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International Dairy Journal 36 (2014) 55e64

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairyj

Caseinomacropeptide modifies the heat-induceddenaturationeaggregation process of b-lactoglobulin

Thomas Croguennec a,b,c,*, Ning Leng a,b,c, Pascaline Hamon a,b, Florence Rousseau a,b,Romain Jeantet a,b,c, Saïd Bouhallab a,b

aAgrocampus Ouest, UMR1253 STLO, F-35042 Rennes, Franceb INRA, UMR1253 STLO, F-35042 Rennes, FrancecUniversité Européenne de Bretagne, France

a r t i c l e i n f o

Article history:Received 25 November 2013Received in revised form6 January 2014Accepted 6 January 2014

* Corresponding author. Tel.: þ33 2 23 48 59 27.E-mail address: Thomas.croguennec@agrocampus

0958-6946/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.idairyj.2014.01.004

a b s t r a c t

The effect of caseinomacropeptide (CMP) concentration on the kinetics of denaturation of b-lactoglobulin(b-Lg) and on the size and structure of the heat-induced aggregates was investigated over a wide range ofpH (3.0e6.7) and temperature (65e95 �C). Irrespective of pH and heating temperature, CMP increasedthe rate of b-Lg denaturation. When b-Lg and CMP were negatively charged (i.e., at pH 6.7), increasingCMP concentration hindered aggregate formation; the aggregates had a smaller hydrodynamic diameterand the protein solution turbidity decreased. However, when b-Lg and CMP were oppositely charged (i.e.,at pH 4.0), CMP promoted aggregate formation and large particles (>5 mm) were formed on heating.Nevertheless, the covalent aggregates constituting these large particles were of smaller size than thoseformed on heating b-Lg in the absence of CMP. At pH 4.0, CMP induced the formation of large particleswhen added in a solution of preformed aggregates of b-Lg.

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1. Introduction

Whey proteins are used in a wide range of food applicationsbecause of the versatility of their functional properties (solubility,gelling, foaming, emulsifying). However, the functional propertiesof commercial whey proteins can be unpredictable due to varia-tions in the protein composition and quality of commercial prod-ucts (Havea & Singh, 2003). These variations are inherent to the rawmaterial (by-products of cheese, acid casein and caseinate manu-facture ormilkmicrofiltration) and to the processes used to preparewhey proteins ingredients (ultrafiltration or ion exchange chro-matography; Wang & Lucey, 2003). Commercial whey proteins in-gredients from cheese whey are predominant on the market ofwhey protein ingredients (IDF, 2013). They contain a significantamount of the C-terminal part of the k-casein, called casein-omacropeptide (CMP), which is released by rennet action duringcheesemanufacture. In contrast, CMP is absent in commercial wheyprotein ingredients from milk microfiltrate and from acid wheyfrom acid casein and caseinate manufacture. When present, theproportion of CMP in commercial whey proteins can reach 20e25%

-ouest.fr (T. Croguennec).

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(w/w) of the total protein content (Thoma-Worringer, Sorensen, &Lopez-Fandino, 2006), and even more when whey proteins un-dergo intensive heat-induced denaturation before cheese makingto retain a higher amount of whey proteins in the cheese forimproving cheese yield.

The CMP fraction includes a variety of peptides having geneticvariability and differing by post-translational modifications (e.g.,glycosylation, phosphorylation). They are intrinsically unfoldedpeptides having no aromatic residues and only one sulphur-containing amino-acid (Met) in its sequence. The pI of the CMPvaries from 4.15 (aglycosylated: aCMP) to 3.15 (glycosylated: gCMP)due to the presence of sialic acid in the glycan moieties (Kreuss,Strixner, & Kulozik, 2009). Typically, gCMP represents about 50%of whole CMP. In the following sections the mixture of aCMP andgCMP is referred to as CMP. In binary systems, CMP would formsupramolecular complexes with b-lactoglobulin (b-Lg) at pH 7 andpH 3.5 (Martinez, Farias, & Pilosof, 2010). In addition, the dena-turation temperature of b-Lg (Martinez et al., 2010) as well as thefunctional properties of b-Lg (Gault & Fauquant, 1992; Martinezet al., 2010; Martinez, Sanchez, Patino, & Pilosof, 2009, 2012),whey protein isolate (WPI) (Outinen & Rantamaki, 2008; Thoma-Worringer, Siegert, & Kulozik, 2007) and whey protein concen-trate (WPC) (Veith & Reynolds, 2004) were changed in the presenceof CMP. These authors indicated that the extent of variations of

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e6456

functional properties is dependent on the ratio between CMP andother proteins in whey protein ingredients, pH, concentration andtype of salts. But the mechanism of action of CMP is not completelyunderstood and could explain the contradictory results on thegelling properties of whey proteins: whereas CMP improved firm-ness of b-Lg gels (Gault & Fauquant, 1992), an opposite trend wasreported for WPI and WPC gels (Outinen & Rantamaki, 2008; Veith& Reynolds, 2004). To progress on the control of whey proteingelling properties, the denaturation process of whey proteins in thepresence of CMP needs to be understood. However, to our knowl-edge the kinetics of heat-denaturation of whey proteins in thepresence of CMP and the structure of the aggregates formed are notaddressed yet. To fill the gap the influence of CMP:b-Lg molar ratioon the kinetics of heat denaturation of b-Lg at different pH valueswas undertaken and the size and some structural characteristics ofthe formed aggregates were analysed.

2. Materials and methods

2.1. Materials

b-Lg was obtained from a confidential industrial source. The b-Lg powder contained 93% protein (determined by Kjeldahl method)comprising 53% b-Lg A and 47% b-Lg B (determined by reversephase-high performance liquid chromatography on a PLRP-S,Phenomenex, Le Pecq, France), 4.3 � 0.2% moisture and about0.89 � 0.04% ash (both determined according to Schuck, Dolivet, &Jeantet, 2012). CMP was prepared in-house according to Gault andFauquant (1992). The CMP powder contained 71 � 0.3% poly-peptides (determined by Kjeldahl method) mainly CMP A variantwith 0, 1 or 2 phosphate residues, determined by mass spectrom-etry according to Molle and Leonil (1995), 12.8� 0.5% moisture andabout 6.8 0.15% ash (both determined according to Schuck et al.,2012).

NaCl was from Grosseron-Labogros (Saint Herblain, France),trifluoroacetic acid (TFA), NaOH, glycine, sodium dodecyl sulphate(SDS) and Tris buffer were from SigmaeAldrich (Saint-Quentin-Fallavier, France), HCl was from VWR international-Prolabo (Fon-tenay sous bois, France), acetonitrile (high performance liquidchromatography grade) and acetic acid were from Fischer scientific(Loughborough, UK), and sodium acetate (trihydrate) was fromPanreac (Castellar de Vallès, Spain). All chemicals for electropho-resis were from Bio-Rad (Marnes-la-coquette, France) except lowand high molecular weight markers that were from AmershamBiosciences (Little Chalfont, UK).

2.2. Protein sample preparation

Stock solutions of b-Lg and CMP solutions were prepared at20 g L�1 in 30 mM NaCl. The pH of the solutions was adjusted at pH3.0, 4.0, 5.0, 6.0 or 6.7 using 0.2 M HCl or 0.1 M NaOH. Protein so-lutions and 30 mM NaCl were subsequently mixed adequately toobtain b-Lg solutions at 10 g L�1 (0.54 mM) containing 0, 0.27, 0.54or 1.08 mM CMP (using an averaged molecular mass of 6.8 kDa)corresponding to CMP:b-Lg molar ratio of w0, 0.5, 1, and 2respectively.

Protein samples at different CMP:b-Lg ratio were dialysedagainst a NaCl (30 mM) stock solution using a 100e500 Da nominalcut-off dialysis bag (Spectra/Por, Rancho Domingez, CA, USA) at25 �C for 2 h and at 4 �C overnight to prevent variability in themineral content between protein samples. Preliminary experi-ments indicated that b-Lg and CMP were fully retained by thedialysis membrane. After dialysis, the concentration of b-Lg wasdetermined by absorbance at 278 nm using 3278 ¼ 0.96 L g�1 cm�1

and it was adjusted in all protein solutions at 0.22mMwith the NaCl

stock solution (30 mM). CMP does not absorb at 280 nm. The pH ofprotein samples were checked and if necessary adjusted to therequired value.

A series of tightly capped tubes containing 1 mL of sample wereheated in a water bath; the holding temperature ranged from 65 to95 �C. At various time periods, tubes were removed and immedi-ately cooled on ice. After cooling a volume of 200 mL of sample wasused to analyse the denaturation of b-Lg. A volume of 20 mL of ac-etate/acid acetic buffer (0.5 M, pH 4.6) was added to the 200 mLtaken above to induce the precipitation of denatured proteins.Denatured proteins were removed by centrifugation for 10 min at12,000� g using an Eppendorf 5415C Microcentrifuge (ScientificSupport, Hayward, CA, USA). The supernatant was analysed byreversed phase-high performance liquid chromatography (RP-HPLC) for quantification of the residual native proteins (soluble atpH 4.6). For aggregate analysis, heating times were chosen toensure denaturation of about 70% of b-Lg molecules.

2.3. Reversed phase-high performance liquid chromatography

Proteins were analysed by RP-HPLC on a PLRP-S column(150 � 4.6 mm) (Polymer laboratories, Amherst, MA, USA). Thecolumn was connected to a Waters chromatography system (Mil-ford, MA, USA), consisting of a Waters 2695 Separation Module, aWaters 2487 Dual l Absorbance Detector and an Empower chro-matography application software. The column was equilibratedwith 80% of solvent A (Milli-Q water containing 0.106%, v/v, TFA)and 20% solvent B (HPLC grade-acetonitrile:Milli-Q water (80:20, v/v) mixture containing 0.1% (v/v) TFA). A volume of 110 mL of thesupernatant prepared as outlined above was diluted with 890 mL ofsolvent A and 20 mL of the mixture were injected in the column. Alinear gradient started with 20% of solvent B to reach 80% over25 minwas used for protein elution. Protein separation was carriedout at 40 �C at a flow rate of 0.8 mL min�1. UV absorption wasmeasured at 214 and 280 nm.

2.4. Turbidity measurements

The change of protein sample turbidity induced by heating wasmeasured at 600 nm on a UV/visible spectrophotometer UV MC2

(SAFAS, Monaco). Sample turbidity (s) was determined using thefollowing relationship:

s ¼ 2:303� OD=l (1)

where OD is the optical density of the sample and l is the light pathlength in the cell (1 cm).

2.5. Dynamic light scattering

Particle size was measured by dynamic light scattering using aZetasizer NanoZS apparatus (Malvern Instruments, Malvern, UK).Unheated samples were filtered through a 20-mm filter to removedust particles while heated samples were analysed without filtra-tion. Protein samples were placed in a 10 � 10 mm disposablepolystyrene cell (Sarstedt, Nümbrecht Germany) equilibrated at20 �C for measurements. Intensity of scattering is detected at 173�

(backscatter detection) to reduce multiple scattering. Experimentaldata were analysed using the general purpose model. Results werethe mean of 13 runs. From the diffusion coefficient extracted fromthe fit of the correlation curve, the hydrodynamic diameter ofparticles was calculated by using the Stockes-Einstein equation. Forvolume-size representation, a refractive index of 1.45 was used. Allthe samples were measured in triplicate.

0.00

0.20

0.40

0.60

0.80

1.00

0 10 20 30

Heating time, t (min)

Frac

tion

of so

lubl

e β-

LG

at p

H 4

.6

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30

Heating time, t (min)

(Ct/C

0)-0

.5

0.00

0.20

0.40

0.60

0.80

1.00

0 10 20 30

Heating time, t (min)

Frac

tion

of so

lubl

e -L

G a

t pH

4.6

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30

Heating time, t (min)

(Ct/C

0)-0

.5

Fig. 1. Fraction of soluble native b-lactoglobulin (b-Lg) variant A (circles) and B (tri-angles) in the absence (filled symbols) and presence (open symbols) of casein-omacropeptide (CMP; CMP:b-Lg molar ratio of 2) as a function of heating time at 80 �C,pH 6.7. Inset: plots of (Ct/C0)(�0.5) against heating time; straight lines represent a re-action order of 1.5.

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e64 57

2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) under non-reducing conditions was performed using aMini Protean II system (Bio-Rad, Marnes-la-coquette, France) usinga 4e15% acrylamide gradient separating gel (Mini-Protean TGXTM,BioRad). Protein samples (10 mL) were diluted 10 folds with thedenaturing buffer (77.975% 0.0625 M TriseHCl pH 6.8; 20% glycerol;2% SDS; 0.025% bromophenol blue). A volume of 10 mL (corre-sponding to 4 mg of b-Lg) was loaded in the sample slots and themolecular entities were separated at 200 V for 30 min using a 0.3%(w/v) Tris, 1.44% (w/v) glycine, 0.1% SDS buffer. Low (14.4e97 kg mol�1) and high (53e220 kg mol�1) molecular weightmarker kits were used for molecular weight calibration. The gelswere stained with Coomassie Brillant Blue G250 and scanned byImage Scan II (Amersham Biosciences, Little Chalfont, UK).

2.7. Determination of kinetic parameters

The kinetic parameters (order of reaction; rate constant) ofdisappearance of native b-Lg A and B (soluble fraction at pH 4.6quantified by RP-HPLC) were determined after integration of thegeneral rate equation (Van Boekel, 1996):

��dCtdt

�¼ kn � Cn

t (2)

which gives for a reaction order ns 1

�CtC0

�¼ ð1� ðn� 1Þ � k� tÞð 1

1�nÞ (3)

or

�CtC0

�ð1�nÞ¼ 1� ðn� 1Þ � k� t (4)

where k ¼ kn � Cn�10 .C0 (g L�1) is the initial concentration of b-Lg

and Ct (g L�1), the concentration of native b-Lg at heating time t; n isthe reaction order; kn (g1�n Ln�1 s�1) is the rate constant that isdependent of the reaction order and k (s�1), the rate constant that isindependent of the reaction order. Equation (3) is commonly usedto extract the rate constant of denaturation of b-Lg from the slope ofthe plot (Ct/C0)(1�n) against t.

The denaturation activation energy of b-Lg (Ea, J mol�1) wasdetermined from the slope of the representation of the evolution ofb-Lg denaturation rate constant logarithm with inverse of tem-perature (Arrhenius plot):

ln k ¼ ln k0 ��

EaR� T

�(5)

where k0 (s�1) is the denaturation frequency factor; R, the universalgas constant (8.314 J mol�1 K�1) and T, temperature (K).

For b-Lg, this representation is characterised by two linear partsseparated by a sharp bend at Arrhenius critical temperature (Tc). Tcwas determined analytically from Equation (5) applied to the twolinear parts of the Arrhenius plot; it corresponded to the inter-ception of the two linear parts. The linear parts refer to 2 differentkinetics limitations: in the high temperature range (T > Tc), thedenaturation kinetics is limited by protein aggregation (aggrega-tion-limited reaction) and in the low temperature range (T < Tc),only a fraction of b-Lg molecules are unfolded and prone to ag-gregation limiting the denaturation kinetics (unfolding-limitedreaction) (Tolkach & Kulozik, 2007).

3. Results

The kinetic parameters of b-Lg denaturation (reaction order andrate constant) in the absence and presence of various concentra-tions of CMP were determined from the reduction in native b-Lg(soluble fraction at pH 4.6) on heating using equation (2). Typicalresults are given on Fig. 1: the reduction in native b-Lg A and B wasaccelerated in the presence of CMP at pH 6.7 (Fig. 1) and this wasthe case for all other pH values tested (3.0, 4.0, 5.0 and 6.0). In thefollowing sections, only results obtained at pH 4.0 and 6.7 will bereported. At pH 4.0 the influence of CMP on the denaturation of b-Lg was the highest. At pH 4.0, which corresponds to pH value ofindustrial acid whey, the net charge of b-Lg is positive and it isnegative for CMP. In contrast, at pH 6.7 the net charges of b-Lg andCMP are negative.

3.1. Influence of caseinomacropeptide on the reaction order of b-lactoglobulin denaturation

In the absence of CMP, the kinetics of disappearance of native b-Lg on heating were best fitted using a reaction order of w1.5 forboth pH 4.0 and 6.7 and both b-Lg variants. At pH 6.7, the reactionorder of disappearance of native b-Lg did not change in the pres-ence of CMP until a CMP:b-Lg molar ratio of 2:1, as indicated by thestraight lines yielded from the plots of (Ct/C0)�0.5 versus t (innerplot on Fig. 1). In contrast, at pH 4.0, the reaction order of the ki-netics of disappearance of native b-Lg increased continuously form

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e6458

w1.5 in the absence of CMP to >2 when the CMP:b-Lg molar ratiowas�1. The increase of the reaction order was observed for both b-Lg variants at all heating temperatures. The evolution of the reac-tion order versus CMP:b-Lg molar ratio at 80 and 85 �C is indicatedon the inner plot of Fig. 2a and c, respectively.

3.2. Influence of caseinomacropeptide on the rate constant ofb-lactoglobulin denaturation

As the reaction order varied according to the experimentalconditions, the rate constant k (s�1), which is independent of thereaction order, was used to characterise the kinetics. In the absenceof CMP, the rate constant of disappearance of native b-Lg issignificantly lower at pH 4.0 than at pH 6.7. The disappearance of

Fig. 2. Rate constants for the heat-induced denaturation of b-lactoglobulin (b-Lg) versus cas(c) 85 �C, pH 4.0; (d) 85 �C, pH 6.7. Inset: reaction order of the kinetics of heat-denaturationsymbols; dotted lines are guide to the eyes.

native b-Lg was accelerated with increasing CMP concentration forboth b-Lg variants and for each experimental condition investi-gated. CMP had a higher impact on b-Lg denaturation at pH 4.0 thanat pH 6.7 (Fig. 2).

At pH 4.0, the rate constants (k) of disappearance of native b-Lg inthe absence of CMPwas almost 10 times higher at 85 �C than at 80 �Cfor both variants A and B. Furthermore the rate constants wereincreased in the presence of CMP. At 80 �CandaCMP:b-Lgmolar ratioof 2:1, k values increased 4 and 2.5 fold for b-Lg A and B, respectively(Fig. 2a). The increase of rate constants was accentuated at 85 �C by afactor 15 for b-Lg A and a factor 8 for b-Lg B in the presence of a two-fold molar excess of CMP over b-Lg, respectively (Fig. 2c).

At pH 6.7, the rate constant of disappearance of native b-Lg A islower than that of b-Lg B (Fig. 2b,d). Increasing CMP concentration

einomacropeptide:b-Lg (CMP:b-Lg) molar ratio at: (a) 80 �C, pH 4.0; (b) 80 �C, pH 6.7;of b-Lg versus CMP:b-Lg molar ratio b-Lg variant A, open symbols; b-Lg variant B, filled

Table 1Denaturation activation energy (Ea, kJ mol�1) of b-lactoglobulin (b-Lg) variant A andB below and above Arrhenius critical temperature at different pH and at variouscaseinomacropeptide:b-lactoglobulin (CMP:b-Lg) molar ratios.a

pH CMP:b-Lg molar ratio b-Lg A b-Lg B

T < Tc T > Tc T < Tc T > Tc

4.0 0 458 144 440 1441 497 99 466 1152 570 62 524 89

6.7 0 318 194 352 1751 308 171 337 1432 294 151 314 137

a T< Tc, below Arrhenius critical temperature (Tc); T> Tc, above Arrhenius criticaltemperature.

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e64 59

had a much more limited effect on the kinetics of disappearance ofnative b-Lg as compared with pH 4.0. At 80 �C, the k valuesincreased only slightly for b-Lg A and doubled for b-Lg B in thepresence of 0.44 mM of CMP (CMP:b-Lg molar ratio of 2:1) (Fig. 2b).A similar trend was observed after heating at 85 �C (Fig. 2d).

The b-Lg denaturation Arrhenius plots at pH 4.0 and 6.7 at 65e95 �C is shown in Fig. 3. For both variants, Arrhenius critical tem-peratures (Tc) that were not affected by the presence of CMP, werearound 88 and 80 �C at pH 4.0 and pH 6.7, respectively. In theaggregation-limited temperature range (T > Tc) the denaturationactivation energy (Ea) of b-Lg variants A and B decreased withincreasing amount of CMP for both pH conditions (Table 1). In thistemperature range, Ea of b-Lg denaturation was higher at pH 6.7than at pH 4.0 and its decrease in the presence of CMPwas higher atpH 4.0 than at pH 6.7, i.e., �57% against �22%, respectively forvariant A with an addition of two fold excess of CMP. In theunfolding-limited temperature range, Ea of b-Lg denaturationincreased with CMP concentration at pH 4.0 while it decreased atpH 6.7. At pH 4.0, addition of two fold excess of CMP induced anincrease of Ea by about þ16 to þ20%. The observed decrease of Eavalue at pH 6.7 in the presence of CMP was slightly higher forvariant B compared with variant A (Table 1).

3.3. Influence of caseinomacropeptide on the appearance of b-lactoglobulin solutions

The visual appearance of b-Lg solutions in the absence andpresence of CMP before and after heating at 85 �C is shown in Fig. 4.Heating times, calculated from to the kinetic parameters deter-mined previously, were chosen to ensure denaturation of w70% of

Fig. 3. Arrhenuis plots for the denaturation of b-lactoglobulin (b-Lg) variant A (a, c) and Bomacropeptide:b-Lg molar ratios of 0, 1, and 2, respectively; T, temperature in Kelvin � 10�

b-Lg in all samples. Heating did not change the turbidity of the CMPcontrol samples. All b-Lg solutions were transparent before heating(tubes 3, 5, 7). After heating, the solution appearance varied ac-cording to the pH and the CMP:b-Lg molar ratio. At pH 4.0 (Fig. 4a),the b-Lg solutions containing CMP (tubes 4, 6) got highly turbid assoon as they were heated for few seconds. Furthermore, a phaseseparation was observed at the end of the heating time and thiscontinued during storage. This resulted in a biphasic systemwith adense phase that looked like a highly hydrated precipitate and atransparent supernatant. In contrast, b-Lg solutions without CMPdeveloped a homogeneous, stable and slightly turbid appearanceon heating (tube 8).

At pH 6.7, the turbidity of the heated solutions decreased withincreasing CMP content. The measured turbidity varied from0.317� 0.015 in the absence of CMP (tube 8) to 0.112� 0.008 with a

(b, d) at pH 4.0 (a, b) and pH 6.7 (c, d). Circles, squares and triangles are for casein-3.

Fig. 4. Photographs of the protein solutions at (a) pH 4 and (b) pH 6.7 before (tubes 1, 3, 5, 7) and after (tubes 2, 4, 6, 8) heating at 85 �C. Heating time was chosen to give 70% heatdenatured b-lactoglobulin (b-Lg). Tubes 1 and 2, control caseinomacropeptide (CMP) samples; tubes 3 and 4, samples with CMP:b-Lg molar ratio of 1; tubes 5 and 6, samples withCMP:b-Lg molar ratio of 2; tubes 7 and 8, control b-Lg solutions without CMP; tube 9, heated and cooled b-Lg solutions before addition of CMP to reach a CMP:b-Lg molar ratio of 1.

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e6460

CMP:b-Lg molar ratio of 1 (tube 4) and 0.035� 0.010 with a CMP:b-Lg molar ratio of 2 (tube 6).

In additional experiments, CMPwas added at room temperaturein previously heated and cooled b-Lg solutions in the absence ofCMP (similar conditions than for tubes 8). After CMP addition, theCMP:b-Lg molar ratio was 1:1 (tubes 9). At pH 4.0, the turbidity ofthe heated and cooled b-Lg sample increased immediately afteraddition of CMP and slow phase separation occurred after fewminutes (tube 9 on Fig. 4a). At pH 6.7, no CMP effect was detectedand the slight turbidity decreasewas due to the dilution effect (tube9 in Fig. 4b).

3.4. Influence of caseinomacropeptide on the size of b-lactoglobulinaggregates formed on heating

Prior to heating, the hydrodynamic diameter (Dh) of the parti-cles in solution, determined from the peak mean intensity on DLS,changed slightly at pH 4.0 and 6.7 in the absence and presence ofCMP; A bimodal distribution was observed but more than 85% ofthe signal intensity corresponded to lower size particles. It has to benoted that area intensity weighs the particles according to their Dh;the area intensity of lower size particles are underestimatedcompared with largest one regarding their weight fraction. Using arepresentation in volume only one peak corresponding to lowersize particles was observed for unheated samples (Fig. 5). At pH 4.0,the predominant peak in the intensity-size representation had amaximum value at 10.7 nm in the absence of CMP and 7.7 nm and7.4 nm in the presence of CMP at CMP:b-Lg molar ratio 1:1 and 2:1,

respectively. At pH 6.7, the predominant peak was at 7.2 nm in theabsence of CMP and 7.2 nm and 8.5 nm in the presence of CMP atCMP:b-Lg molar ratio 1:1 and 2:1, respectively. Solutions of pureCMP used as control showed a bimodal distribution in an intensity-size representation with the peak of higher intensity (>60%) at5.4 nm and 5.2 nm at pH 4.0 and pH 6.7, respectively. This peak wasthe only peak detectable using a volume-size representation(Fig. 5).

The size of the aggregates formed on heating at 85 �C wasanalysed in samples with and without CMP and containing 30% ofresidual native b-Lg. The size of the aggregates depended on the pHand on the CMP concentration in b-Lg solution (Table 2). In theabsence of CMP, two populations of aggregates with Dh of125 � 38 nm (intensity of 41 � 6%) and 861 � 238 nm (intensity of50 � 9%) were detected after heating at pH 4.0. For all otherconditions only one predominant population (>85% in peak in-tensity) was observed (Table 2). At pH 4.0, the Dh of the aggregateswas significantly different with andwithout CMP. In the presence ofCMP, the Dh of the aggregates was too large (>5 mm) to be deter-mined correctly (at the limit of the apparatus resolution). Incontrast, the Dh of the aggregates formed on heating at pH 6.7decreased slightly in the presence of CMP (Table 2).

The addition of CMP to a pre-heated and cooled b-Lg solutionmodified the size distribution of the aggregates at pH 4.0; the so-lution conserved a bimodal distribution but only 15 � 5% of thesignal intensity came from the particles of lower size (121� 26 nm)after addition of CMP. A proportion of 85 � 5% of the signal in-tensity was due to particles of around 5 mm. In contrast, the size

Fig. 5. Volume e size representation for unheated b-lactoglobulin (b-Lg) solutions at (a) pH4 and (b) pH 6.7 in the absence (open square) and presence of caseinomacropeptide(CMP) at a CMP:b-Lg molar ratio of 1 (open triangle) and 2 (open diamond) and for unheated CMP solution (full circle).

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e64 61

distribution of the aggregates was not significantly changed whenthe same experiment was performed at pH 6.7.

Table 2Size distribution parameters of the particles in protein solutions at differentcaseinomacropeptide:b-lactoglobulin (CMP:b-Lg) molar ratios heated at differentpH.a

pH CMP:b-Lgmolar ratio

Dh (nm) Peak intensity (%) Width (nm)

4.0 0 125 � 38 41 � 6 38861 � 238 50 � 9 245

1 5141 � 725 100 e

2 5188 � 219 100 3296.7 0 240 � 7 88 � 5 147

1 193 � 8 85 � 3 1162 178 � 8 90 � 1 79

a Dh, hydrodynamic diameter.

3.5. Influence of caseinomacropeptide on the structure of b-lactoglobulin aggregates

The aggregates formed on heating were dissociated by SDS(under non reducing conditions) and the resulting molecular en-tities that are stabilised by covalent bonds, were analysed by SDS-PAGE. For heated samples (CMP:b-Lg molar ratio of 0, 1, 2),monomers (M) and oligomers (dimers, D; trimers, T; tetramer, t;pentamers, P) of b-Lg, intermediate molecular entities (I) enteringthe resolving gel and large aggregates (L) stuck in sample slot wereobserved (Fig. 6). Additionally, for both pH conditions, the bandcorresponding to CMP was clearly identified in lanes containingsamples with a CMP:b-Lg molar ratio of 1:1 and 2:1. Differences inband intensity were observed according to the pH of heating andthe concentration of CMP. Whatever the amount of CMP, theamount of oligomers was higher at pH 6.7 than at pH 4.0. Increasingthe amount of CMP in b-Lg samples resulted in the formation ofsmaller covalent aggregates as shown by the higher intensity of thebands corresponding to dimers, trimers and tetramers of b-Lg.Additionally, the intensity of the band corresponding to aggregatesstuck in the sample slots decreased with increasing CMP

concentration and at pH 4.0 it almost disappeared completely forCMP:b-Lg molar ratio of 2:1. At pH 6.7, large aggregates were stillpresent but their intensity decreased and the intensity of the smearon the top of the resolving gel, corresponding to intermediatemolecular entities, increased with increasing CMP concentration.Results similar to those described above were obtained at lower(75 �C) and higher (95 �C) heating temperature (data not shown).

4. Discussion

CMP was shown to affect the kinetic of denaturation/aggrega-tion of b-Lg and the nature of formed aggregates. CMP effect wasdependent on the pH of the solution and it acted in the same wayon both b-Lg variants. In the absence of CMP, b-Lg variant A waspreferentially heat-denatured at pH 4.0 while b-Lg variant B waspreferentially heat-denatured at pH 6.7 in agreement with the re-sults reported by O’Kennedy, Mounsey, Murphy, Pesquera, andMehra (2006). The presence of CMP did not change this observedpH-dependence heat-denaturation preference.

4.1. Caseinomacropeptide promotes the heat-denaturation ofb-lactoglobulin

CMP sped up the denaturation kinetics of b-Lg whatever thetemperature and the pH investigated. Similar results were obtainedfor b-Lg heated with other peptides (Kosters, Wierenga, de Vries, &Gruppen, 2011, 2013). This may be explained by an interactionbetween CMP and b-Lg in solution (slight modification of the Dh ofunheated b-Lg) as already suggested by Martinez et al. (2009,2010). These authors indicated that CMP and b-Lg interacted byelectrostatic and hydrogen bonds at pH 7.0 and pH 3.5, pH valuesclose to the ones selected in the present study. However thesemolecular interactions are of different strengths according to pHdue to different net charge for b-Lg and CMP molecules. Never-theless, we failed to detect such interactions at molecular levelusing fluorescence, and a low level of interaction was detected byisothermal titration calorimetry (ITC) at pH 6.7 only and at 25 �C(data not shown). This would suggest that CMP contribution to b-Lg

Fig. 6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of caseinomacropeptide (lane CMP) and b-lactoglobulin samples unheated (lane b-Lg) and heated at 85 �C withvarious CMP:b-Lg molar ratio (lanes CMP:b-Lg 0, 1, 2); heated samples contained 70% of heat-denatured b-Lg. Mononers (M) and oligomers (dimers, D; trimers, T; tetramer, t;pentamers, P) of b-Lg, intermediate molecular entities (I) entering the resolving gel and large aggregates (L) are indicated. Lanes LMW and HMW are low molecular mass (14.4e97 kDa) and high molecular mass markers (53e220 kDa), respectively.

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e6462

denaturation occurred mainly during heating when b-Lg moleculesare in the unfolded state.

The change in the Dh of the aggregates of b-Lg at pH 4.0 after theaddition of CMP supports this hypothesis. The effect of CMP on thekinetics of denaturation of b-Lg was higher at pH 4.0 probablybecause favourable electrostatic-driven associations exist betweenCMP (negatively charged) and b-Lg (positively charged). This couldalso explain the opposite pH-dependent evolution of b-Lg dena-turation Ea in the unfolding-limited temperature range withincreasing amount of CMP even if the rate constant increased forboth pH conditions: Ea increased at pH 4.0 but decrease at pH 6.7with increasing CMP content. The unfolding reaction is reversibleand the rate constant of b-Lg denaturation in the unfolding-limitedtemperature range depends on the barrier to b-Lg unfolding (Ea)but also on the factors affecting the fraction of unfolded b-Lg thatreverses to the native state on cooling (Tolkach & Kulozik, 2007). Inthe absence of CMP, calculated Ea in the unfolding-limited tem-perature range was in the range of values reported in the literature,which vary between 280 kJ mol�1 and 430 kJmol�1 (Le Bon, Nicolai,& Durand, 1999). The decrease of Ea at pH 6.7 indicates a favourableb-Lg unfolding suggesting that when CMP and b-Lg carry the samenet charge, the interaction destabilize the native state of b-Lg. Thisresult is in agreement with the decrease of the denaturation tem-perature of b-Lg at pH 7 from 74.2 � 0.1 to 71.5 � 0.1 (Martinezet al., 2010). In contrast, the increase of b-Lg denaturation Ea inthe unfolded-limited temperature range at pH 4.0 indicated thatCMP stabilised the structure of native b-Lg, which requires moreenergy to unfold. At pH 4.0, b-Lg and CMP are oppositely chargedand the repulsive electrostatic interactions on b-Lg surface werereduced, reducing consequently the tendency of native b-Lg tounfold on heating. Similar structural stabilisation was already putforward for negatively charged b-Lg by increasing ionic strength oradding calcium ions (Petit, Herbig, Moreau, & Delaplace, 2011;Schokker, Singh, Pinder, & Creamer, 2000). Nevertheless at 80 �C(unfolding-limited temperature range) the rate of disappearance ofnative b-Lg was increased as shown on Fig. 2a. An explanation isthat CMP interacts more strongly with b-Lg in an unfolded state asdiscussed above. This interaction traps b-Lg in an unfolded state,preventing its refolding to the native state on cooling. This inter-action also reduces the barrier to protein aggregation due to the

shielding of b-Lg positive charges by the residual negative chargesof CMP at pH 4.0. Hence, the fraction of unfolded b-Lg prone toaggregation would increase in the presence of CMP. As the 2 re-actions (b-Lg unfolding and aggregation of unfolded b-Lg) acts onthe rate constant of b-Lg denaturation in the unfolding-limitedtemperature range due to the strong overlapping of the 2 re-actions, the change in the aggregation behaviour of b-Lg in thepresence of CMP enhances its overall heat-denaturation kinetics atpH 4.0.

In the aggregation-limited temperature range, all b-Lg mole-cules are unfolded and the rate of aggregation of unfolded mole-cules is dependent on the aggregation barrier (Ea), which varieswith the strength of the interactions between b-Lg molecules. CMPinduced a decrease of the b-Lg denaturation Ea (aggregation bar-rier) at pH 4.0 and pH 6.7. This result was expected at pH 4.0 due tothe shielding of b-Lg positive charges by CMP negative charges, butit is rather surprising at pH 6.7 where the negative charges of CMPwould reinforce the repulsive interactions between b-Lg molecules.However, it has to be kept in mind that even in the aggregation-limited temperature range, a fraction of unfolded b-Lg reverses toits native form on cooling (Tolkach & Kulozik, 2007). The in-teractions between CMP and unfolded b-Lg may trap a fraction of b-Lg molecules under irreversibly unfolded forms. This reaction goesin the direction of an apparent higher rate constant of aggregationand a decrease of b-Lg denaturation Ea.

4.2. Caseinomacropeptide affects the structure of b-lactoglobulinaggregates

The structure of the aggregates was affected by the presence ofCMP in a pH-dependentmanner, which is depicted schematically inFig. 7. At pH 6.7 CMP hindered the assembly of unfolded b-Lg andsmall covalent aggregates into larger structures as shown by thedecrease of the Dh of the aggregates. It is well known that caseinsmodify the structure of thewhey protein aggregates by a chaperon-like behaviour (Guyomarc’h, Nono, Nicolai, & Durand, 2009; Kehoe& Foegeding, 2011; O’Kennedy & Mounsey, 2006; Yong &Foegeding, 2008); this behaviour results from the well-separatedhydrophobic and hydrophilic domains and the intrinsically un-structured properties of the caseins (Farrel, Qi, & Uversky, 2006).

Fig. 7. Schematic representation of how caseinomacropeptide (CMP) affects thedenaturation/aggregation process of b-lactoglobulin (b-Lg) as a function of pH: [N],native b-Lg; [U], unfolded b-Lg; [SA], small aggregates; [LA], large aggregates. Grey,negatively charged molecules; white, positively charges molecules.

T. Croguennec et al. / International Dairy Journal 36 (2014) 55e64 63

The hydrophobic patches of the caseins establish hydrophobic in-teractions with the hydrophobic patches exposed on the surface ofwhey proteins during heat-induced denaturation, whereas thecharged residues of the caseins stabilise the aggregates. It is prob-able that the mechanism of action of CMP observed in the presentstudy is similar to the mechanism of action of caseins at pH 6.7despite the less hydrophobic character of CMP molecules due tohigh charge density of the acidic residues and sialic acid moieties(Kreuss et al., 2009). Consequently, the high charge density of CMPwould protect unfolded b-Lg molecules from extensive covalentand non-covalent aggregation at pH 6.7, as supported by the higherfraction of small covalent aggregates, the reduction of Dh of theaggregates and the decrease of b-Lg solution turbidity. Thus, theformed aggregates would be preferentially composed of b-Lgmolecules surrounded with CMP monomers but this needs to beconfirmed.

In contrast, CMP promoted the non-covalent assembly ofunfolded b-Lg molecules and small covalent aggregates at pH 4.0where CMP and b-Lg carry opposite charges. This results in phaseseparation and a drastic increase of the size of the aggregates in thepresence of CMP. This suggests that the electrostatic interactionsbetween CMP and b-Lg contributed to the increasing size of b-Lg/CMP aggregates. However electrostatic interactions are not suffi-cient to explain the large size increase of the aggregates becausesuch structures were not observed without heat treatment. Werather assume that in a first step, CMP establish hydrophobic in-teractions with unfolded b-Lg molecules limiting the growth of thecovalent aggregates (as indicated by SDS-PAGE) as alreadyobserved at pH 6.7. Secondly, the neutralisation of b-Lg charges atthe surface of small aggregates by CMP molecules reduced thestabilising electrostatic barrier favouring further non covalent as-sociation into very large aggregates. The electrostatic-driven ag-gregation reaction was confirmed when CMP was added to asolution of b-Lg aggregates. Under this condition large aggregateswere formed and a phase separation occurred. In contrast to whathappens at pH 6.7, aggregates containing small b-Lg aggregates andunfolded b-Lg molecules connected with CMP are favoured at pH 4.The active contribution of CMP in the building of the aggregatesalso explains the change of the reaction order of b-Lg denaturationat pH 4.0.

5. Conclusions

To conclude, results highlighted in this study could explainchanges observed in the gelling properties of whey protein in-gredients according to their origin and preparation procedures byimpacting on the unfolding, and aggregation of b-Lg. Since CMP

effect occurred on a wide range of pH and temperature, and isdependent on CMP concentration its precise quantification inwheyprotein ingredients would improve the control of whey proteinfunctionalities. In another hand the prevalent interaction of CMPwith unfolded b-Lgmolecules that is observed at room temperatureand pH 4.0 constitutes another probable source of variability ofwhey protein functionalities.

Acknowledgements

The authors thank Valerie Briar-Bion and Julien Jardin for massspectrometry analysis of CMP.

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