoresis or size-exclusion chromatogra-phy later evidenced the significant forma-tion of whey protein/κ-casein complexesin the serum phase of heated milk [70,135, 171, 186]. Comparison of the micelle-bound and serum types of complexes sep-arated from milk at pH 6.7 indicated thatthe two types of complexes had compara-ble sizes and compositions [31] althoughmicelle-bound complexes contained higherproportions of κ- and αs2-casein and lesswhey proteins [70].

2.3. Proposed aggregation pathwaysfor complex formation

2.3.1. Formation of primaryaggregates

The primary formation of heat-inducedβ-lactoglobulin aggregates [43] or ofβ-lactoglobulin/α-lactalbulin aggregates,prior to their binding to κ-casein on thesurface of the casein micelle, has been

proposed in skim milk [36, 37, 53, 54, 70]as well as in model protein mixtures([79] – pathway I.A, Fig. 1). On additionof β-lactoglobulin or α-lactalbumin toskim milk prior to heating at 70–90 ◦C,Corredig and Dalgleish [30] furtherreported that the ratio between the twowhey proteins as bound to the caseinmicelles remained constant and pro-posed that it reflected the compositionof β-lactoglobulin/α-lactalbumin primaryaggregates. After separation of the serumphase of milk using precipitation of thecasein micelles with chymosin, Vasbinderet al. [186] found that the serum com-plexes only contained negligible amountsof κ-casein and further identified thesecomplexes as whey protein aggregates.However, no such whey protein specieshas ever been otherwise clearly identi-fied in milk. Observations that κ-casein,when present, readily affected heat-induced whey protein aggregation inboth milk [53, 135] and model proteinmixtures [45, 72, 128] also disagree withthe primary aggregation of whey protein,devoid of κ-casein (pathway I.B, Fig. 1).Euber and Brunner [55] immobilisednative β-lactoglobulin on the stationaryphase of a size-exclusion chromatographycolumn, heat-treated it to activate thethiol then applied enriched κ-casein ontothe column. As covalent bonding of theκ-casein onto the activated β-lactoglobulinwas possible, the authors concluded thatprimary aggregation of denatured wheyproteins was not a requisite for the forma-tion of heat-induced whey protein/κ-caseincomplexes, at least in the model systemused.

2.3.2. Partition of soluble and micelle-bound complexes: role of thecasein micelle

Various studies have shown that apositive relationship existed between theamount of denatured whey protein and


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the dissociated κ-casein both found inthe serum phase of milk after heat treat-ment [3, 5, 6, 130, 171, 173, 174]. Thisevidence suggests that formation of theheat-induced whey protein/κ-casein serumcomplexes is somewhat related to dissocia-tion of the κ-casein, and the question arisesas to whether the dissociation of κ-caseinoccurs prior to (pathway II.C, Fig. 1), orafter (pathway II.D, Fig. 1), its interactionwith denatured β-lactoglobulin (or withprimary heat-induced whey protein aggre-gates, if they exist). The answer to thisquestion is of key importance to controlthe role of the heat-induced complexes bet-ter in many technological applications ofheated milk. If the complexes indeed re-sult from either pathway I or II as describedin Figure 1, the local interactions betweenκ-casein and the whey proteins may varyand the resulting serum and micelle-boundcomplexes may have significantly differ-ent properties and, possibly, different func-tional properties during the destabilisationof skim milk, e.g., during acid gelation. Todate, the reported research fails to unequiv-ocally rule out either one pathway or theother, or to determine whether both path-ways occur concomitantly.

Results that support the initial dissocia-tion of κ-casein (pathway II.C, Fig. 1) are,first, that isolated soluble κ-casein doesform complexes with whey proteins whenheated in the absence of casein micelles inthe serum phase of milk [51] or in modelconditions [72] (see also Sect. 2.1.1). Inmilk, κ-casein can dissociate from thecasein micelle on heating, even in the ab-sence of whey proteins [10, 174]; the pres-ence of both soluble κ-casein and dena-tured whey proteins in the serum phase ofheated milk is therefore possible. Further-more, in skim milk Anema and Li [12] rea-soned that the casein micelle should re-pel the denatured whey protein when skimmilk is heated at pH values away fromthe isoelectric pH values of the micelles(∼ 4.8). The authors then suggested that

formation of a higher proportion of serumcomplexes at alkaline pH values of milkcould only be possible if κ-casein first dis-sociated off the casein micelle. On ad-dition of soluble κ-casein to skim milk,Anema [4] reported that less whey pro-teins were being attached to the surfaceof the casein micelle as a result of heat-ing, as if the added κ-casein partially de-routed the whey protein away from the ca-sein micelles. Other studies by Anema andKlostermeyer [5], Anema [4] and Anemaet al. [8] furthermore suggested that dis-sociation of the κ-casein and heat denat-uration of the denatured whey protein didnot seem to kinetically coincide. In partic-ular, κ-casein dissociated at temperaturesbelow that of denaturation of the whey pro-teins [5] and reached its maximum at anearlier stage than denaturation of the wheyproteins [4, 8]. On cooling, κ-casein thathad not interacted with denatured wheyprotein in the serum phase of milk wouldpartially re-associate with the casein mi-celles, as would the other caseins [5], thusexplaining the high correlation betweendissociation of the κ-casein and formationof serum complexes.

On the other hand, results that supportthe initial interaction of the whey proteinswith κ-casein on the surface of the ca-sein micelle (pathway II.D, Fig. 1) are,first, that micelle-bound complexes un-equivocally exist (see Sect. 2.2) and areprobably as likely to be in equilibriumwith the serum phase of milk as the othercomponents of the casein micelles (ca-seins and minerals). Donato et al. [51] andParker et al. [145] who, respectively, addedsoluble κ-casein (3 g·kg−1) or caseinate(5–10 g·kg−1) to skim milk reported thatthe added κ-casein or caseinate was not in-volved in the formation of the serum heat-induced complexes, as long as casein mi-celles were present. The authors concludedthat the denatured whey proteins prefer-ably interacted with κ-casein on the surfaceof the casein micelle, rather than with


dissociated κ-casein, to form the serumcomplexes. Despite their conflicting re-sults, both Dalgleish and Anema’s groupshave reasoned that added κ-casein in sol-uble form should show a higher diffusionrate and higher number of collisions withthe denatured whey proteins than micelle-bound κ-casein [3, 4, 51]. Quite likely,other important factors for successful wheyprotein/κ-casein interaction are the surfacecharge of the κ-casein and accessibilityof its disulphide bonds. The material usedby each group may be quite different inthese aspects, considering the purificationmethods involved. In their study, Donatoet al. [51] proposed that κ-casein in solu-ble form probably bears a high density ofcharge and has less accessible hydropho-bic sites than κ-casein, that lies in the mi-cellar porous outer layer. The casein mi-celle is indeed a porous structure throughwhich molecules such as proteins or poly-mers can easily diffuse [111, 163]. Also,the dissociation of micellar κ-casein is af-fected by the medium conditions. At pH6.7–7.1, the heat treatment of skim milkor of whey protein-free milk to whichincreased concentrations of whey proteinwere added yielded larger dissociation ofthe κ-casein than that found in wheyprotein-free milk [10, 174]. Reciprocally,blocking or removal of β-lactoglobulin’sfree thiol group prior to heat treatmentof milk was reported to prevent extensivedissociation of the κ-casein [10, 62]. Us-ing partially renneted skim milk, Renanet al. [161] furthermore showed that al-most all the denatured whey proteins in-teracted with immobilised, hydrophobicpara-κ-casein to form micelle-bound com-plexes, even though up to 66% of thetotal κ-casein had not been hydrolysedand was therefore assumed to be capa-ble of partially dissociating upon heatinginto a soluble form available for interac-tion with denatured whey proteins in theserum phase [174]. However, this interpre-tation ignores the possible implications of

the polymeric distribution of κ-casein onits dissociation rate after limited renneting.

The above section shows that conflict-ing views exist on the actual sequenceof events leading to the partition of thewhey protein/κ-casein complexes betweenthe serum and the colloidal phases of skimmilk. To date, no single experimental ap-proach has allowed conclusive evidence,most likely because of unavoidable bias on,e.g., reactivity of thiols and charge effects(when varying pH), structural changes onisolation of proteins (prior to addition) orchange in hydrophobicity of the κ-casein(on renneting). It is also possible that bothpresented sequences actually co-exist inskim milk, rendering experimental resultseven more confusing. Propositions thatwould, to some extent, restore or main-tain the original conditions of skim milkwould be, e.g., to cycle pH (to investigatereversibility of the dissociation of serumcomplexes), to compartment the milk us-ing heat-proof membranes (in complementto immobilisation of either κ-casein orβ-lactoglobulin), or to use the genetic tool-box in order to modify milk in situ (e.g.alter cysteine distribution on κ-casein orβ-lactoglobulin, or try to vary the dissocia-tion behaviour of κ-casein by changing itscharge through the sugar moiety).

2.4. Kinetic parameters of complexformation as a function ofprotein composition andtemperature

An overview of the literature onthe reaction pathway leading to wheyprotein/κ-casein complexes in heated milkand related kinetics shows that no oronly little research has taken into ac-count the dissociation of κ-casein andits interaction with denatured whey pro-tein, no matter the sequence of the twoevents (Fig. 1). Most kinetic studieswere conducted and analysed from the


viewpoint of β-lactoglobulin, whose denat-uration/aggregation kinetics were thoughtto govern that of all the whey pro-teins. Denaturation of β-lactoglobulin inmilk has essentially been reported to fol-low 1.5-order kinetics. The correspondingArrhenius plot shows a break that sepa-rates two linear domains, below and above90 ◦C [2, 15, 38, 39, 140, 141]. Both thenon-integer order and non-linearity of theArrhenius plot reflect the generally ac-cepted two-step denaturation/aggregationprocess of whey proteins. At temperaturesof 70–90 ◦C, the activation energy, Ea,ranges between 250 and 350 kJ·mol−1 de-pending on studies, and it is believed thatdenaturation is rate-limiting [42]. At 90 ◦Cor higher temperatures, de Jong [42] calcu-lates that the denaturation rate constant ofthe β-lactoglobulin dramatically increasesand that the reaction becomes almost in-stantaneous, so that aggregation becomesthe rate-limiting step. As a consequence,Ea ranges between 30 and 100 kJ·mol−1

at 90–150 ◦C. In their work, Oldfieldet al. [140–142] have proposed distinct ki-netic parameters for the formation of inter-molecular disulphide bonds, of hydropho-bic interactions and for the association ofthe whey proteins with the casein micelles,all three reactions being part of the over-all “aggregation” step mentioned in earlierstudies.

Conversely, changes in the rate ofκ-casein dissociation with heating timeor temperature have hardly been reported(see data by [4, 6, 8]). As a consequence,hardly any kinetic parameter has yet beenproposed for the dissociation of κ-casein,although its relevance to skim milk wasacknowledged [142]. Comparison withβ-lactoglobulin denaturation would, how-ever, help position dissociation of theκ-casein relatively to complex forma-tion between κ-casein and β-lactoglobulin(see Sect. 2.3.2). To the authors’ knowl-edge, the only reported attempt to intro-duce κ-casein into a kinetic description

of aggregation reactions in milk was byde Jong and van der Linden [43], who as-sumed that interaction of heat-aggregatedβ-lactoglobulin with κ-casein was oneunlikely route to termination of the heat-induced complexes. They indeed calcu-lated that the β-lactoglobulin/κ-casein in-teraction reaction involved less than 20%of the total β-lactoglobulin, a conclusionthat does not comply with recent data, asdescribed above (see Sect. 2.3.1).

3. STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES OFTHE COMPLEXES

3.1. Size and shape

In skim milk, heat treatment andthe subsequent formation of wheyprotein/κ-casein complexes on the surfaceof the casein micelle have been shownto significantly affect micelle diameterto extents that help estimate the sizerange of the micelle-bound complexes.Observations using transmission (TEM)or scanning electron microscopy (SEM)have, for instance, respectively, showedthe formation of elongated, over 100-nm-long appendages ([82, 97, 132] – Figs. 2Aand 2D) or of 20- to 100-nm-large roundappendages ([97, 144] – Fig. 2C) on thecasein micelles of heated skim milk. Inagreement with pictures of micelle-boundcomplexes, SEM observation of the serumphase of heated milk shows round-shapedserum complexes of 20–50 nm ([164]– Fig. 2G) while TEM pictures show10−30×100−200 nm noodle-like particles([31, 81, 176] – Figs. 2B, 2E and 2F).TEM noodle-like particles, whose lengthmay exceed the size of the casein micellein both their serum and micelle-boundform, were interpreted as smaller, ∼ 25-nmparticles that artefactually associatedduring sample preparation [96]. In electron


4

6

8

0Serum and micelle-bound complexes in skim milk

100 nm

100 nm

10 nm

100 nm

Micelle-bound complexes

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C D

G

F

EB

Serum complexes

Serum and micelle-bound complexes in skim milk

100 nm

100 nm

10 nm

100 nm

100 nm

10 nm

100 nm

Micelle-bound complexes

A

C D

G

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EB

Serum complexes

Figure 2. Scanning (SEM) or Transmission Electron Micrographs (TEM) of the heat-induced wheyprotein/κ-casein complexes found in skim milk. (A) TEM of skim milk heated at pH 6.5 withessentially micelle-bound complexes, (B) TEM of skim milk heated at pH 6.8 with essentially serumcomplexes [31]; (C) SEM or (D) TEM of one or two casein micelles with protruding micelle-boundcomplexes [81, 97]; (E, F) TEM of isolated serum complexes [81, 96]; (G) SEM of isolated serumcomplexes [164].

microscopy, artefacts indeed occur thatwould affect size such as, e.g., dehydra-tion, projection onto a (repulsive) carbonsurface that favours protein-protein inter-actions in TEM, coating with gold in SEM,etc. For these reasons, light scattering orimproved microscopic techniques (e.g.cryo-TEM) are preferred. Application oflight-scattering techniques on diluted milkgenerally yields smaller size values thanmicroscopic observations. In conditionswhere the formation of micelle-boundcomplexes was favoured, studies havereported that the micellar hydrodynamicdiameter (Dh) increased by up to 40 nmon heating skim milk at 90 ◦C for 10–30 min [3, 11, 13, 14, 161], suggesting

that micelle-bound complexes have aDh close to 20 nm. In the serum phaseof milk, complex sizes of 30 to 100 nmwere reported using either light scatteringor gel electrophoresis [51, 96, 164, 186],corresponding to reported molecularweight values of ∼ 3.5 × 106 [70] to2 × 107 g·mol−1 [96]. Despite possibleartefacts, Creamer et al. [31] observedthat the structure of the serum wheyprotein/κ-casein complexes varied fromglobular to elongated particles as the pHof heat treatment increased, or as moreκ-casein was dissociated and involved inthese complexes. Similarly, unpublishedresults by Guyomarc’h et al. [72] showedthat the whey protein/κ-casein complexes


formed in heated model solution turnedinto elongated structures as the wheyprotein/κ-casein ratio decreased below 2.

3.2. Physico-chemical properties ofthe complexes

3.2.1. Surface charge and solubility

Isolation of the serum complexes fromskim milk using ultracentrifugation andsize-exclusion chromatography [50, 70,171] or rennet precipitation of the caseinmicelle and gel electrophoresis [135, 186]have allowed their specific characterisa-tion. Characterisation of the micelle-boundcomplexes, however, can still only be de-duced from changes in the properties of thecasein micelles on heating.

Whey protein/κ-casein complexes asisolated from the serum of heated skimmilk were reported to bear electronegativezeta potential values of –15 to –20 mV inmilk ultrafiltration permeate at 25 ◦C atpH 6.7. These values compared with thoseof the casein micelles. The complexes pre-cipitated in the pH range 3.5–5.5 in thesame medium and temperature [74, 96].Their apparent isoelectric pH value, or pI,was found to be 4.4–4.5 and was onlyslightly lower than that of the casein mi-celles (∼ pH 4.7).

3.2.2. Surface hydrophobicity

In agreement with previous studies onheated solutions of β-lactoglobulin [160]or on ultracentrifugal fractions of heatedmilk [24, 91], serum heat-induced com-plexes bear a significantly higher surfacehydrophobicity than unheated casein mi-celles [96]. Other results strongly sug-gest that the micelle-bound complexesalso increase surface hydrophobicity of theheated casein micelle [74]. Anema et al. [8]and Mollé et al. [131] have evidenced thatκ-casein involved in the heat-induced whey

protein/κ-casein complexes was cleavedby chymosin, which accounts for the lit-tle or no difference in the final amountsof caseinomacropeptide (CMP) released inheated and unheated skim milk on rennet-ing [8, 188]. However, despite a further in-crease in their surface hydrophobicity as aresult of chymosin action, the serum heat-induced complexes may still be found insoluble form after renneting [131, 186].These results indicate that strong repulsiveinteractions, such as electrostatic repulsionby, e.g., the C-ter end of the (residual un-renneted) κ-casein, ensure stability of thepartially renneted serum complexes [161].

3.2.3. Density

To date, no value of the density ofthe heat-induced complexes is availablein the literature. Assuming that the heat-induced complexes have a spherical shape,the fact that they can be separated fromcasein micelles using ultracentrifugationindicates that their density may be some-what lower than that of the casein mi-celles (1070–1250 g·L−1 [22, 88]). In ourcentrifugal conditions, applied to heatedskim milk, i.e. milk ultrafiltrate (MUF) wasthe solvent phase (ρmuf ≈ 1025 kg·m−3,ηmuf ≈ 1.05 mPa·s at 25 ◦C), a major-ity of the serum complexes (∼ 70 nmaverage hydrodynamic diameter Dh) werepelleted at ω2R = 80 000× g; i.e., cov-ered 4.5 cm (average height of the sam-ple in the ultracentrifugal tube) in 65 min(v = 1.15 × 10−5 m·s−1). Applica-tion of Stokes’ law v = D2

h (ρcomplexes –ρmuf )ω2R/18 ηmuf yields a first, rough es-timation of ∼ 1080 kg·m−3 for the wheyprotein/κ-casein complexes. As this valueis within the range for casein micelles, ul-tracentrifugal separation of the serum com-plexes from casein micelles may thereforealso be a consequence of their smaller size.In the future, it is suggested that volu-minosity of the complexes, following the


approach recalled in [44], is calculated tofurther confirm or not the above value.

Using model mixtures of whey proteinisolate and casein material, Guyomarc’het al. [72] showed that model heat-induced complexes that involved κ-caseinor sodium caseinate were slightly lessdense than those of similar molecularweight that only contained denatured wheyproteins. If extrapolated to heated skimmilk, a low density may be linked withthe high water retention capacity of theheat-induced whey protein/κ-casein com-plexes in dairy gels [40, 73, 157].

4. TECHNOLOGICAL FACTORSCONTROLLING PROPERTIESOF THE COMPLEXES

This section focuses on how heat-treatment conditions, including milk pro-tein concentrations, pH, temperature andsalts, strongly affect the resulting com-plexes both qualitatively, through, e.g.,their composition and structure, and quan-titatively, through their final amount andrepartition between the serum and micellarphases of skim milk.

4.1. Composition of milk prior toheating

The composition of the heated systemaffects formation of the complexes andtheir resulting properties at numerous lev-els. In skim milk, increasing the total pro-tein concentration accelerates denaturationof the whey proteins [7]. The increase intotal solid content of milk conversely in-creases the dissociation rate of κ-casein [6]but retards heat denaturation of the wheyproteins [2] as a consequence of the pro-tective role played by lactose and othersoluble non-protein components againstprotein unfolding [7, 153, 154].

The initial concentrations ofβ-lactoglobulin and α-lactalbumin inskim milk [28–30,37], or of whey proteinsand κ-casein in model mixtures [72],will eventually affect the composition ofthe resulting micelle-bound and solublecomplexes, as the involved proteins seemto aggregate cooperatively. Non-dairyglobular proteins, e.g. from egg [57] orsoy [166], may also be added to milk tosuccessfully modify the composition ofserum complexes. Various studies thatused model protein mixtures also indicatedthat an increased concentration of κ-caseininhibited growth of heat-induced wheyprotein aggregates [45, 128, 135, 176].In similar model mixtures, Guyomarc’het al. [72] confirmed that heat-inducedcomplexes that involved a higher propor-tion of κ-casein had smaller molecularweight and radius. In milk/whey blendsor in micellar casein/whey protein mix-tures heated in the milk solvent phase,larger micelle-bound [21] and serumcomplexes [21, 51, 151] with a higherwhey protein/κ-casein ratio [70] areformed when more whey proteins and/orless casein are present. However, theeffect was negligible when studying wheyprotein/κ-casein serum complexes ofheated skim milks that showed slightgenetic variation in their concentrations ofκ-casein and whey protein at pH 6.7 [51].Organisation of the κ-casein in solubleform or on the surface of the micellealso seems to bear significant importancein routing the denatured whey proteinstowards formation of the micelle-boundand serum complexes ([4, 51, 74, 145] –see Sect. 2.3.2).

For unknown reasons, the presenceof caseins other than κ-casein may alsostrongly affect the heat-induced aggrega-tion of globular proteins, including wheyproteins. O’Kennedy and Mounsey [138],Zhang et al. [194], and Matsudomiet al. [127] have, for instance, reportedthat the addition of αs- and/or β-casein


to either whey or egg proteins inhibitedextensive aggregation of the globularproteins on heat treatment. It has beenproposed that the disordered caseinsmay exert a protective “chaperone-like”effect on unfolding globular proteinsthrough their phosphoserine residuesand/or hydrophobic surface, without for-mation of new heterogeneous heat-inducedcomplexes. Would the latter scheme begeneral, κ-casein may therefore behave inthe same way as αs- or β-casein but, as itcontains cysteine residues, could proceedwith intermolecular thiol/disulphideexchange [72]. Other interesting stud-ies have reported that the addition ofvarious ligands, including denaturantspecies or surface-active phospholipids,to milk or whey protein solutions in-hibited the heat-induced formation ofmodel serum [181, 182] or micelle-boundcomplexes [180].

4.2. pH

Of all technological factors, the ef-fect of pH on the formation of the heat-induced complexes and on their distribu-tion in milk has been widely investigated.From early studies on, it has been ac-cepted that the proportion of heat-inducedserum and micelle-bound complexes re-spectively increase and decrease as the pHof heat treatment is increased from about6.5 to 7.5 [31, 102, 172, 173]. Later studieshave expanded this pH range from 5.2 [29]to 10.5 [71] with similar conclusions andhave also demonstrated the sensitivity ofpH as a factor to control the proportionsof both types of complexes [3]. In skimmilk heated at 80–120 ◦C for several min-utes, 60–85% of the total whey proteinswere co-located with the casein micelleat pH 6.4–6.5, versus 20–60% at naturalpH values (6.6–6.8), 10–15% at pH 6.9 orabove [3, 50, 71, 103, 142, 171, 187] andclose to 100% at pH 6.2 [29, 103]. Con-versely, 10–20% of the total κ-casein was

found in dissociated form as a result ofheat treatment at pH 6.3–6.5, versus 30–40% at the natural pH of milk and 60–70% at pH 7.1 or above [3–5, 8, 50, 130].These results are the direct consequenceof the heat-induced reaction between de-natured whey proteins and κ-casein, lead-ing to their co-location in both the serumand the micellar phases of milk, with re-spective proportions that depend on thedissociation rate of κ-casein. This conclu-sion is in line with that of Anema [3],who reported that the amounts of non-sedimentable κ-casein and denatured wheyproteins were highly correlated through-out pH 6.5–7.1. Although conflicting viewsare still opposed on its local organisation,current models have generally describedthe internal structure of the casein micelleas an expanded, porous network of caseinmolecules held together by, essentially, hy-drophobic interactions and colloidal cal-cium phosphate (CCP [44, 89, 168, 192]).In a recent paper, Anema [4], however,reasoned that this proposition fails to ac-count for the increased dissociation of ca-seins, especially κ-casein, since hydropho-bic interaction and CCP should reinforceas temperature and/or pH are increased.As Horne [89] also pointed out, reflec-tion on the structure of the casein mi-celle should probably take better accountof other types of interactions, especiallyelectrostatic, in order to explain some ofits heat-induced changes. Adjusting thepH away from pI, for instance, increaseselectrostatic repulsion between the caseinmolecules, while increasing temperature isexpected to weaken hydrogen bonds. Aokiet al. [16] and Fox et al. [60] have also pro-posed that the heat-induced dissociation ofcaseins related to the part taken by citratein the mineral equilibrium of calcium andphosphate, as the precipitation of calciumphosphate into irreversible forms at hightemperatures would leave soluble citrateions free to dissociate the natural cementof casein micelles.


Of all whey proteins, it has beenshown that heat denaturation of theβ-lactoglobulin in model solution is af-fected by pH [159]. However, Law andLeaver [106] rather reported that thedenaturation rate of most of the whey pro-teins in skim milk changed only mod-erately throughout pH 6–9. Despite this,significant variations exist in the result-ing complexes in terms of size, shapeand composition, as a function of heat-treatment pH of skim milk. Chromato-graphic studies have, for instance, shownthat the size and whey protein/κ-casein ra-tio of the serum complexes of skim milkdecreased as the pH of heat treatment in-creased from about 6.3 to 7.3 [50, 162,164] while light-scattering analysis andprotein mass distribution in milk indicatedthat the size of the micelle-bound com-plexes also decreased with increasing pHfrom 6.3 to 7.1 [11, 187]. The increasedamount of dissociated κ-casein as pH in-creased probably accounts for the smallersize of the serum complexes (see the ef-fect of the whey protein/κ-casein ratio inSect. 4.1) but not of the micelle-boundones. Regarding the latter, Vasbinder andde Kruif [187] have proposed that the de-natured whey proteins associated with asmaller number of κ-casein sites at pH6.35–6.45 than at pH 6.55–6.70, yieldinga less homogenous coating of the caseinmicelle with larger complexes of a highwhey protein/κ-casein ratio. Although un-reacted κ-casein has been reported in thecolloidal phase of heated milk [70], thereason why there would be more of it at pH6.35–6.45 is unknown. As the pH of heattreatment increases, the serum complexesalso involve a higher proportion of caseinsother than κ-casein, possibly through for-mation of a different population of par-ticles [50] and turn into noodle-like par-ticles [31]. Changes in the pH of milkhave important consequences on, e.g., themineral balance between the colloidal andserum phases (e.g. [112]), activity of the

thiol groups (e.g. [52]), and conformation,charge distribution and net charge of theproteins [63]; all of which being likely toaffect the nature, orientation and strengthof protein-protein interactions (e.g. [99]).To date, the biochemical investigation ofhow pH may control protein-protein inter-actions and thus direct size, shape, com-position and dissociation of the wheyprotein/κ-casein complexes is lacking.

4.3. Temperature

For a constant duration of heat treatment(10, 15 or 30 min), increasing the temper-ature of heat treatment of skim milk in therange 70–95 ◦C increases denaturation ofwhey proteins [38], aggregation into heat-induced serum and micelle-bound com-plexes [186] and in fine the gelation pH andelasticity of resulting acid gels [123, 186].However, for a given degree of whey pro-tein denaturation, for which any value canbe obtained through an infinite numberof time-temperature loads, heating temper-ature in the range 70–110 ◦C does notdramatically affect the formation of theheat-induced whey protein/κ-casein com-plexes, despite the occurrence of a well-documented break at 80 ◦C or 90 ◦C inthe Arrhenius plot for the denaturationof α-lactalbumin and β-lactoglobulin, re-spectively [38]. Denaturation of the wheyprotein as measured by Dannenberg andKessler [38], and Lucey et al. [123] alsotakes aggregation into account, albeit theresulting complexes were not positively as-sayed. As methods have now been devel-oped to isolate micelle-bound or serumheat-induced whey protein/κ-casein com-plexes from skim milk (see e.g. [70, 186]),it would be interesting to dedicate fu-ture research to quantitatively exploringthe contribution of the complexes (size,number, etc.) to heat-induced changes inmilk.


Various authors have, however, indi-cated that ultra-high temperature treat-ments (≥ 120 ◦C) or long incubation times(> 30 min) yielded changes in the prop-erties of dairy gels made from heatedmilk [41, 58, 101, 123, 146], possibly as aresult of changes in the structure of thewhey protein/κ-casein complexes. Mottaret al. [132] proposed that micelle-boundcomplexes formed longer appendages inUHT-treated than in batch-heated milks,which prevented interactions between thecasein micelles on acid gel formation.At ultra-high temperatures, however, otherchanges occur in skim milk that may alsoaffect the properties of the resulting gels in-dependently of the whey protein/κ-caseincomplexes, such as casein aggregation, de-phosphorylation or proteolysis [170].

4.4. Addition of salts

Because their presence favours attrac-tive interactions between protein particlesby screening the surface, salts and espe-cially sodium and calcium cations havelong been demonstrated to play a role inthe heat-induced aggregation of whey pro-teins in model systems (see e.g. the reviewby Foegeding et al. [59]). Comparatively,little research has investigated the effectsof salts on the heat-induced formation ofwhey protein/κ-casein complexes in skimmilk. By analogy with the behaviour ofheated whey protein isolates, the fact thatthe concentration in soluble ions, hencethe ionic strength, decreases as the pH ofskim milk increases [112] may, however,be an explanation for the pH-dependentchanges in the final size, and possiblyshape, of the heat-induced serum com-plexes. On decreasing the ionic strengthof lactose-free skim milk model systems,Smits and van Brouwershaven [176] ob-served that the heat-induced associationof β-lactoglobulin with the casein mi-celle was reduced. Whether or not serum

complexes were actually formed was notindicated. The effects of calcium or sodiumcations on heat-induced protein associa-tion were similar. Similarly, the increasein soluble calcium or the decrease inphosphate concentration in skim milkreduced the amount of heat-dissociatedκ-casein [175], suggesting that less serumand more micelle-bound complexes mayhave been formed, or that less complexeswere formed altogether.

Although neither the whey proteinsnor κ-casein contain as many calcium-binding sites as αs- or β-caseins, onehas to remember that native α-lactalbumindoes contain calcium and that κ-caseinhas one phosphoseryl residue. Smits andvan Brouwershaven [176] have varied theconcentrations of calcium ions in lactose-free skim milk and reported that less cal-cium ions may induce association of somesolubilised αs- and β-caseins with the heat-induced serum complexes, albeit the pres-ence of dissociated caseins in the super-natant of Ca2+-depleted systems may onlybe a consequence of a distinct effect ofthe reduction of calcium ions in milk sys-tems on the structure and equilibrium ofthe colloidal casein fraction [64]. However,Guyomarc’h et al. [72], who comparedcomplex formation in model systems ofwhey protein isolate in the presence of ei-ther isolated κ-casein or sodium caseinate,suggested that αs- and β-casein do inter-fere with formation of model heat-inducedserum complexes (see also Sect. 2.1.1).In that respect, calcium ions may be onecause for the protective role of αs- andβ-caseins in preventing heat-induced ag-gregation and precipitation of whey proteinisolate in simulated milk systems [138].It has indeed been shown that caseins,especially αs- and β-caseins, e.g. in theircasein micelle form, bind to calcium phos-phate crystals [184] and that more calciumphosphate is being transferred to the caseinmicelles as the temperature increases [64,155]. It may therefore be that, in the


presence of caseins other than κ-casein, alarge part of the soluble calcium is routedaway from the denatured whey proteins,hence increasing electrostatic repulsionand promoting unfavourable conditions forheat aggregation, as already reported formodel systems of whey proteins in thepresence of varying ionic strength [125].

5. FUNCTIONALITY OF THECOMPLEXES IN DAIRYTECHNOLOGY

5.1. Favouring acid-induced gelationof milk

5.1.1. Increasing gel strength and pHof gelation

Because of its technological interest inyoghurt-making, the positive effect of theheat treatment of milk prior to acidifica-tion has been widely documented. Com-pared with unheated milk, heating milk attemperatures up to ∼ 100 ◦C for severalminutes before acidification increases thepH of gelation from ∼ 4.9 to ∼ 5.4 [82,120, 123] and leads to acid gels withfinal higher viscosity and firmness [41,120, 123, 146], more homogenous mi-crostructure with higher connectivity andlower porosity of the network [98, 147]and higher whey retention capacity [40,157], although protein rearrangements andsubsequent syneresis may occur [124].These heat-induced changes correlatedwell with the denaturation rate of wheyproteins [40,41,123,146] and formation ofheat-induced whey protein/κ-casein com-plexes [98,120]. These results were furthersupported by recent studies [1,9,13,49,73,110, 186, 189, 190] and have been widelyreviewed [113, 117–119, 177].

5.1.2. Proposed mechanism ofinteraction between caseinmicelles and complexes duringacidification

The respective roles of the micelle-bound and serum heat-induced wheyprotein/κ-casein complexes in acid gela-tion have been investigated using the ef-fect of the pH of heat treatment in therange 6.5 to 7.2 to vary the proportions ofserum and micelle-bound complexes (seeSect. 4.2). On increasing the pH of heattreatment to 7.1, the proportion of serumcomplexes is increased and higher valuesof the gelation pH, faster development andhigher final elasticity of the acid gels havebeen reported [9, 13, 103, 187], althoughthese responses decreased on further in-crease in the pH to 7.2 [164]. Lakemondand van Vliet [103, 104] proposed that alower proportion of micelle-bound com-plexes would reduce steric hindrance andthus favour early interaction between thecasein micelles, hence the higher pH ofgelation. As a consequence, the forminggel would be allowed more time for rear-rangement in the course of acidification,yielding stiffer gels with larger pores andstraighter strands. However, changes in thepH of heat treatment also induce changesin the composition, thiol reactivity andsize of the heat-induced complexes that areas likely to account for these effects asthe larger proportion of serum complexesat mild alkaline pH values. Formation ofmore disulphide bonds as heat-treatmentpH is increased may, for instance, also ac-count for higher rigidity of the resultingacid gel [103, 104].

In other studies, model milk systemsat pH 6.7 were reconstituted out of theultracentrifugal separation of the serumand colloidal fractions of heated and un-heated skim milk [49, 73, 98, 120]. Al-ternatively, isolated whey proteins wereadded to skim milk prior to, or after, heattreatment [137, 169]. As a matter of fact,


no clear relationship could be found whenthe proportions of in situ micelle-bound,in situ serum and/or added κ-casein-freewhey protein serum complexes were var-ied in skim milk systems at pH 6.7. Var-ious studies reported that acid gelationstarted earlier as more serum complexeswere present in the system, whether theserum complexes were formed in situ [49,73] or added to milk as whey proteincomplexes [137, 169]. Some studies re-ported the positive effect of an increasedproportion of in situ or added serum com-plexes on the final increase in elasticityand whey retention of the acid gel [73,137]. However, other research converselyreported that the occurrence of micelle-bound complexes, rather than serum ones,induced the formation of acid gels withhigher final firmness [49, 120, 169] orthat further heat treatment of unheated ca-sein micelles in the presence of eitherin situ or added serum heat-induced com-plexes yielded an additional increase inthe final firmness and homogeneity ofthe acid gel [98, 137, 169], most likelythrough the binding of the serum com-plexes to the casein micelles on heatingand hence, through the increase in the pro-portion of micelle-bound complexes. How-ever, in these reconstituted systems thefinal amount of heat-induced complexeswas not constant. In a third approach,the micelle/serum partition of the heat-induced whey protein/κ-casein complexeswas modulated in situ at pH 6.7 using par-tially renneted casein micelles [161] andno change in the gelation behaviour ofheated skim milk was reported that de-pended on that partition [74]. The factthat the above approaches to controllingchanges in the proportions of micelle-bound and serum complexes do not yieldconverging results strongly suggests thatthe micelle/serum partition of complexesis not one or the only factor to accountfor changes in the acid gelation propertiesof heated skim milk. In fact, the primary

factor for changes in the acid gelationbehaviour of heated milk may rather bethe total amount in whey protein/κ-caseincomplexes, as suggested by Vasbinderet al. [186] and Donato et al. [49]. Recentstudies furthermore indicated that the heat-induced serum complexes associate withthe surface of the casein micelle very earlyin the course of acidification, i.e. turninginto micelle-bound complexes before gela-tion starts [1, 49], although it seems thatnot all of them are eventually incorporatedin the gel [103]. Whether or not the serumcomplexes also interact with each other onacidification is unclear, but when caseinmicelles are present they do not seem toform a separate gel [1].

Incidentally, these observations rule outthe hypothesis that the heat-induced serumcomplexes would first gel separately as thepH of milk reached ∼ 5.4 (i.e. about the pIof β-lactoglobulin), followed by gelationof the whey protein-coated casein micellesat pH ∼ 5.1. This hypothesis accounted forthe reported two-phase formation of acidgels made from heated milk [73, 119, 123]but is not supported by the fact that pIof the serum complexes is 4.5 rather than5.4 [96]. In fact, a similar two-phase gelformation occurs whenever acidified milkstarts to gel at pH higher than ∼ 5.1, inde-pendently of the presence of heat-inducedcomplexes (e.g. [90, 121, 122]), and hasbeen attributed to dissolution of the col-loidal calcium phosphate [143].

Considering the higher surface hy-drophobicity and only slightly higher pIof the whey protein/κ-casein complexesas compared with those of the casein mi-celles, Famelart et al. [57], Jean et al. [96]and Guyomarc’h et al. [74] proposed thatthe early onset of gelation of heated skimmilk accounted for changes in the pH-dependent attractive/repulsive balance ofinteractions between the milk protein par-ticles on acidification.

Regarding gel formation and tex-ture development, the increased surface


hydrophobicity may help enhance connec-tions between particles on acidification,and possibly allow some thiol/disulphideexchange on acidification [189]. The im-proved texture of acid gels of heated milkcould also be attributed to the increasednumber of proteins involved in the acidnetwork [73], hence a more connected anddenser network; involving covalent disul-phide bonds in the gel structure [104,120],whereas casein acid gels are essentiallybased on low-energy interactions be-tween casein particles [185]. Coating ofthe casein micelles by denatured wheyproteins may help inhibit syneresis dueto prevented fusion of the aggregatedcasein micelles [98, 147] or to the highwater-binding capacity of the denaturedwhey proteins [40, 73, 157].

5.2. Preventing rennet-inducedgelation of milk

The heat treatment of skim milk, or itsfractions, has long been applied as a meansto recover the whey protein fraction asmicelle-bound or separated complexes inthe cheese curd, thus increasing both theprotein and total cheese yields since thedenatured whey proteins also retain wa-ter [17–19,93,94,105,107,126,196]. How-ever, coating of the casein micelles by heat-induced whey protein/κ-casein complexesaffects the rennet coagulation propertiesof the heated milk, yielding slow-forming,soft, humid and crumbly curds.

Steric hindrance of the κ-casein, whichis the substrate of chymosin, as a resultof its interaction with the denatured wheyproteins seems to inhibit the primary phaseof renneting [26, 58, 183] although closerinvestigations have only reported slight ornon-significant effects [8, 188]. In fact, in-teraction of the denatured whey proteinswith the surface of the casein micelle onheating more noticeably affects the sec-ondary phase of renneting, as it preventsmicelle fusion and therefore, cohesion and

syneresis of the rennet curd [126, 144,183, 188]. As a consequence, the heat-ing of milk as a means to increase thecheese yield is more appropriate to pro-cesses where acid coagulation is dominantover rennet action and where extensivedrainage is not required, i.e. to the manu-facture of lactic, fresh cheeses rather thansoft or hard ones [69].

5.3. Implications of the complexesin other processes

Besides the largely investigated effectsof heat treatment in either the acid or rennetcoagulation of milk, denaturation of thewhey proteins and interaction with the ca-sein fraction have also been used to in-crease yields in preparation of milk pro-tein isolates (e.g. [66]). Furthermore, heattreatment in conditions that favour the for-mation of small, serum complexes pro-duces isolates with somewhat improvedrehydration properties and viscosity, andmodified emulsifying and foaming prop-erties as compared with sodium caseinateor conventional milk protein isolate [67,68]. Bohoua-Guichard et al. [23] have alsoreported improved emulsifying propertiesof model heat-induced protein complexeswhen both β-lactoglobulin and κ-casein areinvolved in the complex, rather than eitherone protein or the other.

On the other hand, the pH-dependentdissociation of the κ-casein, in rela-tion to the formation of serum, ratherthan micelle-bound, heat-induced wheyprotein/κ-casein complexes has also beenheld responsible for the decrease in theheat stability of UHT milk as the pH ofheating increased from 6.7 to 6.9 [172–174]. At lower pH values where micelle-bound complexes are formed, it wassuggested that the complexes protected thecasein from heat precipitation. At pH val-ues higher than 7.0, repulsive electrostaticinteractions between the casein micelleswere thought to take over the adverse effect


of κ-casein depletion and to stabilise thecasein micelle. McMahon [129] also pro-posed that extensive dissociation of theheat-induced whey protein/κ-casein com-plexes, and subsequent aggregation, weresomewhat involved in the age-gelation ofUHT milk on long storage.

6. CONCLUSION:FUTURE AND PERSPECTIVES

Heat-induced milk protein complexesdo have potential interest for current andfuture research aiming at optimisationof industrial dairy processes. However,one major obstacle to their applicationis the absence of an economically sus-tainable process that would allow isola-tion or production of the heat-inducedwhey protein/κ-casein complexes on alarge scale. Only little research has beendone on the formation of these complexesunder other processes than heating (e.g.ultra-high pressure [27]). Better knowl-edge of the properties of the complexes,and of the means to control these prop-erties, is also necessary to improve theirfunctional potential as ingredients and toenlarge their possible application to food-stuff other than dairy gels. This includes,for instance, their intrinsic viscosity andsurface properties; stability and phase di-agram in various media, or flavour. In-teraction of these complexes with min-erals and/or other food components suchas polysaccharides could also be a greatasset for the future value of these com-plexes. Eventually, their potential nutri-tional properties, such as their digestibil-ity, as well as their potential applicationas nanoscopic carriers of minerals or otherligands, should be examined in comparisonwith other milk protein fractions.

Acknowledgements: The authors wish tothank D. Dalgleish (University of Guelph,Canada) and M.-H. Famelart (UMR STLO,

Inra-Agrocampus Ouest, France) for valuablesupport.

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