ntrate (WPC), whey protein isolate and skim milk has been explored by

of processing milk proteins. This review examines the differences in the denaturation pathways that give rise to different final products.Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. -lactoglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3.1. Structure of -LG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2. Effect of heat on -LG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.3. Effect of pressure on -LG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.4. Comparison of heat and pressure treatments of -LG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.5. Effects of ligands on heat- or pressure-induced changes in -LG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. -lactalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.1. Structure of -LA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9many groups using a wide range of techniques. In general terms, heat treatment and pressure treatment have similar effects: denaturing andaggregating the whey proteins and diminishing the number of viable microorganisms. However, there are significant differences between theeffects of the two treatments on protein unfolding and the subsequent thiol-catalysed disulfide-bond interchanges that lead to different structuresand product characteristics. Application of a range of techniques has given insight into the subtle differences between the pathways from nativeproteins to the final product mix. This review covers some of the techniques used and their strengths, and the probable pathways from nativeprotein to the final products. -LG is one of the most pressure-sensitive proteins and -lactalbumin (-LA) is one of the most pressure resistant. Ina heated WPC system, bovine serum albumin is very sensitive and -LG is more resistant. In a heated milk system, -LG reacts with -casein (-CN) and not with S2-CN, but, in pressure-treated milk, -LG forms adducts with either -CN or S2-CN. In both treatments, the role of -LG iscentral to the ongoing reactions, involving -LA and -CN in heated systems but involving -CN, S2-CN and -LA in pressurized systems. 2006 Elsevier Ltd. All rights reserved.

Keywords: High pressure; Heat; Milk proteins

Industrial relevance: High hydrostatic pressure (HHP) processing, as opposed to heat treatment, has received much attention recently as a meansPressure treatment of -lactoglobulin (-LG), whey protein conceReview

Interactions of milk proteins during heat and highhydrostatic pressure treatments A Review

T. Considine a,b,, H.A. Patel a,b,c, S.G. Anema a,b, H. Singh b, L.K. Creamer a,b

a Fonterra Research Centre, Private Bag 11 029, Palmerston North, New Zealandb Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand

c Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11 222, Palmerston North, New Zealand

Received 30 May 2006; accepted 15 August 2006

Abstract

Innovative Food Science and Emerging Technologies 8 (2007) 123www.elsevier.com/locate/ifset4.2. Effect of heat on -LA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.3. Effect of pressure on -LA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Corresponding author. Fonterra Research Centre, Private Bag 11 029, Palmerston North, New Zealand. Tel.: +64 6 350 4600x7561; fax: +64 6 356 1476.E-mail address: Therese.Considine@fonterra.com (T. Considine).

1466-8564/$ - see front matter 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.ifset.2006.08.003

5. Bovine serum albumin. . . . . . . . . . . . . . . . . . . . . . .5.1. Structure of BSA . . . . . . . . . . . . . . . . . . . . . .5.2. Effect of heat on BSA . . . . . . . . . . . . . . . . . . .5.3. Effect of pressure on BSA . . . . . . . . . . . . . . . . .

6. Mixtures of whey proteins. . . . . . . . . . . . . . . . . . . . .6.1. -LA and -LG . . . . . . . . . . . . . . . . . . . . . .6.2. BSA and -LG. . . . . . . . . . . . . . . . . . . . . . .6.3. BSA and -LA. . . . . . . . . . . . . . . . . . . . . . .6.4. Whey protein concentrate . . . . . . . . . . . . . . . . .

7. Milk systems . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1. Appearance of milk . . . . . . . . . . . . . . . . . . . .7.2. Particle size changes . . . . . . . . . . . . . . . . . . . .7.3. Dissociation of casein from the casein micelles . . . . . .7.4. Whey protein denaturation . . . . . . . . . . . . . . . . .7.5. Whey protein interactions with the casein micelles . . . .7.6. Interactions in whole milk . . . . . . . . . . . . . . . . .7.7. Effects in acid gel systems . . . . . . . . . . . . . . . . .7.8. Effects on the renneting properties of milk . . . . . . . .

8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

neutral pH foods at room temperature, the various microorgan-isms have to be reduced to negligible numbers to give storage

the traditional heat processing of foods, which often may havenegative side effects such as heat-induced changes of flavour

2 T. Considine et al. / Innovative Food Science aand colour (Balny & Masson, 1993; Datta & Deeth, 1999).However, heat treatments of dairy products have remainedcommercially dominant.

High hydrostatic pressure (HHP) is known to induce proteindenaturation by altering the delicate equilibrium between theinteractions that stabilize the folded conformation of nativeproteins (Masson, 1992). Protein compressibility is governed bythe compression of the internal cavities, which depends on theinternal packing. Application of HHP induces either local orglobal changes in protein structure and finally may lead todenaturation (Mozhaev, Heremans, Frank, Masson, & Balny,1996). The hydrophobic core of a protein is less stable at highpressure, because of a larger loss of partial molar volume uponits local unfolding. The preferential melting of the hydrophobiccore region with a large loss of partial molar volume is likely tobe a consequence of its proximity to the large water-accessiblecavities. In general, the loss of cavity volumes represents a majorfactor contributing to the partial molar volume change upon theunfolding of a protein (Frye & Royer, 1998; Weber &Drickamer, 1983). Pressures of 100200 MPa are sufficient tocause dissociation of oligomeric proteins and of multi-protein

Fig. 1. Size exclusion chromatography combined with multi-angle laser lightscattering (Superdex 75): estimated molar mass profile of -lactoglobulinaggregates after a heat treatment of 5 min at 78.5 C (). Elution profiles for UVabsorption and light scattering at 90 C included. Arrows indicate -lactoglobulin aggregates (1), tetramers (2), trimers (3), dimers (4), native -1. Introduction

Preservation of foods in a temperate climate to capture thesummer surplus for the winter deficit was the original purposeof seeking for more preservation techniques. Currently, theability to move food long distances has given us the ability toaccess out-of-season foods. Nevertheless, it is still desirable tohave ingredients or foodstuffs that are stable over time. Forlactoglobulin (5) and non-native monomers (6). (Reproduced with thepermission of Schokker et al. (1999). Copyright 1999 International DairyJournal, Elsevier).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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stability. Initially, heat pasteurization was very valuable and thiswas complemented by sterilized tin-plated steel cans ofconcentrated milk, meat, fruit and vegetables. About a centuryago, a study performed at the West Virginia University Agri-cultural Experiment Station indicated that pressure affectedmilk and other food materials, destroying microorganisms anddenaturing proteins (Hite, 1899; Hite, Giddings, & Weakly,1914). Pressure treatment of foods is an appealing alternative to

nd Emerging Technologies 8 (2007) 123complexes (Silva & Weber, 1993), whereas small monomericproteins are usually denatured between 400 and 800 MPa(Heremans, 1982). Localized unfolding and subunit dissociation

(Havea, Singh, Creamer, & Campanella, 1998; Manderson,Hardman, & Creamer, 1998; Patel, Singh, Anema, & Creamer,2004). The sodium dodecyl sulfate (SDS)-PAGE technique isused in many protein and proteomic studies to determine themolecular weight or size of proteins or protein subunits, if thedisulfide bonds of a protein aggregate are chemically reducedprior to PAGE analysis. The potentially very high resolution ofthe method and the wide range of molecular sizes, from 5 to150 kDa, on a single gel have led to its widespread use. The gelsthat are used have pore sizes that are governed by the method ofgel formation and most proteins have a mobility in an SDS-containing buffer that is correlated to the molecular mass of theprotein (Andrews, 1986). Traditionally, casein proteins areanalysed in 4 or 5M urea solutions and often the disulfide bondsare reduced using 2-mercaptoethanol or other reducing agentsprior to analysis. Whey proteins are usually analysed withoutreduction and with neither SDS nor urea in the system the so-called alkalineor nativePAGE system.

Protein aggregation can be studied using a combination oftwo different types of electrophoresis, to give a two-dimen-sional (2D) pattern that can be related back to known mobilitiesin pH 8 buffer containing 5 M urea or 3.5 mM SDS (Fig. 2).This technique was developed by Havea et al. (1998) andManderson et al. (1998) and was shown to be useful forunderstanding heat-induced changes in complex protein systemssuch as whey proteins or mixtures of -lactoglobulin (-LG), -

3ce and Emerging Technologies 8 (2007) 123Table 1Techniques commonly used for analysing heat and pressure induced changes inmilk proteins

Technique Reference

ScatteringLight scattering, small-angle X-rayscattering (SAXS)

Holt, de Kruif, Tuinier, & Timmins,2003; Pessen, Kumosinski, & Farrell,1989

Size exclusion chromatography (SEC) Schokker et al., 1999 (Fig. 1)Polyacrylamide gel electrophoresis(PAGE)

Andrews, 1986; Havea et al., 2001,1998; Manderson, 1998; Mandersonet al., 1998; Patel Singh, Anema, &Creamer, 2004

Secondary structure determinationX-ray crystallography, Far-UV circulardichroism (CD), Fourier transforminfrared (FTIR) spectroscopy,Fourier transform of Raman spectra,H/D exchange using nuclearmagnetic resonance (NMR) methods

Belloque et al., 2000; Edwards, Jameson,Palmano, & Creamer, 2002; Kelly &Price, 2000; Ngarize, Adams, & Howell,2004; Tanaka & Kunugi, 1996; Ngarize,Herman, Adams, & Howell, 2004;Panick et al., 1999; Subirade, Loupil,Allain, & Paquin, 1998

Tertiary structure determinationIntrinsic fluorescence, Extrinsicfluorescence, Induced circulardichroism (CD)

Collini et al., 2003; Dufour, Hui BonHoa, & Haertle, 1994; Heremans et al.,1997; Kontopidis, Holt, & Sawyer,2004; Masson, 1992; Narayan &Berliner, 1997, 1998; Pearce, 1975

T. Considine et al. / Innovative Food Sciencan result in the unmasking of buried groups that are able to pairwith other newly exposed groups and thus lead to aggregation(Masson, 1992).

The exposure of buried hydrophobic residues to water andtheir resulting hydration, which occurs during unfolding, causesa decrease in the partial molar volume of a protein. Hydration ofexposed hydrophobic groups plays a decisive role in thethermodynamic stability of a protein and can therefore be re-garded as an important factor for understanding protein un-folding, dynamics, stability and functions.

In this paper, we briefly catalogue the features of some of themany techniques, including less common types of polyacryl-amide gel electrophoresis (PAGE), used to assess the changes inthe protein structures as a consequence of heat and pressuretreatments. This is followed by descriptions of the effects of heatand pressure treatment of the individual whey proteins, wheyprotein mixtures and milk.

2. Techniques

There are a number of techniques that are suitable for studyingthe system at elevated temperatures and pressures and others thatare restricted to room temperature and pressure. These techniquescan be used to compare the properties of the protein mixturesbefore and after heat or pressure treatment. Table 1 aims tosummarize the most common techniques. The only technique thatwill be described in detail in this review is PAGE.

Using PAGEmethods, it is possible to differentiate native andnon-native proteins and hydrophobically bonded aggregates andto identify the components of disulfide-bonded aggregatesFig. 2. Diagram showing the preparation and running of a sample of heated milk.The milk sample is dispersed in sodium dodecyl sulfate (SDS) buffer, mixedwith acrylamide solution and set in two sample wells of a standard SDS-polyacrylamide gel electrophoresis (PAGE) gel to give two sample gels. Afterelectrophoresis of the samples, one of the gel columns is stained and the other isimmersed in a solution to reduce the covalent disulfide bonds. After washing,the treated gel is clamped into the electrophoresis cell and the resolving gel is setunder it, and then the stacking gel is set around it. All of the buffers contain SDS.After electrophoresis and staining, the previously stained gel column is placedabove and parallel to the column that had been set into the gel. This set-up is thenscanned and photographed. This procedure allows the relationship between the

stained spots/bands of the disulfide-bonded proteins and the reduced proteins tobe established. (Reproduced with the permission of Patel et al. (2004).Copyright 2004 Food New Zealand, Scientific Supplement, June 2004).

lactalbumin (-LA) and bovine serum albumin (BSA) (Havea,Singh, & Creamer, 2001). In the case of SDS-PAGE and thenreduced SDS-PAGE (Fig. 3), specific aggregation via disulfide-bond interchange can be examined without the complication ofchanged hydrophobic interactions. This 2D PAGE techniqueseparates the initial mixture of the proteins, using SDS-PAGE,with all the native and process-induced disulfide bonds intact.Once these proteins and aggregates are separated on a gel strip, itis treated with a disulfide-bond reducing agent, such as 2-

mercaptoethanol. This strip is then used as the sample source forthe second dimension PAGE analysis, which separates theproteins as reduced SDS-monomeric protein species, thusallowing complete identification of the various disulfide-bondedaggregates.

Fig. 3 shows the difference between untreated, heat-treatedand pressure-treated -LG. Untreated -LG shows no aggre-gates (Fig. 3A), whereas heating (Fig. 3B) or pressurizing (Fig.3C) -LG leads to the production of dimers, trimers and largeraggregates. 2D analysis of heat-treated (75 C for 8 min) -LGproduced a series of spots corresponding to monomer, dimer,

4 T. Considine et al. / Innovative Food Science and Emerging Technologies 8 (2007) 123trimer and larger molecular aggregates before 2-mercaptoethanoltreatment. A number of faint spots had mobilities correspondingto -LG dimer after reduction, indicating that, after heat treat-ment, a proportion of -LG existed as dimers and trimers linkedby non-disulfide covalent bonds. However, there is a possibilitythat dimerization of the monomeric protein may have occurredthrough oxidation of thiol groups during sample manipulation orelectrophoresis (Havea et al., 2001).

3. -lactoglobulin

3.1. Structure of -LG

The first medium resolution three-dimensional structure of-LG at 2.8 was reported by Papiz et al. (1986). The structurehas subsequently been refined considerably by Brownlow et al.(1997) and Jayat et al. (2004), who reported resolution to 1.8 and 1.95 respectively. Native -LG (Fig. 4) has nine strandsthat are folded into two -sheets: sheet 1 contains strands B, Cand D, and part of strand A (A1); sheet 2 contains strands D, E,F, G and H, part of strand A (A2) and strand I. One side of sheet1 is hydrophobic and the other side is hydrophilic. Sheet 2 isalso hydrophobic on one side and faces the hydrophobic side ofsheet 1, thus creating a very hydrophobic cavity, which isnevertheless filled with water. There is also another hydropho-bic region on the side of sheet 2, where the three-turn helix liesabove it and along strands F, G and H. -LG has two disulfidebonds and one free Cys (CysH). The -helix covers the CysHresidue, providing it remains packed against the exterior of thecalyx. Subjecting -LG to different denaturing conditions, e.g.heat, pressure, urea etc., exposes these Cys residues and initiatesa chain of reactions involving SH/SS exchange (Fig. 5).

Fig. 3. Two-dimensional electrophoretic patterns of (A) untreated-lactoglobulin (-LG) (1.5 mg/mL), (B) a heat-treated (75 C for 8 min) solution of -LG in permeateprepared from a solution of whey protein concentrate powder in water (100 g/kg) and(C) a -LG solution (1.5 mg/mL) pressure treated at 600 MPa for 30 min. Firstdimension, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)without treatment with 2-mercaptoethanol; second dimension, SDS-PAGE ofsamples within a gel strip that was treated with 2-mercaptoethanol. At the top ofeach gel, there is a stained gel strip showing the protein bands from the first dimensionseparation running from left to right. Immediately below this strip is a second strip,which had contained the same protein bands thatwere used as samples for the second-dimension separation. On the left-hand side, a portion of the corresponding sample

was run from top to bottom, to help to identify the protein bands. (Centre photographreproduced with the permission of Havea et al. (1998). Copyright 1998 Journal ofDairy Research).

Schokker, Singh, Pinder, Norris and Creamer (1999) usedSEC to monitor the heat-induced aggregation of -LG AB atneutral pH (Fig. 1). These authors showed that aggregationoccurs via many intermediates, and mainly via disulfidebonding and to a much lesser extent via non-covalentinteraction. Thus, the protein can be considered to be in oneof two states, namely the native fold or one of a range of non-native folds and containing non-native disulfide bonds. Thekinetics (loss of native protein) do not need to be an integralorder. Sava, Van der Plancken, Claeys and Hendrickx (2005)

the relative positions of the five Cys residues and the bound palmitate (Wu, Perez,are also labelled. The diagram was drawn from the PDB file 1GXA using RASMOL

5ce and Emerging Technologies 8 (2007) 1233.2. Effect of heat on -LG

Heat treatment of -LG at neutral pH causes the dimericnative protein to dissociate, partially unfold, denature and ag-gregate; the rates and pathways are dependent on the proteinconcentration, pH, temperature and other factors. Two majoraggregation features, or possibly mechanisms, are related tohydrophobic association and disulfide-bond interchange reac-tions (Havea, Carr, & Creamer, 2004).

At temperatures up to about 65 C, the overall tertiarystructure of -LG in neutral solution changes reversibly. At thistemperature, some irreversible reactions involving the disulfidebonds that are important for maintaining the native structuretake place. The thermal energy available at higher temperaturesallows the helix to lift away from the sheet to a small extent andallows this side chain to interact with the neighbouring disulfidebond within the confines of a hydrophobic cage (Creamer, et al.,2004; Lowe et al., 2004) (Fig. 5A). Increased exposure ofpreviously buried hydrophobic groups and the free Cys121initiates aggregation via thiol-disulfide exchange that can then

Fig. 4. Diagram of the three-dimensional structure of -lactoglobulin that showsPuyol, & Sawyer, 1999). The helix and the strands that constitute sheets 1 and 2Ver 2.6.

T. Considine et al. / Innovative Food Scienresult in further aggregation via hydrophobic association.The concept is that heat treatment of -LG allows the thiol of

Cys121 to react with the Cys106Cys119 disulfide bond to givea free Cys119 (reaction 1) and Cys121Cys106 (Fig. 5A). Thefree Cys119 (reaction 2) reacts with another -LG disulfidebond, Cys66Cys160, leading to the formation of Cys119Cys66 and a free Cys160 (Fig. 5B). This free Cys160 has thepotential to react with other proteins if present (e.g. BSA or -casein (-CN)). Creamer et al. (2004) have recently reportedthat approximately 35% of Cys160, which is disulfide bondedto Cys66 in the native protein, is present with a free thiol afterheat treatment. This possible pathway is supported by manystudies (Croguennec, Bouhallab, Molle, O'Kennedy, & Mehra,2003; Croguennec, Molle, Mehra & Bouhallab, 2004; Livney &Dalgleish, 2004; Surroca, Haverkamp, & Heck, 2002). Anotherpossibility is the interaction of two -LG molecules (Fig. 5C)through a disulfide-bond interchange reaction, as outlined inFig. 5A, to form a disulfide-bonded dimer. Exactly which di-sulfide bonds are involved is uncertain, but Cys66 and Cys160are less likely to be involved.Fig. 5. Thiol-disulfide interchange in -lactoglobulin (-LG). A. The intermo-lecular interchange that precedes all other interchange reactions (Croguennec et al.,2003; Creamer et al., 2004). B. The possible second interchange that involves theCys66Cys160 -LG disulfide bond, which is spatially distant in native -LG.C. A plausible interchange involving two -LG molecules to form a disulfide-bonded dimer without involving the interchange shown in B.

. T,

ce arecently investigated the kinetics of heat-induced structuralchanges of -LG. The kinetics were monitored by following thesolubility, turbidity, surface hydrophobicity and sulfhydrylgroup of -LG when heated between 67.5 and 82.5 C at pH7.5 for various times. A first-order fractional conversion modelwas applied to describe the heat-induced changes in the surfacehydrophobicity of -LG and the decrease in the slow reactingSH groups. These showed a temperature dependence of the kvalues (rate constants).

In contrast, the kinetics describing the heat-induced changes

Fig. 6. Proposed model of the heat and pressure denaturation of -lactoglobulin Bpress). Copyright 2006 Food Chemistry).

6 T. Considine et al. / Innovative Food Scienin surface SH groups of -LG solutions showed a break in theArrhenius plot at 80 C. The authors explained this as arisingfrom the complexity of the irreversible thermal denaturationprocess of -LG, which involves a number of successive re-action steps. The model of Roefs and De Kruif (1994) wassuggested as the possible mode for thermal behaviour of -LGin buffer solutions.

Sava et al. (2005) suggested different rate-determining stepsinvolving the participation of two consecutive reactions in thedenaturation process. At lower temperatures (67.578 C), therate-determining step is unfolding of the molecules, whereas, athigher temperatures (7882.5 C), the aggregation processinvolving unfolded molecules becomes rate determining.

Our group recently proposed calling these steps Stage I T(native protein) and Stage II T (heat-denatured protein), asdefined by the absence of native disulfide bonding after the heattreatment (Fig. 6; Considine, Patel, Singh, & Creamer, in press).For simplicity, Stage I T signifies at least 90% native -LGstructure whereas the -LG in Stage II T may include up to 20%native protein.

3.3. Effect of pressure on -LG

Of all the major whey proteins, -LG appears to be the mostsensitive to hydrostatic pressure (Patel, Singh, Havea, Con-sidine, & Creamer, 2005; Stapelfeldt, Petersen, Kristiansen,Qvist, & Skibsted, 1996); consequently, the effect of pressuretreatment on -LG has been explored by many authors, eitherduring the pressure process or after pressure release. Gaininginsight into the mechanisms that occur during pressurization orafter pressure release will help to unravel the pressure de-naturation of -LG in terms of unfolding and aggregation,which has been explored extensively for heat-treated -LG(Singh & Havea, 2003).

Belloque, Lpez-Fandio and Smith (2000) measured H-D1

temperature; P, pressure. (Reproduced with the permission of Considine et al. (in

nd Emerging Technologies 8 (2007) 123exchange using H NMR to determine the denaturation of -LGAB after a defined high pressure treatment. Pressures of100 MPa resulted in some unfolding of -LG but allowed thecore to remain structured. Increasing the pressure between 100and 200 MPa initiated structural changes (Belloque et al., 2000;Mller, Stapelfeldt & Skibsted, 1998). -Sheets formed byFGH strands were the strongest portion of -LG afterpressurization at 200 MPa for 5 min (Belloque et al., 2000),thus indicating that they are the last part to unfold duringpressure processing. Subsequent increases in pressure (300 and400 MPa) showed high flexibility of the entire structure of -LG. The exposure of the amide of the indole group of Trp19 andthe backbone amides from several residues belonging to FGHstrands upon pressurization demonstrated that the increasedflexibility involves the entire structure of -LG, including thestrongest region. Pressure treatment of the individual variantsindicated that the structure of the core of -LG A becomesflexible more rapidly than that of -LG B (Belloque et al.,2000).

Kuwata et al. (2001) used 1H/15N 2D NMR to investigateconformer fluctuations and folding/unfolding equilibria ofpressure-treated -LG at pH 2. It was suggested that the twosides of the barrel fluctuate independently of each other, withthe non-core (strands BE, Fig. 4) fluctuating relatively easilyat low pressure (0.1100 MPa) and producing an intermediate

ce aconformer I1. However, for I1 to denature completely, a pressureabove 200 MPa is required. In contrast, the core side is morestable at low pressure, but denatures easily at medium pressure( 150 MPa), producing another conformer I2 (strands FH,Fig. 4). This instability at high pressure is due to a single largeloss of partial molar volume upon local unfolding and is likelyto be a consequence of the proximity of the hydrophobic core tothe large water-accessible cavities. At pressures greater than200 MPa, I1 and I2 are likely to unfold even further and producean unfolded conformer. Releasing the pressure restores thenative structure of -LG, because no thiol-disulfide interchangewill occur at pH 2.

Subirade, Loupil, Allain and Paquin (1998), using FTIRspectroscopy, monitored the conformation of -LG after pressuretreatment. Pressures ranging between 0 and 140 MPa did notaffect -sheets. Yang, Dunker, Powers, Clark and Swanson(2001) used combinations of higher temperature (50 C), higherpressure (600 MPa) and longer pressurizing times (up to 64 min)to monitor the conversion of-sheets to non-native -helices andthus the loss of tertiary structure. Pressure-treated moleculesunfolded non-cooperatively and were stable for 3 months at 5 Cbecause of the formation of a hydrophobic molten globule. Thisstability suggests that refolding involves a substantial energybarrier. Aouzelleg, Bull, Price and Kelly (2004), using CD, fur-thered this work using a combination of pressure, temperature andtime. This yielded a molecular structure with a less rigid tertiarystructure in the vicinity of the aromatic amino acids and anincrease in -helix content as the intensity of all the treatmentsincreased. Pressure was found to be the most important parameterin bringing about the molecular changes.

Hummer, Garde, Garca, Paulaitis and Pratt (1998) examinedthe pressure dependence of hydrophobic interactions withpressure denaturation of proteins. They focused on pressure-dependent transfer of water into the protein interior, graduallyfilling cavities and eventually breaking the native proteinstructure. Thus, they examined the effects of pressure on theassociation of non-polar residues in water. The pressuredestabilization of hydrophobic aggregates was explainedusing an information theory model of hydrophobic interactions,which accounts for the primitive hydrophobic effects ofsolvation, association and conformational equilibria of smallnon-polar solutes in water (Hummer, Garde, Garca, Pohorille,& Pratt, 1996). Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure, with watermolecules penetrating their core.

The renaturation of pressure-denatured -LG is thus anentropy-controlled process, and the remarkable negative valuefor the entropy of activation reflects the high degree of order inthe transition state for the refolding, which probably involvesseveral hydrogen-bonded water molecules. Refolding of -LG(variants A and B and mixed variants AB) after higher pressure(400 MPa for 5 min) release indicated that the structure of thecore refolded back to its native conformation after the pressurewas released (Belloque et al., 2000). These results are in

T. Considine et al. / Innovative Food Scienagreement with Iametti et al. (1997), who found that only 10%of the structure was lost at 600 or 900 MPa. Mller et al. (1998)reported that the level of exposed thiol groups that occurred ontreatments at 150 MPa decreased after storage for 2 days; thiswas attributed to the refolding of the molecule. If the proteinwas completely unfolded, it is very unlikely that the structurewould be rebuilt in the same manner as the native structure. AsFTIR studies have shown that pressures up to 1000 MPa do notresult in complete unfolding of -LG, Panick, Malessa andWinter (1999) suggested the existence of a flexible but stillpartially structured core of -LG, which allows the protein torefold back to the original structure (Kuwata et al., 2001).

As described for temperature, when -LG is in an unfoldingenvironment such as pressure, the free SH group of Cys121(CysH121) has the ability to interact irreversibly with disulfidebonds. It was speculated by Belloque et al. (2000) that theintermolecular SS bonds, formed by SH/SS exchange reaction,are likely to involve Cys66Cys160 and Cys121 rather thanCys106Cys119. Inter- and intramolecular reactions of SHgroups were suggested by Tanaka, Tsurui, Kobayashi and Kunugi(1996) as the main cause for pressure-induced irreversibledenaturation of -LG at 400 MPa and pH 6.8. It was also sug-gested that most SS bonds resulted from SH/SS interchangereactions rather than oxidation from SH groups (450MPa, 25 C,pH 7.0; Funtenberger, Dumay, & Cheftel, 1997). This is inagreement with Valente-Mesquita, Botelho and Ferreira (1998),who suggested that the correct refolding of the -LG dimer washampered by the formation of non-native disulfide bonds. Re-cently, Considine, Singh, Patel and Creamer (2005) suggestedthat pressures below 100 MPa do not allow exposure ofCysH121 and therefore that disulfide-bond exchange reactions donot occur. Pressures greater than 500 MPa allow this exchangereaction to occur readily.

Tanaka and Kunugi (1996) used H/D exchange reactionsunder pressure (200 MPa) and analysed samples by FTIR andNMR spectroscopy after pressure release. They suggested anintermediate unfolding stage under moderately high pressures.Previously, denaturation by pressure was described with a simpletwo-state process. However, there were indications (Jonas &Jonas, 1994) of the existence of pressure-induced pre-denatur-ation transitions, which in turn pointed to step-wise processes.Stapelfeldt and Skibsted (1999) also proposed a three-stagepressure denaturation of -LG. However, with Stage I at a lowpressure level (b50 MPa), Stage I induced pressure melting,increased thiol activity and induced collapse of the inner calyx.Stage II, 200 MPa, showed some reversibility after pressurerelease, whereas Stage III, high pressure, led to irreversiblechanges with aggregation and gel formation. Considine, Singhet al. (2005) proposed a three-stagemodel (Fig. 7) that can be usedto describe the denaturation and aggregation of pressure-treated-LG. In Stage I P (0.1150 MPa), the native structure is thestable structure after pressure treatment. In Stage II P (200450 MPa), the native monomers interchange reversibly with anon-native monomer and a disulfide-bonded dimer (Fig. 5). Fi-nally, in Stage III P (beyond about 500MPa), the stable state afterpressure treatment is unfolded. Stages II P and III P are looselyencompassed by Stage II T in the heated systems (Fig. 6), indi-

7nd Emerging Technologies 8 (2007) 123cating that some of the interchange reactions that occur in systemsheated at lower Stage II T temperatures, e.g. 75 C, do not occur atintermediate pressures (e.g. 250 MPa).

linCon

ce a3.4. Comparison of heat and pressure treatments of -LG

Comparisons between heat and pressure treatments of -LGhave not been extensively reported. Panick, Malessa and Winter(1999), using SAXS and FTIR spectroscopy, assessed thequaternary, tertiary and secondary structures of heat- andpressure-treated -LG. Above 60 C, the aggregation of -LG(1%) was evident, as seen by an increase in intermolecular -sheet content and an increase in the radius of gyration. Sub-sequent cooling showed that this aggregation process wasmostly reversible. The different -LG variants were shown tohave dissimilar denaturation and aggregation temperatures: -LG AB, 62 C; -LG A, 65 C; and -LG B, 70 C. Whenidentical samples to those used for heat treatment werepressurized (b150 MPa), the monomerdimer equilibrium of-LG was similar to that of heat denaturation. At pressures of 130 MPa, unfolding and aggregation of -LG were observed.-Sheet structure decreased during pressurization, which is incontrast to heat denaturation. The pressure stabilities of thedifferent variants were similar, but this is in contrast to a laterreport (Belloque et al., 2000). These observations led theauthors to conclude that there is an absence of extensive hydro-phobic interactions during pressure-induced aggregation andthat pressure-induced denaturation occurs through one aggre-gation mechanism (Panick et al., 1999).

Comparison of the effect of heat treatment and pressuretreatment of -LG B for 30 min (Considine et al., in press)showed that no modified -LG was seen at 100 MPa. At higher

Fig. 7. Proposed three-stage model of the pressure denaturation of -lactogloburetinol or sodium dodecyl sulfate (SDS). (Reproduced with the permission ofChemistry).

8 T. Considine et al. / Innovative Food Scienpressures (about 150450 MPa) at 22 C and neutral pH, themajor products were non-native monomers, dimers and trimers innative-PAGE and only monomer and dimer species could be seenin SDS-PAGE. It would appear that there was some feature of theaggregation at pressures between 150 and 450 MPa thatprevented further disulfide bond formation because, at higherpressures, particularly above 600 MPa; Fig. 7, native protein(Considine, Singh et al., 2005, in press), the full range ofaggregates was apparent in the SDS-PAGE patterns.

In contrast, upon heat treatment of -LG, unfoldedmonomers and non-covalently linked and disulfide-linked ag-gregates are formed. The concentration of non-native-like -LGspecies withMr values less than approximately 200,000 Da alsodecreases with increasing heat treatment time after 10 min(Manderson, 1998). These non-native-like species are reactionintermediates during heat treatment, with large disulfide-linkedaggregates becoming the end product of the -LG aggregationpathway.

The pressure denaturation of -LG is thus different from heatdenaturation, as the latter gives rise to the full range of disulfide-bonded polymers at temperaturesN72 C.One possibility is that thehydrophobic environments available at high temperatures (N65 C)are not available at unfolding pressures. Pressure diminishes thehydrophobic effect; thus the protection of CysH121 by the largehydrophobic amino acid side chains becomes less effective as thepressure is increased. Consequently, moderate pressures mayfavour the formation of various monomers and dimers and not theformation of larger aggregates. At higher pressures, -LG formslarger aggregates, most probably because of the lack of thehydrophobic effect that stabilizes the helical and sheet structures of-LG by exposingCysH121. Ikeuchi et al. (2001)monitored in situintrinsic fluorescence and 8-anilino-1-naphthalenesulfonate (ANS)binding by fluorescence of -LG under pressure (0400 MPa) atpH 2 or pH 7. -LG was irreversibly denatured at pH 7 whereas itwas reversibly denatured at pH 2. This irreversibility was attributedto novel intermolecular disulfide bonds through SH/SS inter-change reactions, as previously discussed (Tanaka&Kunugi, 1996;Valente-Mesquita et al., 1998).

Many ligands can be used to probe the structure of -LGafter treatment. After exposure to pressure at 400 MPa at pH 7or pH 2, -LG showed considerable loss of retinol bindingability at pH 7. This was also shown by others (e.g. Dufour, HuiBon Hoa, & Haertle, 1994; Valente-Mesquita et al., 1998). Incontrast, cis-parinaric acid (cPA) binding to pressurized -LG

B (-LG B), and -LG B with added 8-anilino-1-naphthalenesulfonate (ANS),sidine, Singh et al. (2005). Copyright 2005 Journal of Agricultural and Food

nd Emerging Technologies 8 (2007) 123was lost at both pHs (Dufour et al., 1994; Ikeuchi et al., 2001).Stapelfeldt and Skibsted (1999) found a decrease in the fluo-rescence quantum yield of cPA when it was pressurized in thepresence of -LG. The largest effect was seen for moderatepressures, corresponding to the pressure region in which theysuggested that pressure melting of -LG preceded pressureunfolding. These authors speculated that the binding of cPAmay have affected the pressure stability of -LG.

3.5. Effects of ligands on heat- or pressure-induced changes in-LG

Ligands such as all-trans retinol, palmitic acid, SDS and ANSwill bind hydrophobically to -LG. These interactions have beenfound to delay the heat-induced unfolding and aggregation of -LG B (Considine, Patel, Singh, & Creamer, 2005). The addition

ce aof some ligands to -LG prior to heat treatment delays the firststep (loss of native structure) of the initial reaction by stabilizingthe native state (Considine, Patel et al., 2005). Recently,Considine, Singh et al. (2005; Fig. 7) showed that ligands suchas SDS can delay the initial stages of -LG unfolding andaggregation under pressure (Stage I P to Stage II P; Fig. 7),whereas ANS and retinol postpone the later stages of -LGdenaturation (Stage II P to Stage III P; Fig. 7). A comparisonstudy by Considine et al. (in press) on the addition of conjugatedlinoleic acid (CLA) or myristic acid to -LG before heat orpressure treatment confirmed that the presence of ligands inhibitsthe transition from Stage I T to Stage II T in heat-treated samplesand both from Stage I P to Stage II P and from Stage II P to StageIII P in pressure-treated samples. CLA appears to be the mostversatile ligand and inhibits both thermal and pressuretransitions.

4. -lactalbumin

4.1. Structure of -LA

-LA has a molar mass of 14.2 kDa, is stabilized by fourdisulfide bonds and does not contain a free thiol group (Brew &Grobler, 1992). However, one of the disulfide bonds (Cys6-Cys120) is more sensitive to cleavage than the other threebecause of its lower inherent stability (Kuwajima, Ikeguchi,Sugawara, Hiraoka, & Sugai, 1990). The protein also exists in anumber of environment-dependent conformations, including theholo (native, calcium-bound) form, which is the major form inmilk.

A molten globule (MG) state of a protein is a partially foldedcompact state having a high content of native-like secondarystructure but lacking specific tertiary structure (Dolgikh et al.,1981, 1985; Ohgushi & Wada, 1983). Some MG states conformto the original description, but a range of proteins have beenshown to form MG states that contain specific tertiary inter-actions (Dolgikh et al., 1981; Marmorino, Lehti, & Pielak,1998; Ohgushi & Wada, 1983; Ptitsyn, 1995). Although mostglobular proteins can form MG states, these are generally un-stable so that analytical assessment is difficult. However, -LA,under defined pH and temperature conditions, can form asufficiently stable MG state for investigation.

4.2. Effect of heat on -LA

-LA has been studied extensively in recent years as a modelfor protein folding studies mainly because of its ability to form aMG (e.g. Wijesinha-Bettoni, Dobson, & Redfield, 2001). When-LA is heated in solution, it can repeatedly undergo thermaltransition. This transition is at a lower temperature (b 66 C)than that of -LG ( 73 C; Regg, Moor, & Blanc, 1977).Reports have shown that, when -LA binds less than 1 mole ofcalcium (or other divalent cation) per mole of protein, thethermal transition temperature of this apo--LA decreases to

T. Considine et al. / Innovative Food Scienabout 35 C (Relkin, 1996). Addition of calcium ions to the apo--LA increases the transition temperature to about 66 C(Relkin, Launay, & Eynard, 1993). Extensive heating (100 Cfor at least 10 min) in the absence of extra calcium gives rise todisulfide-bonded -LA dimers, trimers etc. as well as some non-native monomeric protein (Chaplin & Lyster, 1986).

It is generally accepted that -LA does not polymerize byitself when heated above 70 C (Dalgleish, Senaratne, &Francois, 1997; Gezimati, Creamer, & Singh, 1997; Schokker,Singh, & Creamer, 2000). Heat treatment of -LA has very littleeffect on aggregation and disulfide-bond interchange, partlybecause no free thiol is available (Calvo, Leaver, & Banks,1993).

4.3. Effect of pressure on -LA

Kobashigawa, Sakurai and Nitta (1999) suggested that thedifference in delta(V) (V) values between apo- and holo--LAmust be caused by the release of bound Ca2+ from -LA andaccompanying solvation, which substantially decreases thepartial molar volume (Kauzmann, 1959). Results from Koba-shigawa et al. (1999) indicated that -LA changes itsconformation from MG to the unfolded state without volumechanges. It was shown that the tight packing is lost in the MG of-LA, but a hydrophobic core exists, which disappears at thetransition from MG to the unfolded state (Alexandrescu et al.,1992; Alexandrescu, Evans, Pitkeathly, Baum, & Dobson, 1993;Baum, Dobson, Evans, & Hanley, 1989; Semisotnov et al.,1987). Further, it has also been demonstrated that the interior ofthe MG state of -LA is highly hydrated (Kharakoz &Bychkova, 1997). -LA has been shown to be resistant todenaturation at very high pressures up to 400 MPa (Huppertz,Fox, & Kelly, 2004a; Lpez-Fandio, Carrascosa, & Olano,1996; Scollard, Beresford, Needs, Murphy, & Kelly, 2000;Tanaka & Kunugi, 1996). One apparent difference between heatand pressure is that -LA heated to high temperature (Lyster,1970) can form dimers and larger aggregates, but, in pressure-treated solutions of pure -LA, no transformation of monomer-LA to larger disulfide-bonded aggregates is observed (Patelet al., 2004), unless the pressure is very high, 1000 MPa(Jegouic, Grinberg, Guingant, & Haertle, 1996).

5. Bovine serum albumin

5.1. Structure of BSA

BSA is a single polypeptide of 582 amino acid residueswith a molecular weight of 66,433 Da and exists in a multi-domain structure with complex ligand-binding specificities(Hsia et al., 1984). It is characterized by an overall oblateshape and consists of three domains (I, II and III), eachstabilized by an internal network of disulfide bonds (Carter &Ho, 1994). The primary structure has 17 disulfide bridges thathold the molecule in a structure consisting of nine loops. Itcontains one free thiol group, Cys34 (Carter & Ho, 1994). Thesecondary structure is composed of 76% helix, 10% turn and23% extended chain, and no -sheet (Carter & Ho, 1994;

9nd Emerging Technologies 8 (2007) 123Gelamo, Silva, Imasato, & Tabak, 2002; Gelamo & Tabak,2000; Reed, Feldhoff, Clute, & Peters, 1975; Wetzel et al.,1980).

bonds and intermediate-sized aggregates that were held togethermainly by disulfide bonds and to a lesser extent by non-covalentbonding (Dalgleish et al., 1997; Havea et al., 1998).

Hines and Foegeding (1993) showed evidence for stronginteractions between unfolded molecules of -LA and -LGthrough analysis of the kinetics of the thermal aggregation ofthese proteins at pH 7.0. They observed that the aggregation rateof -LA at 80 C increased an order of magnitude in thepresence of -LG. This was attributed to an interaction betweendenatured -LA and -LG molecules. There is evidence that -LA is readily heat denatured but has a much greater tendency torenature rather than to form aggregates (Regg et al., 1977).Hines and Foegeding (1993) suggested that the interaction ofdenatured -LA with denatured -LG molecules was kineti-cally or thermodynamically more favourable than renaturation.

Hinrichs and Rademacher (2004) monitored the effect of highpressure and temperature on the thermal denaturation of -LG A,-LG B and -LA in whey protein isolate solutions. They used anon-linear regression method in which all data at all temperaturesand pressures for each protein were analysed simultaneously.-LA

ce and Emerging Technologies 8 (2007) 1235.2. Effect of heat on BSA

Numerous studies have been carried out to explore heat-induced denaturation of BSA (Clark, Judge, Richards, Stubbs, &Suggett, 1981; Takeda, Wada, Yamamoto, Moriyama, & Aoki,1989; Yamasaki, Yano, & Aoki, 1991). These authors concludedthat BSA does not denature up to 40 C. Conformational changesare reversible between 42 and 50 C, but unfolding of the -helices of BSA is irreversible between 52 and 60 C.Temperatures from 60 C and above show unfolding of BSAand thiol-catalysed aggregation begins. Above 70 C, gelformation occurs if the concentration is sufficiently high. Asthe temperature increases, some molecular regions becomeaccessible to new intermolecular interactions, producing solubleaggregates through disulfide and non-covalent bonds (Wang,1999). Kang and Singh (2003) described the differential scanningcalorimetry profile of intact BSA, which did not adequately fit atwo-state model, with two peaks at 61 and 66 C. Recently,Murayama and Tomida (2004) explored the heat-inducedstructure and conformation of monomeric BSA by FTIRspectroscopy. These authors reported that intermolecular -sheet formation of BSA is irreversible on heating above 70 C.The conformational changes in BSA occur at both 57 and 75 C.

5.3. Effect of pressure on BSA

Very few investigations on the effect of pressure on pureBSA have been reported. Pressure resistance (100400 MPa)has been demonstrated by Hayakawa, Kajihara, Morikawa, Odaand Fujio (1992), using spectrofluorometry. They reported thatpressure could not change the protein's -helix and that it didnot impart sufficient energy to disrupt SS bonds, thus main-taining the molecular structure of BSA.

6. Mixtures of whey proteins

6.1. -LA and -LG

In the presence of -LG, which has a free thiol group that cantrigger aggregation by SH/SS interchange, -LA aggregationoccurs readily (Calvo et al., 1993; Dalgleish et al., 1997). Duringthe heating of mixtures of -LA and -LG at low ionic strength,soluble aggregates form via disulfide and hydrophobic interac-tions (Calvo et al., 1993; Dalgleish et al., 1997; Schokker et al.,2000). Homopolymers of each protein, aswell as heteropolymers,which are formed from disulfide-bonded -LA dimers, 1:1aggregates of -LG:-LA dimer and non-native monomers anddimers etc., of both -LA and -LG (Havea et al., 2001; Hong &Creamer, 2002) have been observed. The presence of -LAdiminished the proportion of smaller aggregates and increased thenumber of very large aggregates within both variant proteinmixtures. Preheating -LG decreased the extent of loss of -LAfrom the mixtures, suggesting that the bond shuffling that occursduring heat treatment is enhanced by the formation of MG

10 T. Considine et al. / Innovative Food Scienintermediates and by thiol catalysis (Hong & Creamer, 2002).Heat-induced interactions within mixtures of -LA and -LGgenerated large aggregates that were held together by disulfideFig. 8. One-dimensional (1D) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) pattern of a 5% whey protein concentrate (WPC)solution, control (Lane T0), heated at 72 C for 15 s (Lane T1) and heated at140 C for 5 s (Lane T2), and a 12% WPC solution, control (Lane P0), pressuretreated at 200 MPa for 30 min (P1) and pressure treated at 800 MPa for 30 min(Lane P2). The SDS-PAGE patterns of the respective reduced samples arepresented: control (TR0), heated at 72 C for 15 s (TR1) and at 140 C for 5 s(TR2), control (PR0), pressure treated at 200 MPa for 30 min (PR1) and at800MPa for 30 min (PR2). All the major whey protein bands were identified andmarked appropriately. The protein aggregates formed as a result of heat orpressure treatment are marked as X2, X3, X4, X5 and X6. These protein

aggregates were subsequently characterized using two-dimensional (2D) PAGE.(Reproduced with the permission of Patel et al. (2004). Copyright 2004 FoodNew Zealand, Scientific Supplement, June 2004).

denatured more slowly than -LG at all temperatures. Theactivation energy of either -LG A or -LG B increased withincreasing pressure whereas the activation energy of -LA showeda distinctive maximum at 400 MPa, probably because of the V/P change. As mentioned earlier, thermal denaturation of -LAoccurs only in the presence of reactive thiol groups (Jegouic et al.,1996; Jegouic, Grinberg, Guingant, &Haertle, 1997). Hinrichs andRademacher (2004) suggested that the maximum activation energyat 400 MPa may be due to the stabilization of the protein in itstertiary structure by pressure and because disulfide-bond exchangeis hindered. Therefore, less reactive -LA is available to aggregatewith the unfolded-LGby disulfide-bond exchange.A critical partof the process is for the SS bond of -LA to physically contactone of the CysH residues of another protein, generally -LG, andfor the -LA to obtain a CysH, which could then, in effect, createnew SS bonds with -LA, -LA aggregates and -LA:-LG

dimers. It is very likely that the critical -LA:-LG interactionstake place in a hydrophobic environment. However, in a highpressure situation, very few hydrophobic environments will arise,in part, because V/P is much less for -LA (Lassalle, Li,Yamada, Akasaka, & Redfield, 2003) than for -LG (Royer,2002) and thus-LA retains most of its structure at high pressure.On increasing the pressure to 800 MPa, -LG was shown to beN80% irreversibly denatured whereas 40% of -LA wasdenatured. This was attributed to the conformational stabilizingbonds inherent to each protein's structure (Hinrichs, Rademacher,& Kessler, 1996; Messens, Van Camp, & Huyghebaert, 1997).

6.2. BSA and -LG

BSA responds to heat treatment similarly to -LG, except thatBSA aggregates at a lower temperature (Gezimati, Singh, &

rn (l strnd af the

11T. Considine et al. / Innovative Food Science and Emerging Technologies 8 (2007) 123Fig. 9. Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) pattemercaptoethanol; second dimension SDS-PAGE of reduced sample within a getreatment (72 C for 15 s (T1)) and severe heat treatment (140 C for 5 s (T2)), a(P1)) and severe pressure treatment (800 MPa for 30 min (P2)). The preparation o

bands were identified and marked appropriately. The SDS-PAGE pattern of the origreduced SDS-monomeric protein spots dissociated from the high molecular weight prof Patel et al. (2004). Copyright 2004 Food New Zealand, Scientific Supplement, Jufirst dimension sodium dodecyl sulfate (SDS)-PAGE without treatment with 2-ip) of a 5% whey protein concentrate (WPC) solution, control (T0), mild heat12% WPC solution, control (P0), mild pressure treatment (200 MPa for 30 min2D gel is explained in detail schematically in Fig. 2. All the major whey protein

inal sample after reduction was run in the left-hand lane to help to identify theotein complexes after reduction of the gel strip. (Reproduced with the permissionne 2004).

interchange with the disulfides of -LA, thus generating -LA:BSA adducts and -LA dimers (Havea et al., 2000).

No pressure studies on the interaction between purified BSAand -LA have been reported to date.

6.4. Whey protein concentrate

Whey protein concentrate WPC contains many proteinsother than -LG and denaturing -LA and heat denatures mostof them readily; for example, BSA can induce -LA todenature. It is likely that pressure treatment of immunoglobulins(Igs), for example, unfolds them and that they react with oneanother or with other proteins to give disulfide-bondedaggregates, but such minor protein aggregates have not yetbeen identified. It is noticeable that BSA, a helical protein, isquite resistant to irreversible (i.e. disulfide-bond interchange)

Table 2Quantity (g/L skim milk) of major proteins in Scottish milk (Davies & Law,1980)

Protein component Quantity

s1-Casein 10.25s2-Casein 2.74-Casein 10.48

12 T. Considine et al. / Innovative Food Science and Emerging Technologies 8 (2007) 123Creamer, 1996). deWit and Klarenbeek (1984) have reported thatthe thermal transition of BSA is lower than that of-LG, plus both-LG andBSAhave a free cysteine (Carter&Ho, 1994).Gezimatiet al. (1996) reported that, in mixtures of -LG and BSA, BSAseemed to influence the loss of native-LG, whereas the presenceof -LG did not seem to influence the loss of native BSA from theprotein mixtures. Similar findings were reported by Havea et al.(2001). Hines and Foegeding (1993) examined the effect of addedBSA on the aggregation of -LG at 80 C. They reported thatnative -LG aggregated with a second-order rate constant withBSA, when they were mixed in a 1:1 molar ratio. The rate wasseven times greater than when -LG was alone in solution and itwas also reported that aggregation of BSA was unaltered by thepresence of -LG.

No studies on pressure for mixed -LG-BSA systems havebeen found to date.

6.3. BSA and -LA

Havea, Singh and Creamer (2000) studied the formation of newprotein structures in heatedmixtures ofBSAand-LA.Heating theindividual proteins alone showed BSA only containing largedisulfide-bonded BSA aggregates. Combination of-LA and BSA(1:1 w/w) led to the production of larger disulfide-bondedaggregates and SDS-monomeric BSA and -LA. On loweringthe concentration from 5 to 2%, fewer larger disulfide-bondedaggregates were evident and there was less SDS-monomeric -LAorBSA.These authors concluded that BSA forms disulfide-bondedaggregates that contain available thiol groups that can catalyse theformation of differently structured-LAmonomers, dimers, higherpolymers and adducts of -LA with BSA. When all three wheyproteins are present (-LA,-LG andBSA), BSA ismore effectivethan -LG in catalysing the formation of -LA polymers (Haveaet al., 2001). This may be related to the differences in thermal

-Casein 3.45Whey proteins 5.80denaturation between the protein species (Section 6.2). Thus BSAwill begin to unfold and aggregate before -LG. The exposed thiolgroup of BSA molecules/aggregates can react via thiol-disulfide

Table 3Relative composition (g/100 g) of a typical acid whey protein concentrate on adry basis (Havea et al., 1998)

Protein Quantity

Total protein 84.5-Lactoglobulin 45.6-Lactalbumin 16.0Bovine serum albumin 5.2pressure-induced denaturation, which is in contrast to its readydenaturation by heat.

Heated WPC contains 1:1 disulfide-bonded adducts of -LAand -LG, which become more obvious at low concentrations(Havea et al., 1998). In these systems, the aggregation rate of -LA was lower than that of -LG and -LG B aggregatedslightly faster than -LG A. Almost all of the -LG was in-corporated into the aggregates via disulfide bonds and to a lesserextent via hydrophobic interactions (Havea et al., 1998).However, when similar samples were pressure-treated -LGdimer was predominant (Patel et al., 2005).

The rate of loss of the three major proteins in WPC solutionunder pressure treatment was -LGNBSAN-LA (Patel et al.,2005), which is quite different from that obtained using heattreatment (BSAN-LG-LA) (Havea et al., 1998). Thisconfirms and supports the view that there are few similaritiesbetween heat- and high-pressure-induced aggregation of wheyproteins (Fig. 8). The most significant differences in the de-naturation and aggregation of proteins are mainly related to therupture of non-covalent interactions and the subsequent re-formation of intra- and intermolecular bonds by heat comparedwith high pressure (Heremans, Van Camp, & Huyghebaert,Fig. 10. Changes to the appearance of milk on heat treatment and pressuretreatment. A: untreated milk; B: heated milk (100 C/3 min); C: untreated milk;D: high-pressure-treated milk (600 MPa/30 min).

1997). Similar differences have been found when gels havebeen induced by either heat or pressure treatments (Dumay,Kalichevsky, & Cheftel, 1998; Van Camp, Feys, & Huyghe-baert, 1996; Van Camp & Huyghebaert, 1995).

2D PAGE patterns of control, heat-treated and pressure-treatedWPC (from Fig. 8) are shown in Fig. 9. A band identifiedas Igs in one-dimensional (1D) PAGE (control) gave rise to threebands in 2D PAGE. Several faint spots were resolved from thematerial caught up at the top of the stacking gel (marked as X1 inFig. 9, Po) of the 1D stained strip and were identified aslactoferrin (Lf) and other minor whey proteins. Some changesthat could not be observed in 1D PAGE of either heat-treated orpressure-treated samples due to overlapping bands were moreeasily observed in 2D gels. The aggregation of -LG in mildpressure-treated samples was shown by 2D PAGE (Fig. 9, P1) to

the core of the native micelle (see Figs. 12 and 13) in a milkenvironment ( 39 C; pH 6.66.7) but can move to the surfaceor into the whey phase when the micelle is in a non-nativeenvironment, e.g. b10 C. An important stabilizing factor is thepresence of the so-called colloidal calcium phosphate (CCP)component and removal of CCP results in the micellesdissociating into smaller particles, which have sometimes beencalled subunits or submicelles.

7.1. Appearance of milk

The first and most striking difference between heat andpressure treatment of skim milk is the appearance of the milkdirectly after treatment (Fig. 10b). Skim milk is a white turbidliquid, and on heat treatment the milk appears whiter. On

13T. Considine et al. / Innovative Food Science and Emerging Technologies 8 (2007) 123be due to the formation of disulfide-linked dimers, trimers ortetramers (labelled as d, d, d). A more extensive discussion ofthe various spots can be found in Patel et al. (2004).

7. Milk systems

The heat stability of milk has been extensively studied overthe past 100 years and it is not the purpose of this paper to reviewin detail the literature devoted to this topic. Interested readers aredirected to the numerous reviews on the subject (Fox, 1995;Singh & Creamer, 1992). However, there has been someresearch that can be used to compare and contrast the effects ofheat and pressure on milk and these effects will be brieflysummarized. Because of the limited information on pressure-treated milks, the review is confined to milk at concentrationsclose to those naturally found.

Bovine skimmilk contains all the proteins of whey and all theproteins that associate to become casein micelles (Tables 2and 3). The casein micelles are colloidal particles and thestructure of these particles has not been unequivocally es-tablished. However, there are many models of the casein micellethat attempt to explain the known facts (Holt, 1992; Horne,1998). The s-CNs (s1- and s2-CN) are in the core region ofthe micelle and -CN is on the surface of the micelle. -CN is inFig. 11. Particle size changes on (A) heat and (B) pressure treatment of skim milk formilk at pH 6.9; open triangle: milk at pH 7.1.storage, the milk remains white regardless of the storage con-ditions. In contrast, on pressure treatment skim milk becomestranslucent or semi-transparent with a slightly yellow hue(Fig. 10d). If the milk is held at refrigerator temperatures (about5 C), it will retain the semi-transparent appearance for longperiods (several days). However, if the milk is held at roomtemperature, it becomes progressively more turbid, but does notreturn to the original appearance of the untreated milk. Heattreatment of pressure-treated milk at elevated temperatures( 70 C) causes it to return to an appearance similar to that ofthe original untreated skim milk.

7.2. Particle size changes

When milk is heated, the change in particle size is dependenton the temperature and duration of heat treatment, as well asother factors such as small changes the pH of the milk atheating. Mild heat treatment (b70 C/20 min) of the milk at thenatural pH causes a small decrease in the size of the caseinmicelles (Anema & Li, 2003a,b). On heating milk attemperatures up to 100 C, the change in size was markedlydependent on the pH of the milk at heating (Anema & Li, 2003a,b). At pH 6.5, a marked increase in size of the casein micelleswas observed, with the average size increasing by about 35 nm.30 min. Filled circle: milk at pH 6.5; open circle: milk at pH 6.7; filled triangle:

ce aAs the pH was increased, smaller changes in particle size wereobserved so that, at pH 6.7, the size increase was only about5 nm. At pH above about 6.7, the casein micelle size decreasedon mild heat treatments (Fig. 11A).

These changes in casein micelle size could be related to theinteraction of denatured whey proteins with serum or colloidalphase components in the milk. At pH 6.5, about 70% of thedenatured whey proteins are associated with the casein micelles.The level of association decreases with increasing pH so thatonly about 30% are associated at pH 6.7. As the pH of the milkis increased above about 6.7 before heating, the -CN prog-ressively dissociates from the casein micelles and only lowlevels of denatured whey proteins associate with the caseinmicelles at these elevated pH values, whereas significant levelsof casein are found in the serum phase. This change in caseinmicelle size also has an effect on the viscosity and turbidity ofthe milk (Anema & Li, 2003a,b; Anema, Lowe, & Li, 2004).

Severe heat treatment (130 C/several minutes) of milkcauses substantial increases in the size of the particles in milk.These changes are due to the aggregation of the casein micelles,and the degree of aggregation is also dependent on the initial pHof the milk at heating (Dalgleish, Pouliot, & Paquin, 1987).

The pressure treatment of milk at the natural pH and atpressures above about 300 MPa causes a substantial decrease inthe size of the particles in skim milk, with the average sizedecreasing from about 200 to 100 nm, regardless of the temper-ature at pressurization (Anema, Lowe, & Stockmann, 2005;Huppertz et al., 2004a; Gaucheron et al., 1997). This change inparticle size occurred rapidly, and was accompanied by asignificant increase in the level of serum phase casein, and thechanges in size and serum phase casein appeared to be cor-related (Anema, Lowe et al., 2005). At pressures between about200 and 300 MPa, an increase in particle size could be observeddepending on the pH of the milk, and the temperature, pressureand duration of the pressure treatment (Fig. 11B; Anema, Loweet al., 2005; Gaucheron et al., 1997; Huppertz et al., 2004a).This increase in size is a consequence of the aggregation of thecasein micelles. This aggregation was more pronounced as thetemperature at pressurization was increased and the duration ofpressure treatment was increased (Anema, Lowe et al., 2005;Huppertz et al., 2004a).

Initial reports suggested that the denaturation of the wheyproteins may be involved in this aggregation phenomenon(Huppertz et al., 2004a) but subsequent studies on whey-protein-depleted systems indicate that the aggregation is dueentirely to the casein micelles (Anema, Lowe et al., 2005;Huppertz, Fox, & Kelly, 2004b). Preheat treatment of the milkat 90 C/10 min produced particle size changes similar to thosein unheated milk over the entire pressure range (Huppertz, Fox,& Kelly, 2004c), indicating that the pre-denaturation of thewhey proteins has little effect on the aggregation or dissag-gregation phenomenon in pressure-treated milk.

Microstructural examination of the casein micelles beforeand after pressure treatments has also been completed. Trans-

14 T. Considine et al. / Innovative Food Scienmission electron microscopy (TEM) has generally shown thesame effects as observed by the particle size measurements. Areduction in particle size to about half the initial value wasobserved when a pressure of about 300 MPa or above wasapplied (Gaucheron et al., 1997; Schmidt & Buchheim, 1970;Schrader, Buchheim, & Morr, 1997). However, at pressures ofabout 250 MPa and at temperatures above 20 C, two distinctpopulations of sizes were observed, with the formation of par-ticles larger and smaller than the original casein micelles(Gaucheron et al., 1997).

Some contrasting results have been obtained by atomic forcemicroscopy (AFM). In one study, pressure treatment of caseinmicelle suspensions in water at 300 MPa for 15 min reduced thecasein micelle size to about half the initial value (Regnault,Thiebaud, Dumay, & Cheftel, 2004), in agreement with theTEM and particle size results. However, more recently, an AFMstudy of casein micelles in an MES/TRIS-HCl buffer system atpH 7.3 showed that the casein micelles were disrupted into verysmall particles of only about 520 nm diameter, or less thanabout 10% of the initial casein micelle size (Gebhardt, Doster,Friedrich, & Kulozik, 2006). The differences in these twostudies may be due to sample preparation methods of the caseinmicelle suspensions for pressure treatment, and the preparativemethodology of the samples for analysis by AFM.

There is some effect of small changes in the pH of milk on thechange in casein micelle size on pressure treatment as shown inFig. 11B and also reported by Huppertz et al., 2004a. When thepH of the milk was reduced from the natural pH ( pH 6.7) to6.5, the particle size decreased as the pressure increased, with amarked decrease in size above about 200 MPa. A similar effectwas observed at pH 6.7, although the size decrease at 250 MPawas not as pronounced as that at pH 6.5. When the pH wasincreased to pH 6.9 and pH 7.1, the particle size decreased ontreatment up to 200 MPa, and then increased when the pressurewas increased to 250 MPa. On increasing the pressure above250 MPa, the size decreased again with increasing pressure sothat, at 500MPa or above, samples at all pH had a similar particlesize after pressure treatment. Above 250MPa, the size decreasedmore rapidly as the pH of the milk was decreased.

In situ measurements of the changes in light transmission ofskim milk (Huppertz, Kelly, & De Kruif, 2006; Kromkamp,Moreira, Langeveld, & van Mil, 1996; Orlien, Knudsen, Colon,& Skibsted, 2006) and measurements of the changes in theparticle size of casein micelle suspensions in an MES/TRIS-HCl buffer system at pH 7.3 (Gebhardt et al., 2006) on pres-surization and subsequent depressurization have been made.These studies used specially designed high pressure cells withoptical windows that allowed a light source to monitor changesin the system on pressurization, holding and depressurization.

There are considerable changes in light transmission/particlesize during pressure treatment, which can be partially orcompletely reversed on subsequent pressure release. The extentof the reversibility is dependent on the pressure applied, withalmost complete restoration of the light transmission/size to initiallevels on treatment at low pressures (generally b250 MPa,although some variability in results exists between variousstudies, which is again probably due to the sample preparation

nd Emerging Technologies 8 (2007) 123methodology) and only partial reversibility at higher pressures(250MPa or above). These results suggest that the caseinmicellesare dissociated into small particles under pressure, probably as a

ce aconsequence of the diminished hydrophobic effect and thedissolution of CCP (Anema, Lee, Schrader, & Buchheim, 1997;Schrader & Buchheim, 1998). However, once the pressure isreleased, the highly perturbed casein micelles partially reassoci-ate, presumably through hydrophobic interactions and CCP, withthe extent of reassociation being dependent on the pressureapplied and the temperature of the system (Orlien et al., 2006).

7.3. Dissociation of casein from the casein micelles

The heat treatment of milk can cause a pH-dependent dis-sociation of casein from the casein micelles (Anema &Klostermeyer, 1997; Anema & Li, 2000; Singh & Creamer,1992; Singh & Fox, 1985). This dissociation is also dependenton the temperature of heating, the duration of the heat treatmentand the composition of the milk. In milk at its natural compo-sition, very little dissociation occurs at pH 6.7 or belowregardless of the temperature or duration of heat treatment(Anema & Klostermeyer, 1997; Anema & Li, 2000; Singh &Creamer, 1992; Singh & Fox, 1985). As the pH is increased, thedissociation of casein increases, so that, at about pH 7.1,significant levels of the casein can be dissociated from thecasein micelles.

The composition of the dissociated casein is dependent onthe temperature of heating. At temperatures up to about 70 C,all the caseins are found in the dissociated protein; however, theproportions are altered from that naturally found, with -CN athigher levels, -CN at about the same level and s-CN at lowerlevels than that of the whole casein (Anema & Klostermeyer,1997; Anema & Li, 2000). As the temperature at heating israised above 70 C, the level of -CN in the dissociated caseincontinues to increase whereas the level of s-CN and -CNdecreases so that a very -CN-rich protein is found in the milkserum on heating milk at slightly elevated pH ( pH 7.1) and athigh temperatures (Anema & Klostermeyer, 1997; Anema & Li,2000; Singh & Creamer, 1992; Singh & Fox, 1985). Thischange in dissociation behaviour at temperatures above 70 Cappears to be due to the denaturation of the whey proteins andtheir interactions with -CN, as removal of the whey proteinsincreases the level of s-CN and -CN dissociating at tem-peratures above about 70 C (Anema & Li, 2000).

Unlike heated milk, there are only limited studies on thedissociation of casein micelles in pressure-treated milk. Whenseparated under mild centrifuging conditions (about 25,000 g),pressure treatment of skim milk causes a substantial dissociationof casein from the casein micelles, with up to 50% of the totalcasein transferred to the serum (Anema, Lowe et al., 2005). Thisdissociation of casein has been related to the decrease in particlesize in the pressure-treated milks (Anema, Lowe et al., 2005).At the natural pH of milk, and under relatively severecentrifuging conditions (100,000 g), all caseins were foundin the dissociated protein (Arias, Lpez-Fandio, & Olano,2000; Huppertz et al., 2004c; Lpez-Fandio, de la Fuente,Ramos, & Olano, 1998), although the proportion of -CN

T. Considine et al. / Innovative Food Scienappeared to be increased when compared with the whole casein(Arias et al., 2000; Lpez-Fandio et al., 1998). Increasing thepH of the milk to pH 7.1 increased the level of all caseinsdissociated from the micelles when compared with the samplesat the natural pH ( ~ pH 6.7). Decreasing the pH to 6.0 increasedthe level of s-CN and -CN dissociated, but decreased thelevel of -CN dissociated, whereas lowering the pH furtherincreased the level of all caseins dissociating (Arias et al.,2000).

7.4. Whey protein denaturation

When milk is heated, the whey proteins denature. Consid-erable research has been conducted on the denaturation of thewhey proteins on heating milk as this reaction, along withsubsequent aggregation processes, is key in manipulating thefunctional properties of the milk. Because of the extensiveliterature on the denaturation of the whey proteins, only keypoints, where both pressure and temperature reactions can becompared, will be discussed.

The minor whey proteins such as Lf, BSA and the Igs beginto denature at temperatures as low as 65 C and thereforesignificant denaturation is observed under pasteurizationconditions (about 72 C/15 s). The major whey proteins, -LG and -LA, are more heat stable and significant denaturationoccurs only at temperatures above about 7075 C (Fox, 1995).

Several studies on full kinetic evaluation of the irreversibledenaturation reactions of-LG and-LA in heated skimmilk havebeen completed, with generally the same conclusions (Anema &McKenna, 1996; Dannenberg & Kessler, 1988a; Oldfield, Singh,Taylor, & Pearce, 1998). The irreversible denaturation reactionsare not a simple process. There are marked changes in temperaturedependence of the rate constants at a temperature of about 90 C for-LG and at about 80 C for -LA. This unusual temperaturedependence of the rate constants has been discussed in terms ofdifferent rate-determining steps in the two temperature ranges. Thekinetic and thermodynamic parameters are consistent with thedenaturation or unfolding reactions being rate determining in thelow temperature ranges, whereas these parameters are consistentwith the aggregation reaction being rate determining in the highertemperature ranges (Anema & McKenna, 1996; Dannenberg &Kessler, 1988a; Oldfield et al., 1998).

For pressure-treated milks, studies on whey protein dena-turation are rather limited. However, it is known that wheyprotein denaturation is also observed at sufficiently high pres-sures, and, like heating, denaturation levels increase with in-creasing pressure or duration of treatment once a thresholdpressure is exceeded (Anema, Stockmann, & Lowe, 2005;Hinrichs & Rademacher, 2005; Huppertz et al., 2004a; Lpez-Fandio et al., 1996; Lpez-Fandio & Olano, 1998). Unlikethe heat treatment of milk, where the minor whey proteins (Lf,BSA and the Igs) are most labile, -LG is the most pressure-sensitive milk protein, denaturing at pressures as low as200 MPa. In contrast, -LA and BSA are stable to pressuresup to about 400500 MPa (Anema, Stockmann et al., 2005;Hinrichs & Rademacher, 2005; Huppertz et al., 2004a; Lpez-Fandio et al., 1996; Lpez-Fandio & Olano, 1998). There are

15nd Emerging Technologies 8 (2007) 123no reports on the pressure sensitivity of the Igs or Lf.There are two recent studies on the kinetic evaluation of the

pressure-induced irreversible denaturation of -LG in pressure-

ce a16 T. Considine et al. / Innovative Food Scientreated milk; both examined the combined effects of pressure andtemperature (Anema, Stockmann et al., 2005; Hinrichs &Rademacher, 2005). In one study (Hinrichs & Rademacher,2005), the kinetic parameters for both -LG and -LA wereobtained by a one-step non-linear regression process for pressuresup to 800 MPa and temperatures up to 70 C. The activationvolumes of -LG and -LAwere found to decrease (denaturationmore favoured) as the temperature at pressurization wasincreased, indicating that temperature and pressure act synergis-tically on these proteins. This study suggested that, for both-LGand -LA, the aggregation reactions were the rate-limiting step atall pressures, as no change in pressure dependence was observed.In another study on -LG only (Anema, Stockmann et al., 2005),the activation volume was also found to decrease (denaturation

Fig. 12. Pictorial representation of the likely effect of heating milk at 90 C. Thenative -lactoglobulin (-LG) dimer dissociates and the monomer undergoesinternal disulfide-bond interchange to give reactive monomers that react with -casein (-CN) at the surface (outer region) of the casein micelle. Native -LGmonomers can also form an adduct with -lactalbumin (-LA), which thengives rise to -LA dimers and -LA:-LG dimers. In the severely heat-treatedsamples, s2-CN also forms disulfide bonds with other proteins. (Reproducedwith the permission of Patel et al. (2006). Copyright 2006 Journal ofAgricultural and Food Chemistry).more favoured); however, a change in pressure dependence wasobserved at about 300MPa, and this becamemore pronounced asthe temperature at pressurization was increased from 10 to 40 C.Analysis of the kinetic and thermodynamic parameters of thislatter study suggested a transition from aggregation reactions asrate determining at low pressures, to denaturation or unfoldingreactions as rate determining as the pressure was increased.

7.5. Whey protein interactions with the casein micelles

On heatingmilk, the denatured whey proteins can interact witheach other andwith the caseinmicelles. Considerable research hasbeen conducted on deciphering the specific reactions that occur,the composition of the reaction products and the sequence ofevents involved in the aggregation reactions. Non-covalentinteractions and disulfide bonds (via thiol-disulfide exchangereactions) are known to be involved in the interactions. Earlystudies showed that there were specific reactions between -CNon the casein micelle surface and denatured -LG, and that thesereactions involved thiol-disulfide exchange (Lowe et al., 2004;Sawyer, Coulter, & Jenness, 1963). Recent studies have shownthat the interactions of the denatured whey proteins with thecasein micelles are strongly pH dependent (Anema & Kloster-meyer, 1997; Anema & Li, 2003a,b; Anema, Lowe et al., 2004).High levels, about 80% of the total, of the denatured whey proteinassociate with the casein micelles at pH 6.5, and this leveldecreases with increasing pH so that, at pH 6.7, about 30% of thetotal is associatedwith the caseinmicelles, and, at higher pH, evenlower levels are observed.

The interactions between -LG and -CN in heat-treatedmodel systems have been reported and these results showed thattwo disulfide bridges and a free sulfhydryl group present in thenative structure of -LG play an important role in its heat-induced interactions with -CN (Cho, Singh, & Creamer, 2003;Corredig &Dalgleish, 1999; Dalgleish, 1990; Hill, 1989; Jang&Swaisgood, 1990; Singh, 1995). Interestingly, s2-CN occurs atthe same concentration as -CN, and has one SS bond, but doesnot normally interact with -LG in milk systems. It can bespeculated that s2-CN is inside the micelle and therefore that -LG cannot reach it, but equally valid is that s2-CN is aparticularly stable entity, especially as the dimer. Fig. 12 shows adiagrammatic summary of the proteinprotein interactions thatoccur during heat treatment of milk at 8090 C. It has beenreported that s2-CN reacts with -LG via disulfide bonding inUHT milks (Patel, Singh, Anema & Creamer, 2006).

In contrast to the numerous studies on the distribution ofwhey proteins between the colloidal and serum phases in heatedmilk, only two studies have examined this distribution inpressure-treated milk. Huppertz et al. (2004a) suggested that themajority of the denatured whey protein was sedimentable, andpresumably associated with the casein micelles, under moresevere centrifuging conditions than used in the more recentheating experiments. Arias et al. (2000) examined the effect ofthe pH of the milk after pressurization (400MPa/15 min). Under

nd Emerging Technologies 8 (2007) 123these conditions, not all the -LG and very little of the -LAwillbe denatured (Anema, Stockmann et al., 2005; Hinrichs &Rademacher, 2005), and therefore conclusive information on

ce aT. Considine et al. / Innovative Food Scienassociation with the micelle cannot be obtained. The level ofwhey protein associated with the casein micelles appeared todecrease with decreasing pH at pressurization; however, thismay simply reflect the reduced denaturation of the proteins atthese lower pH values.

Both s2-CN and -CN contain disulfide bonds and s2-CNis more resistant to reduction than -CN polymers. It is un-common to find s2-CN disulfide bonded to any of the wheyproteins under mild heat (b100 C) or pressure (b150 MPa)treatments (Patel et al., 2006). The very clear reaction betweens2-CN and the other Cys-containing proteins in the pressure-treated milk shows several important features. Pressure eitherdisrupts the casein micelle structure sufficiently to expose S2-CN or modifies the structural arrangement of S2-CN within the

Fig. 13. Pictorial representation of the likely effects of medium ( 250MPa) and high (and the -lactoglobulin (-LG) unfolds and aggregates via disulfide bonds. -LG form(-CN), but does not form larger -LG aggregates. The proportion of -lactalbumin (readily unfold. At pressures N600MPa,s2-CN becomes available for thiol interchangethe calcium phosphate. Also the-LGmolecules can polymerize into larger aggregates tJournal of Agricultural and Food Chemistry).17nd Emerging Technologies 8 (2007) 123micelle to the extent that the disulfide bonds become accessibleto the SH of -LG (or its aggregates) or the aggregates of otherproteins.

Using PAGE, large quantities of very large aggregates thatcould not enter the gel were present to a greater extent in heat-treated milk than in pressure-treated milk (Patel et al., 2006),indicating that the sizes of the aggregates were comparativelysmaller in pressure-treated samples than in heat-treated samples.Such differences could be attributed to different effects of heatand pressure treatments on the structure of the proteins, whichmay ultimately lead to different textures of the final products. Thesensitivity of the different proteins to heat (LfN IgNBSAN-LGN-LA) and pressure (LfN-LGN IgNBSAN-LA) mayalso be responsible for the different protein complexes (Patel

N600MPa) pressure treatment at 22 C. The casein micelle swells at 250MPas disulfide-bonded dimers at lower pressure and probably aggregates with -casein-LA) that is included in the aggregates is less than that of -LG because it does notreactions, assisted by the permeation of water into themicelle and the dissolution ofhan dimers. (Reproduced with the permission of Patel et al. (2006). Copyright 2006

ce aet al., 2006). Some complexes are formed readily at low pressure(200 MPa for 30 min), such as disulfide-linked complexes of -LG and -CN, whereas interaction with s2-CN is apparent onlyat higher pressures (400MPa for 30 min; Fig. 13) or temperatures(120 C for 120 s) (Patel et al., 2006).

7.6. Interactions in whole milk

When whole milk is heated, there are a number of changes tothe milk fat globule membrane (MFGM) proteins. At suffi-ciently high temperatures, both -LG and -LA interact with theMFGM, and the amounts increase with increasing temperatureor duration of heat treatment up to a certain maximum level (Ye,Singh, Oldfield, & Anema, 2004). These proteins wereassociated via disulfide bonding. In addition, heat treatmentcaused some changes to the MFGM proteins with a decrease inPAS 6 and PAS 7 as the milk was heated.

In a similar study on pressure-treated whole milk, -LG wasobserved to interact with the MFGM proteins on pressuretreatment between 100 and 800 MPa. The level associatingincreased with the pressure and the duration of pressuretreatment (Ye, Anema, & Singh, 2004). However, the maximumlevel associating with the MFGM was substantially lower thanobserved on heat treatment. Low levels of -LA and -CN wereobserved only at pressures above about 600 MPa. Unlike inheated whole milk, PAS 6 and PAS 7 were stable to pressuretreatment and remained associated with the MFGM; however,xanthine oxidase appeared to decrease with pressure above400 MPa. Subsequent heat treatment of pressure-treated wholemilk caused the levels of -LG and -LA to increase to thelevels observed in heated milk, and some loss of PAS 6 and PAS7 was observed.

7.7. Effects in acid gel systems

The slow acidic destabilization of milk by bacterial culturesto form an acid-induced gel is the basis of yoghurt manufacture.Heat treatment of milk is used to modify the textural propertiesof the acid gels. The denaturation of the whey proteins, and theirinteractions with the casein micelles, when milk is heated attemperatures above about 70 C produces markedly firmer acidgels than observed for unheated milk. In addition, the pH atwhich gelation commences is increased and the time for ge-lation is decreased. Other properties, such as the decreasedporosity and decreased syneresis of the acid gels, are induced bythe heat treatment of milk prior to acidification.

There is a positive correlation between the level ofdenatured whey protein, particularly -LG, and the firmnessof acid set gels (Dannenberg & Kessler, 1988b; McKenna &Anema, 1993), and a negative correlation between thesedenaturation levels and the degree of syneresis of the acid gels(Dannenberg & Kessler, 1988c; McKenna & Anema, 1993). Ithas been proposed that the incorporation of the denatured wheyproteins in the acid gel structure produces a structure with a

18 T. Considine et al. / Innovative Food Scienhigher protein concentration and an increased number of cross-links (Anema, Lee, Lowe, & Klostermeyer, 2004; Lucey &Singh, 1998; Lucey, Teo, Munro, & Singh, 1997).Pressure treatment of milk also affects the properties of acidgels. Acid gels prepared from pressure-treated milk had in-creased rigidity and breaking strength and decreased syneresiswhen compared with acid gels prepared from untreated milk(Johnston, Austin, & Murphy, 1993; Harte, Luedecke, Swan-son, & Barbosa-Canovas, 2003). A recent study showed that therheological properties of acid gels prepared from pressure-treated milk samples were very similar to those of acid gelsprepared from heated milks (Anema, Lauber, Lee, Henle, &Klostermeyer, 2005). The increase in gel strength for acid gelsprepared from pressure-treated milk may be related to thepressure-induced denaturation of the whey proteins, and mayinvolve a similar mechanism to that observed when heated milkis acidified to form a gel (Anema, Lee et al., 2004; Lucey &Singh, 1998; Lucey et al., 1997).

7.8. Effects on the renneting properties of milk

Proteolytic destabilization of the casein micelles is the basisof the cheesemaking process. Traditionally, the enzyme extractused is rennet, obtained from the fourth stomach of the youngcalf; it contains a number of enzymes, of which chymosin (E.C.3.4.23.4) is the major en