Structure and surface properties of the serum heat-induced protein aggregates isolated from heated skim milk

Structure and surface properties of the serum heat-induced protein aggregates isolated from heated skim milk

ARTICLE IN PRESS International Dairy Journal 16 (2006) 303–315 www.elsevier.com/locate/idairyj Structure and surface properties of the serum heat-in...

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International Dairy Journal 16 (2006) 303–315 www.elsevier.com/locate/idairyj

Structure and surface properties of the serum heat-induced protein aggregates isolated from heated skim milk Karine Jean, Marie Renan, Marie-He´le`ne Famelart, Fanny Guyomarc’h UMR 1253 Science et Technologie du Lait et de l’Œuf, INRA-Agrocampus Rennes, 65 route de St. Brieuc, 35 042 Rennes Cedex, France Received 29 September 2004; accepted 10 April 2005

Abstract Serum heat-induced milk protein aggregates were isolated from heated skim milk. Protein analysis showed that the aggregates were essentially composed of whey protein and k-casein linked mainly by disulfide bridges. The hydrodynamic diameter of the aggregates, as measured by dynamic light scattering (DLS), was about 70 nm. Multi-angle DLS analysis and electron microscopy indicated that they were almost of spherical shape. Measurement of the turbidity, size and zeta potential of the aggregates in milk permeate at pH 2–9 showed that their apparent isoelectric point was 4.5. Surface charge in milk permeate at pH 7.0 was 17 mV. 8-anilino-1-naphthalene sulphonic acid (ANS)-binding indicated that surface hydrophobicity of the aggregates was higher than those of native casein micelles, which suggested that their precipitation on acidification may be initiated before reaching pH 4.5. r 2005 Published by Elsevier Ltd. Keywords: Milk protein; Denaturation; Aggregation; Properties

1. Introduction In most dairy processes, thermal treatment of milk is an essential operation aiming at increasing shelf life and improving food safety of the final product. In the specific case of acid gels such as yoghurt and Quarg-type cheeses, it also improves functional properties of the whey protein. It is now known that heat treating milk above 60 1C leads to the denaturation of the whey proteins, resulting in their interaction with each other and with k-casein to form heat-induced serum and micelle-bound protein aggregates, respectively, in the serum phase of milk, and on the surface of the casein micelles (Anema & Li, 2000, 2003; Corredig & Dalgleish, 1999; Dannenberg & Kessler, 1988; Pearse, Linklater, Hall, & McKinlay, 1985; Smits & Van Brouwershaven, 1980; Vasbinder et al., 2003). The occurrence of heat-induced aggregates Corresponding author. Tel.: +33 223 48 53 32; fax: +33 223 48 53 50. E-mail address: [email protected] (F. Guyomarc’h).

0958-6946/$ - see front matter r 2005 Published by Elsevier Ltd. doi:10.1016/j.idairyj.2005.04.001

in milk has been related to high gelation pH (5.3), high firmness, low porosity and low syneresis of acid gels (Anema, Lee, Lowe, & Klostermeyer, 2004; Famelart, Tomazewski, Piot, & Pezennec, 2004; Lucey, Tamehana, Singh, & Munro, 1998; Lucey, Teo, Munro, & Singh, 1997; Vasbinder, van Mil, Bot, & de Kruif, 2001). These effects were mostly studied in complex systems and only little knowledge of the actual interactions between aggregates and/or with casein micelles during gelation could be concluded from varying the milk composition (Anema et al., 2004; Famelart et al., 2004; Guyomarc’h, Queguiner, Law, Horne, & Dalgleish, 2003a; Lucey et al., 1998; Schorsch, Wilkins, Jones, & Norton, 2001; Vasbinder et al., 2003). In contrast to most of the results in the previous literature, it has recently been shown, using a separation procedure that does not underestimate the serum aggregates, that the latter appear to have a larger effect on both gelation pH and final gel strength than micelle-bound aggregates (Anema et al., 2004). As a possible explanation on the positive impact that heat-induced serum and micelle-bound aggregates have

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on acid gelation, it has been speculated that they have a higher isoelectric point than that of micelles (5.4 versus 4.7) and would induce an early destabilisation of the milk protein system (Guyomarc’h et al., 2003a; Vasbinder et al., 2001). Lucey et al. (1998) suggested that the two-step profile of the increase in elastic modulus, G’, of heated skim milk during acidification described a transition from an early, high-pH gelation of the denatured whey proteins to gelation of the casein fraction. It has also been proposed that micelle-bound aggregates hinder the stabilising action of k-casein (Horne & Davidson, 1993) or aid in micelle coalescence (Mottar, Bassier, Joniau, & Baert, 1989). The occurrence of heat-induced aggregates as a new significant group of precipitable protein particles in the milk may increase network connectivity and water retention, and introduce rigid covalent bonds in the gel structure (Guyomarc’h et al., 2003a). In the present study, heatinduced protein aggregates were isolated from the serum phase of heated skim milk and some of their properties were characterised. Using this approach, we hope to provide a better understanding of the role of these aggregates on some heat-induced changes in milk and milk processing.

2. Materials and methods 2.1. Reconstituted skim milk Milk was reconstituted using ultralow heat skim milk powder at a level of 100 g L1 in distilled water at 40 1C, and sodium azide (NaN3) was added as preservative at a level of 0.5 g L1. The ultralow heat skim milk powder had a Whey Protein Nitrogen Index (WPNI) of 9.5 and was prepared as described by Schuck et al. (1994a). 2.2. Milk ultrafiltration (MUF) permeate MUF was prepared by ultrafiltration of fresh pasteurised milk on a 8 kDa TAMI membrane (Tami Industries, Nyons, France) and stored at 5 1C after addition of 0.5 g L1 sodium azide. In order to measure turbidity, size and zeta potential of the heat-induced serum aggregates at various pH values, the pH of the MUF (originally 6.7) was adjusted to values ranging from 2 to 9 using 5 M NaOH and 5 M HCl. At pH 8 and 9, permeate salts precipitated and the solutions were filtered with further pH adjustment before use. 2.3. Imidazole buffer Imidazole buffer was prepared according to Dalgleish (1984) to final concentrations of 20 mM imidazole,

50 mM NaCl, 5 mM CaCl2 and 10 mM NaN3. The pH was adjusted to 7.0 using 5 M NaOH and 5 M HCl. In order to measure the size and zeta potential of artificial or milk heat-induced serum protein aggregates at various pH values, the pH of the buffer was adjusted to values ranging from 2 to 9 using 5 M NaOH and 5 M HCl. Further adjustment was made when necessary on the day of analysis, at least 30 min before use. 2.4. Preparation of ‘‘artificial’’ heat-induced protein aggregates In order to have heat-induced protein aggregates without caseins other than k, aggregates were produced by heating a dispersion of standard k-casein, alactalbumin (a-La) and b-lactoglobulin (b-Lg). Starting solutions of 9 g L1 of k-casein, 9 g L1 of b-Lg and 4.5 g L1 of a-La were prepared in imidazole buffer and left with constant stirring overnight at 5 1C. A mixture of the three species was prepared by mixing equal volumes of each solution in a glass tube, and diluting to final concentrations of 1.8, 1.8 and 0.9 g L1 for kcasein, b-Lg and a-La, respectively. The final concentrations of these proteins were at the ratio found in milk, and were sufficiently diluted to slow aggregation down. The clear solution was split into two aliquots, one of which was heated at 80 1C for 2 min (+90 s of come-up time) and became whitish during heating. Imidazole buffer was used for this experiment as MUF salts readily precipitate on heating, at temperatures X60 1C. 2.5. Other materials The whey protein isolate (WPI), Protarmor 90, was purchased from Armor Prote´ines (St. Brice en Cogle`s, France). Native calcium phosphocaseinate (NPPC) was prepared as described by Schuck et al. (1994b). Standard k-casein was purchased from Charis Innovative Food Service Ltd. (Ayr, UK) and other standard proteins were from Sigma (St. Quentin Fallavier, France). 2.6. Heat treatment Two litres of reconstituted skim milk were heat treated at 90 1C for 10 min in a recirculating tubular heat-exchanger composed of an inox tubular coil (8 mm outer diameter, 7 m length) located in a thermostatically controlled water-bath and connected to a centrifuge pump (Micropump Inc., Vancouver, Canada). The flowrate was 135 L h1 and the flow was turbulent to ensure uniform heat transfer (Re3200). After heat-treatment, the milk was rapidly cooled to room temperature in ice water.

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The isolation of heat-induced protein aggregates from the serum phase of heated skim milk was performed at room temperature by loading 3.2 mL of filtered (1.2 mm, Pall Life Science, St. Germain en Laye, France) concentrated milk serum onto a Sephacryl S-500 HiPrep 26/70 350 mL-column (Amersham Biosciences, Orsay, France). The separation was performed under isocratic conditions using 0.1 M Tris, 0.5 M NaCl and 10 mM NaN3, pH 7, as the mobile phase. The flow-rate was 0.9 mL min1, and the absorbance was monitored at 280 nm. Fractions were collected at 5 min intervals between 170 and 240 min, corresponding to the elution of the heat-induced serum aggregate peak as indicated in Fig. 1a (interpreted after Guyomarc’h et al., 2003b). The eluate fractions were transferred to SpectraPor 1 dialysis tubing with a 6–8 kDa molecular mass cut-off (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). One volume of samples was dialysed extensively against three time 100 volumes of distilled water, at 5 1C for 12 h each time. The pH values at the end of the dialysis process were 6.1 in the dialysate and 5.4 in the dialysed fractions; as a consequence, uncontrolled precipitation sometimes occurred. The contents of the dialysis sacks were poured into plastic containers and freeze-dried in a RP2 V freeze-drier (SGD Se´rail, Argenteuil, France) to give aggregate powder. Fig. 1b shows typical elution profiles of 5 g L1 dispersions of the aggregate powder in milk ultrafiltrate. The late eluting peaks (orotic acid and other dialysable solutes) originated from the MUF (profile not shown). The results showed that some growth of the aggregates occurred between their separation from the milk serum by FPLC and their reconstitution, probably as a result of the dramatic environmental changes on dialysis and freeze-drying. The reconstituted suspensions were

0.8 0.4 0.0 50

100

150

50

100

150

(a)

200 250 300 Retention time (min)

0.3

400

450

dialysable solutes

reconstituted heatinduced milk serum aggregates

0.4

350

orotic acid

0.5

Opticaldensity at 280 nm (Absorbance Units)

2.8. Isolation of heat-induced protein aggregates from the serum phase of milk using size exclusion Fast Protein Liquid Chromatography (FPLC)

1.2

residual individual proteins

1.6

micellar material

Milk serum phase was prepared by ultracentrifugation of 15 mL-aliquots of heated skim milk at 33 000 g for 65 min at 20 1C in a Sorvall Discovery 90 SE centrifuge (Kendro Laboratory Products, Courtaboeuf, France). The supernatant was designated as ‘‘milk serum’’. The protein material of the serum was then concentrated six times by tangential filtration using a 10 kDa molecular mass cut-off cellulose Amicon membrane (Millipore, Molsheim, France) installed on a Filtron apparatus (Pall Life Science, St. Germain en Laye, France). Extensive diafiltration using imidazole buffer allowed reduction of the lactose content of the retentate to a negligible level.

305 heat-induced milk serum aggregates

2.0

Optical densityat 280 nm (Absorbance Units)

2.7. Separation and concentration of milk serum

exclusion peak

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0.2 0.1 0.0

(b)

200

250

300

350

400

450

Retention time (min)

Fig. 1. Typical size exclusion fast protein liquid chromatography (SEFPLC) elution profiles of (a) a supernatant prepared by ultracentrifugation of heat-treated (90 1C/10 min), ultrafiltered skim milk, and (b) freeze-dried isolated heat-induced milk serum aggregates dispersed at 5 g L1 in milk ultrafiltrate. The peak eluted between 170 and 240 min in Fig. 1a corresponds to heat-induced milk serum k-casein/ whey protein aggregates. See Section 2.8. for experimental details.

stable, except for the occasional occurrence of some visible particles that sedimented on standing (samples not used). 2.9. Reverse-phase high-performance liquid chromatography (RP-HPLC) The protein composition of the fractions was determined by RP-HPLC on an Apex wide-pore C18 column (25 cm length, 0.46 cm inner diameter, 7 mm bead diameter—Jones Chromatography, Hengoed, UK) using an adaptation of the method of Visser, Slangen, and Rollema (1986, 1991). Samples were prepared by diluting the samples to 20 mL 100 mL1 with denaturing buffer (7 M urea+20 mM bis–Tris propane; pH 7.5) to which 5 mL mL1 of b-mercaptoethanol was added. The samples were incubated at 37 1C for 1 h. Buffer A was 1.06 mL L1 trifluoroacetic acid (TFA) in ultrapure milliQ water (Millipore, Molsheim, France). Buffer B was 1 mL L1 TFA in 800 mL L1 acetonitrile. The analysis was performed at 46 1C with a gradient of

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Buffer B increasing from 43 to 100 mL 100 mL1. Detection was at 214 nm. 2.10. Sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS– PAGE) Samples of serum heat-induced protein aggregates, kcasein and other milk protein fractions (suspensions of lactoferrin, NPPC or WPI) were analysed by 120 g L1 SDS–PAGE, using the Hoefer system (Amersham Bioscience). Samples were diluted in Laemmli buffer with, or without, dithiothreitol (DTT), at the concentrations shown in Table 1, incubated at 100 1C for 1 h, and loaded at a level of 20 mL. A prestained low molecular mass standard mixture (14.4–97 kDa, Amersham Biosciences) was also applied to the gels. Each sample was analysed in duplicate. Staining was performed with Coomassie Blue. 2.11. Determination of the size and shape of the serum aggregates using light scattering Particle size analysis was performed using dynamic light scattering (DLS) at a fixed angle of 901 on a Zetasizer Malvern 3000 HS (Malvern Instruments, Orsay, France). The laser was a He–Ne laser, with 633 nm wavelength. Both the fresh FPLC aggregate peak eluate and 5 g L1 suspensions of isolated aggregates in MUF were analysed. Samples were equilibrated at 25 1C, diluted in MUF to meet the Zetasizer operating range, and allowed to stand at 25 1C for a standardised period of 20 min to ensure proper equilibration of the diluted system prior to analysis. Presented results are the average of 10 readings, and each sample was analysed 2–3 times. The data were computerised into a particle size distribution using a CONTIN modelling routine (Provencher, 1982). Particle shape was estimated using multi-angle static and dynamic light scattering (MADLS) measurements made using an ALV-5000 multi-bit multi-t correlator Table 1 Sample preparation for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) protein analysis Sample

Aggregate powder Aggregate powder k-Casein k-Casein Casein micelles (NPPC) Whey protein isolate Lactoferrin

Initial dilution Concentration Addition after dilution of DTT in MUF in Laemmli (g L1) (g L1) 1.75 1.75 0.10 0.10 1.87 1.75 0.10

0.88 0.88 0.05 0.05 0.93 0.88 0.05

Yes No Yes No Yes Yes Yes

(ALV-Laser vertriebgesellschaft, Langen, Germany) in combination with a Malvern goniometer and a Spectra Physics laser (Newport Corporation, Irvine, USA) operating with vertically polarised light with a wavelength, l, of 532 nm. Data were collected at scattering angles y between 101 and 1401, which correspond to scattering wave vectors, q, of 2:7  103 oqo3:0  102 nm1 where q ¼ ð4pn=lÞ sinðy=2Þ, n being the refractive index (1.333 for aqueous buffers and 1.3416 for MUF) and l the wavelength of the laser. The temperature was set at 20 1C and controlled by a thermostatically controlled water-bath. The relative scattering intensity ðI r Þ of the particles was determined by subtracting the solvent scattering intensity from the total scattering intensity and dividing by the scattering intensity of toluene. In general I r can be written as I r ¼ KCM w SðqÞ, where M w is the weight-average molar mass of the particle, K is a contrast factor that depends on the refractive index increment and the experimental set-up, and SðqÞ is the q-dependent static structure factor (e.g. Brown, 1996; Higgins & Benoit, 1994). We have used 0.189 for the refractive index increment of casein and we have used a toluene standard with a Rayleigh ratio of 2.79  105 cm1. Plotting I r as a function of q2 leads to a distribution of radii of gyration, Rg. The intensity autocorrelation function measured in DLS is related to the electric field autocorrelation function (g1(t)) using the so-called Siegert relation (Berne & Peckora, 1993); g1 ðtÞ was analysed in terms of distribution of relaxation times using the Laplace inversion routine REPES (Stepanek, 1993). The relaxation times ðtÞ were found to be q2-dependent and at low concentrations they may be related to the diffusion coefficient of individual species in the dispersions: D ¼ ðq2 tÞ1 . The relaxation time distribution was transformed into a distribution of hydrodynamic radii, Rh, using the Stokes–Einstein relation: D ¼ kT=ð6pZRh Þ, where k is Boltzman’s constant and Z is the viscosity. The ratio Rh/Rg, also called the polydispersity index, is an indicator of the particle sphericity. As in DLS, a suspension of isolated serum aggregates and FPLC aggregate peak eluates were analysed. 2.12. Transmission electron microscopy (TEM) The microstructure of the aggregates was examined using a CM 12 transmission electron microscope (Philips Electron Optics, FEI Company, LimeilBre´vannes, France). FPLC eluates of concentrated, or control, serum from heated milk were desalted by dialysis against 100 volumes of distilled water at 5 1C for 4 h. Grids supporting ionised carbon films were wetted with the samples to allow deposition of the protein material (without fixation), rinsed with distilled

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water, and wetted with uranyl acetate (20 g L1) as a contrasting agent. Excess uranyl acetate was removed by contact of the edge of the grid with absorbing paper. The microscope was operated under a tension of 120 kV. Nominal magnification was 28 000  .

imidazole, 16.7 mM NaCl, 1.7 mM CaCl2, and 3.3 mM NaN3) to test the effect of the ionic strength on the zeta potential of these aggregates.

2.13. Zeta potential

The optical density, at 600 nm, of the aggregate suspensions in MUF, or in imidazole buffer, adjusted to various pH values was measured on a double-beam Uvikon 922 spectrophotometer (Kontron Instruments, UVK-Services, Trappes, France). The reference sample was the same solvent phase at the same pH value as in the tested sample.

The zeta potential of a particle in equilibrium with a defined medium expresses the net charge density at its plane of shear, located at the surface of the ionic double layer of the particle and taken as stable on Brownian motion and solvent drag. Motion of the charged particle submitted to an electric field and solvent drag counteraction determine the constant electrophoretic mobility (Ue) of the particle in its medium; Ue can be expressed by the Henry equation Ue ¼

zf ðkaÞ , 6pZ

where  is the dielectric constant (79 for water at 25 1C); z, the zeta potential of the particle; Z, the viscosity of the solvent phase (0.89 mPa s for aqueous buffers and 0.99 mPa s for MUF, at 25 1C); and ka, a factor related to the geometry of the particle and of its ionic double layer. The electrophoretic mobility of a charged particle submitted to an electric field is deduced from the Doppler shift of the laser light scattered by the particle. The zeta potential is then calculated from the Henry equation, assuming spherical particles and ka ¼ 1:5, i.e., the radius of the particle largely exceeds the thickness of the ionic double layer. Uncertainties on the calculation of the zeta potential of such protein particles may arise from the values taken for  and Z, and from the assumption that the isolated heat-induced aggregates are spherical. Dalgleish (1984) further debates the independency of the zeta potential, or rather the position of the plane of shear, on the particle size, or on changes of the particle surface, e.g., the k-casein hairy layer of casein micelles. The zeta potential of the isolated heat-induced serum aggregates suspended in MUF was measured on a Zetasizer Malvern 3000 HS. The aggregate powder was dissolved at a level of 5 g L1 in MUF adjusted to pH values in the range 2–9, and left overnight at 5 1C with continuous stirring to allow complete equilibration. Aliquots of the samples equilibrated at 25 1C were diluted with the corresponding pH-adjusted MUF to meet the Zetasizer operating range and left at 25 1C for a standardised period of 20 min to ensure equilibration prior to analysis. The zeta potential of artificial heatinduced aggregates was also measured according to the same protocol except that the pH-adjusted medium was imidazole buffer, as the heat-treatment medium. The isolated heat-induced serum aggregates were also suspended in three-fold diluted imidazole buffer (6.7 mM

2.14. Turbidity

2.15. Surface hydrophobicity The surface hydrophobicity of the heat-induced serum aggregates, standard b-Lg and NPPC (as micellar casein) was estimated using the 8-anilino-1-naphthalene sulphonic acid (ANS)-binding fluorimetric assay, in a LS 50B spectrophotometer (Perkin Elmer, Saint Quentin-en-Yvelines, France). A solution of 2 mM of ANS was prepared daily in imidazole buffer and filtered on 1 mm. Starting dispersions of b-Lg, serum aggregates and NPPC were prepared in imidazole buffer, left under continuous stirring overnight at 5 1C to ensure complete hydration and filtered on 5 mm. The molarities of the protein dispersions were: 27 mM b-Lg (0.5 g L1, molecular mass 18.6 kDa); 5 nM NPPC (5 g L1), assuming a molecular mass of 106 kDa for hydrated micelles (Mahaut, Jeantet, & Brule´, 2000); and 1.6 mM (dispersion 1) or 0.15 mM (dispersion 2) of heat-induced serum aggregates. The concentrations of the dispersions 1 and 2 of heat-induced serum aggregates were, respectively, 4.8 g L1, assuming a molecular mass of 3  103 kDa (Guyomarc’h et al., 2003b), and 3 g L1, assuming a molecular mass of 2  104 kDa, based on preliminary MADLS results. For each protein sample, the concentrations of ANS and the protein species were diluted so that the ANS: protein molar ratio was 1, 2, 6 or 10 and that absorbance at 390 nm was 0.1–0.15 absorbance units (as the upper limit of the linear range for absorbance by the ANS-b-Lg interaction). Excitation wavelength was 390 nm, and the fluorescence spectrum was observed between 440 and 510 nm. The emission and excitation slits were both set at a bandwith of 5 nm.

3. Results and discussion 3.1. Purity of the aggregate powder The protein composition of FPLC fractions collected during elution of the aggregate peak was analysed using RP-HPLC; a typical RP-HPLC profile is shown on Fig. 2.

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whey proteins

2.4

0.9

β-casein

1.4

αs1-casein

1.9

κ-casein

Optical density at 214 nm (Absorbance Units)

308

0.4

-0.1 0

5

10 15 20 Retention time (min)

25

30

Fig. 2. Typical reverse-phase high-pressure liquid chromatography (RP-HPLC) elution profile of the heat-induced serum k-casein/whey protein aggregate peak fraction defined in Fig. 1a. Peak identification was according to Visser et al. (1986, 1991). See Section 2.9 for experimental details.

Fig. 3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of heat-induced serum milk protein aggregates in dissociating conditions with SDS (lane 2, the circle indicates large material at the top of the stacking gel) or dissociating/reducing conditions with DTT and SDS (lane 3). The other lanes correspond to different proteins in dissociating/reducing conditions (unless otherwise stated): 1, low molecular mass protein standards (Amersham Biosciences, Orsay, France); 4, native phosphocaseinate as a positive standard for caseins; 5, standard k-casein in dissociating conditions; 6, standard k-casein; 7, standard lactoferrin; 8, whey protein isolate as a positive standard for whey proteins. See Section 2.10 for experimental details.

The results consistently showed that the fractions isolated by FPLC from concentrated serum of heated milk were composed of whey proteins (445% of total peak area), k-casein (437.5% of total peak area), and as and b-caseins (o 17% of total peak area). The heatinduced interaction between denatured whey proteins and k-casein in milk is widely documented and the composition found in the present work was in agreement with previous reports (e.g. Corredig & Dalgleish, 1996; Guyomarc’h et al., 2003b; Jang & Swaisgood, 1990; Noh, Creamer, & Richardson, 1989b; Noh, Richardson, & Creamer, 1989a; Singh & Creamer, 1991a). The significant amounts of as - and b-caseins probably originated from dissociated micellar material, usually eluting after the heatinduced serum aggregate peak (Guyomarc’h et al., 2003b). Singh and Creamer (1991a, b) have shown that all the casein species dissociate, at various extents, from the casein micelles as a result of the heat-treatment of milk. The heatinduced non-sedimentable casein fraction is in the form of large particles that can be fully dissociated by SDS–PAGE analysis (Singh & Creamer, 1991a, b). It is therefore possible that the as - and b-caseins found in the aggregate peak fraction were part of the k-casein/whey protein aggregates, e.g. though hydrophobic interactions. It is also possible that some resolution between the aggregate peak and the peak of micellar material was lost as a consequence of the high protein load while using concentrated serum (Fig. 1). This assumption was supported by the higher proportion of as - and b-caseins in the eluate fractions collected at the end of the aggregate peak.

with, or without DTT, as reducing agent is shown on Fig. 3. When the aggregates were only submitted to SDS, a high molecular mass material that did not enter the stacking gel was present. Only faint bands of a-La, b-Lg and caseins were identified. Some of the latter casein bands were probably as - and b-caseins, as identified by RP-HPLC. After reduction with DTT, the aggregates were totally separated into individual proteins. As expected, they were composed essentially of a-La, b-Lg and k-casein. Minor whey proteins (as identified by comparison with WPI and lactoferrin) were also present. Caseins other than k-casein were not visible, except for the faint bands seen under dissociating conditions. These results indicated that a large majority of the bonds involved in the heat-induced aggregation process were DTT-sensitive disulfide bridges. This conclusion is in agreement with previous findings, e.g., Hoffmann and van Mil (1997) and Oldfield, Singh, Taylor, & Pearce (1998). As no analysis was performed under native conditions, it is not possible to conclude whether the proteins found in dissociating conditions (especially caseins) were actually bound to the aggregates or simply isolated with the aggregates during FPLC separation.

3.2. Type of bonds involved in the aggregates

3.3. Size and shape of the aggregates

A typical SDS–PAGE gel electropherogram of the aggregate powder treated in dissociating conditions

The particle size of a 5 g L1 dispersion of reconstituted heat-induced serum aggregates in MUF was

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measured by DLS. In order to check for possible size changes due to dialysis and freeze-drying, the serum of heated milk was analysed by FPLC and the eluate fraction corresponding to the aggregate peak was directly analysed by DLS. Particles with a hydrodynamic diameter of 100–110 nm were found in suspensions of aggregate powder, while particles with a diameter of 65–80 nm were found in the FPLC eluate of the aggregate peak. The larger average diameter of the reconstituted heatinduced serum aggregates indicated that some overaggregation had occurred during dialysis and freezedrying, in accordance with the FPLC profiles presented earlier (Fig. 1). The serum of unheated milk was also analysed by FPLC and the eluate collected between 170 and 240 min was directly analysed by DLS, in order to check for the possible presence of small fat globules in the isolated aggregates. This elution time corresponded to the elution of the aggregate peak (Fig. 1), except that no peak was visible on the FPLC profile of the serum of unheated milk during that time (Guyomarc’h et al., 2003b). Only a very low concentration of particles with a hydrodynamic diameter of 100 nm was detected, close to the lower signal limit of the Zetasizer. This result indicated that the particles detected in samples from sera of heated milk were essentially heat-induced protein species, i.e., the isolated serum aggregates. MADLS measurements were also performed on the same reconstituted heat-induced serum aggregate and FPLC eluate samples. Analysis of the aggregate powder suspension gave a hydrodynamic radius, Rh, of 70 nm (i.e., diameter of 140 nm) and a radius of gyration, Rg, of 88 nm. The hydrodynamic radius, measured by

309

MADLS, was somewhat larger than that observed using the Zetasizer. The polydispersity index, Rh/Rg, of the reconstituted serum aggregates was 0.79, which indicated that these particles were spherical rather than elongated, but it is noteworthy that polydispersity also depends on distribution and drainage. The calculated weight average molecular mass was 2  107 g mol1. Analysis of the FPLC eluate of the serum of heated milk (aggregate peak) gave an Rh of 35 nm, an Rg of 47 nm, a polydispersity index of 0.74 and a molecular mass of 107 g mol1. Estimations of the particle shape and molecular mass were therefore similar in the two types of samples. TEM analysis was also performed on an aggregate peak eluate isolated from serum of heated milk by FPLC and on an aggregate peak eluate isolated from concentrated serum of heated milk. Fig. 4 shows typical micrographs of the two samples. On both micrographs, clusters of well-defined, round, or oval-shaped, particles were clearly visible. At a high aggregate concentration (Fig. 4a), large clusters or chains of particles were formed. In diluted samples (Fig. 4b), a more even distribution of short-chain clusters containing a few particles were formed. Individual particles were visible in places. Size measurements of 22 random particles taken from different micrographs (at both concentrations) indicated an average diameter of 2475 nm. Cluster formation appeared to be a concentration-dependent artefact of the TEM sample preparation, as an even more diluted sample gave numerous individual particles (not shown). The size of heat-induced milk serum aggregates, as presented in this study, or previously reported by other authors, are summarised in Table 2.

Fig. 4. Transmission electron micrographs of the heat-induced serum k-casein/whey protein aggregates isolated in the peak fraction, as defined in Fig. 1a, of the FPLC elution profiles of (a) concentrated, diafiltered serum of heated milk, and (b) untreated serum of the same heated milk. Both samples were desalted and stained with uranyl acetate prior to observation. See Section 2.12 for experimental details.

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Table 2 Summary of reported sizes of heat-induced milk serum aggregates. The experimental approach and type of sample are also indicated Number

Reported diameter (nm)

Method used

References

1.

100–110

Present work

2.

65–80

3. 4. 5.

140 70 60–100

6. 7.

410 20–30

8.

10–20

Dynamic light scattering (DLS) on reconstituted heat-induced milk serum aggregates isolated by Fast Protein Liquid Chromatography (FPLC) DLS on fresh eluted heat-induced milk serum aggregate peak as defined in Fig. 1 Multi-angle DLS on same sample as 1. Multi-angle DLS on same sample as 2. Agarose gel electrophoresis on supernatants of renneted heated skim milks Fast Protein Liquid Chromatography on same samples as 2. Transmission Electron Microscopy (TEM) on same samples as 1. and 2. TEM on the interphase layer of centrifuged heated skim milk

Vasbinder et al. (2003), using agarose gel electrophoresis, reported that the heat-induced protein aggregates found in the serum phase of milk had a diameter in the range 60–100 nm. Guyomarc’h et al. (2003b) estimated that the size of heat-induced serum aggregates isolated from skim milk by the same chromatographic method as in the present work could exceed 10 nm. The present results agree with those of the latter studies. Smits and Van Brouwershaven (1980), using TEM, observed that the interphase layer of centrifuged heated skim milk was rich in k-casein and b-Lg and consisted of short-chain clusters comprised of round particles of 10–20 nm diameter. The aspect of these clusters was very similar to those observed in the present study. However, the micrographs for the serum phase of heated milk differed from those generally obtained for heated milk, as shown by, e.g. Kala´b, Emmons, and Sargant (1976) or Kala´b, Allan-Wojtas, and PhippsTodd (1983), where filaments could be seen protruding from the surface of casein micelle. It is possible that heat-induced micelle-bound aggregates have a different shape to that of the heat-induced serum aggregates characterised in this study. It is also possible that the filaments reported by Kala´b et al. (1976, 1983) were composed of round sub-components, a few nanometres in diameter, and that these could not be seen at the magnification level used. The diameter of the heat-induced milk serum aggregates as determined by DLS, differed from that obtained by TEM analysis by about 40 nm (Table 2). DLS measures the hydrodynamic diameter, i.e., the particle as well as bound water and counter-ions, while TEM measures the actual contours of the particle. A possible explanation for the difference observed between the two methods could therefore be accounted for by the high water retention of the serum k-casein/whey protein aggregates (e.g., Guyomarc’h et al., 2003a). Considering

Present work Present work Present work Vasbinder, Alting, & de Kruif (2003) Guyomarc’h, Law, & Dalgleish (2003b) Present work Smits & Van Brouwershaven (1980)

the k-casein content of the aggregates, it is also possible that the glycosylated C-terminal end of the protein is protruding and is in contact with the solvent, as has been shown for micelles where the ‘‘hairy layer’’ of kcasein has a thickness of 12 nm (Holt & Horne, 1996). 3.4. Isoelectric point of the aggregates Turbidity, DLS size measurement and zeta potential of the heat-induced milk serum aggregates reconstituted in MUF at pH values ranging from 2 to 9 are shown in Fig. 5. Visible flocculation was observed in the samples at pH 4 and 5, in agreement with turbidity and size measurements (Figs. 5a and b). The zeta potential of the aggregates, which was 17 mV at pH 6.7 in MUF, increased with decreasing pH and became positive at pHp4. The pH value where zeta potential became zero was referred to as ‘‘isoelectric point’’ (Ip), albeit this term usually refers to isoelectrofocusing. The Ip of the aggregates in MUF at 25 1C was pH 4.5. This value was close to the apparent isoelectric pH of casein micelles in milk (4.7) but lower than the Ip of the individual component proteins (i.e. 5.3 for b-Lg, 5.4 for k-casein and 4.8 for a-La). It was, however, in accordance with the data of Zhu and Damodaran (1994), who reported that the lowest solubility of heated whey proteins was at pH 4.5, compared with 5.0 for native whey protein. De Wit (1981), de Wit, HonteleckBackx, and Adamse (1988) and other authors also used isoelectric precipitation at pH 4.6, rather than 5.2, under conditions similar to those in milk (especially ionic strength), to assay the level of whey protein denaturation. Kella, Yang, and Kinsella (1989) also showed that the cleavage of disulfide bonds in native whey proteins (i.e., directed denaturation) reduced the pH at which maximum precipitation occurred, from 5.1 to 4.0.

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2.00 1.50 1.00 0.50

(a)

0.00

Average hydrodynamic diameter (nm)

Optical density at 600 nm (Absorbance Units)

2.50

2500

2000

1500 1000

500

0

(b)

25 20

Zeta potential (mV)

15 10 5 0 -5 0

1

2

3

4

5

6

7

8

9

10

-10 -15 -20 -25

(c)

pH

Fig. 5. Turbidity (a), hydrodynamic diameter as measured by dynamic light scattering (b) and zeta potential (c) of: (K) a suspension of purified heat-induced milk serum aggregates in milk ultrafiltrate, (J) a suspension of artificial heat-induced k-casein/whey protein aggregates in imidazole buffer, and ( ) a suspension of purified heat-induced milk serum aggregates in three-fold diluted imidazole buffer, as a function of pH. Data are the mean of two replicate samples where error bars show standard deviation. For light scattering measurements at pH 4 and 5, flocculated aggregates exceeded the upper limit of the Malvern Zetasizer (2000 nm) and absolute values are meaningless. See Sections 2.1–2.3, for experimental details.

To check for the possible effect of phosphorylcontaining as - and b-caseins in the isolated heat-induced milk serum aggregates, artificial aggregates were prepared by heating standard k-casein, a-La and b-Lg together in imidazole buffer. The solution became whitish on heating, as opposed to imidazole buffer without protein that remained clear. This indicated that

311

the heated mixture of proteins gave a suspension of large particles. DLS measurements showed that the heated mixture contained particles that had a hydrodynamic diameter of 550756 nm. The unheated mixture contained particles with a hydrodynamic diameter of 35.572.7 nm due to standard k-casein polymers. Isolation of the artificial aggregates using FPLC gave a fast eluting aggregate peak that confirmed a size greater than that of the natural milk serum aggregates (data not shown). The protein composition of the peak determined by RP-HPLC analysis was k-casein (44.5% of total peak area) and whey proteins (55.5% of total peak area. The whey protein: k-casein area ratio of the artificial aggregates was 1.3, versus 1.2–1.8 in natural aggregates. Dilution of the suspension of artificial aggregates at a level of 2 mL 10 mL1 in imidazole buffer, in 7 M urea+20 mM bis–Tris propane at pH 7.5, or in 7 M urea+20 mM bis–Tris propane at pH 7.5+bmercaptoethanol, showed that they were partly composed of disulfide covalent bonds (data not shown). The heat-induced k-casein/whey protein aggregates formed under these conditions were therefore different from those formed in milk. However, it can be speculated that both types of aggregates are comparable with respect to physicochemical characteristics that depend on protein–protein and protein–solvent interactions. Figs. 5b and c shows the changes in hydrodynamic diameter and zeta potential of the artificial aggregates suspended in imidazole buffer, as a function of pH. The zeta potential of the artificial aggregates in imidazole buffer was about 20 mV at pH 6.7 and 25 1C, and increased with decreasing pH to 0 mV at pH 4.7. This pH for the isoelectric point of the artificial aggregates agreed with that for the reconstituted heat-induced milk serum aggregates. At pH values o4 and 45, absolute values of the zeta potential of the artificial aggregates seemed consistently higher than those of the natural milk serum aggregates. Direct comparison of the two systems used should, however, only be made with caution because of differences (e.g., distribution of the proteins and charges on the surface of the aggregates; density, thickness, or composition of the ionic double layer of the aggregates) between the artificial aggregates in imidazole buffer and natural aggregates in milk ultrafiltrate. The effect of ionic strength on the Ip value of the heatinduced serum aggregates was also investigated. The Ip of the whey proteins and k-casein which form the aggregates, are not expected to be very sensitive to variations of the ionic strength. However, the high phosphoryl content of the as - and b-caseins make their Ip value sensitive to changes in ionic strength. Fig. 5c shows the zeta potential of heat-induced serum aggregates reconstituted in diluted imidazole buffer, with an ionic strength about three-times lower than that of MUF, as a function of pH. The variation in zeta

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potential with pH was similar to that reported above for the artificial aggregates in imidazole buffer and for the natural aggregates in milk ultrafiltrate, and was 0 at pH 4.5. This suggested that the Ip of the heat-induced serum aggregates was not sensitive to ionic strength. This result, as well as the demonstration of an Ip of 4.7 for the artificial aggregates, showed that the value of 4.5 as the Ip of the heat-induced serum aggregates was not influenced by the presence of as - and b-caseins. Measurements of the zeta potential of natural heatinduced serum aggregates in MUF, or in diluted imidazole buffer, and of artificial aggregates in imidazole buffer all showed that the isoelectric point of heatinduced k-casein/whey protein aggregates was 4.5–4.7. This value does not support the hypotheses which suggest that the gelation of heated milk at a pH higher than that of unheated milk (5.4 versus 4.8) is due to the destabilisation of the heat-denatured whey proteins at a high pH. The Ip found in the present study may, however, be affected by the slight over-aggregation of the heat-induced serum aggregates that occurs on isolation, and by the experimental conditions. Hence, no conclusion on the actual behaviour of these aggregates during acidification of milk can be drawn until further measurements of zeta potential are performed in milk permeate extracted from heated skim milk adjusted to various pH values. Alting, de Jongh, Visschers, and Simons (2002) found a positive relationship between the Ip of b-Lg (modified by succinylation) and the pH at which turbidity of the medium started to increase. Therefore, it seems clear that the Ip of the heatinduced aggregated protein plays a role in determining gelation pH. However, the acid gelation of high-heated treated milk at a higher than normal pH (e.g. 5.4 versus 4.8) may also be attributable to other factors. Famelart et al. (2004) studied texture formation in acid gels made with co-heated mixtures of globular proteins and casein micelles in milk ultrafitrate. These authors reported a higher gelation pH and final gel strength when the globular protein was ovalbumin rather than b-Lg, despite the lower isoelectric point of ovalbumin (Ip4.8 versus 5.3 for b-Lg). These results indicate that electrostatic interaction properties of the heatinduced protein aggregates may not be the only factor responsible for the higher acid-induced gelation pH of heat-treated milk.

environment with hydrophobic characteristics. Fig. 6 shows the fluorescence intensity taken at the maximum of the emission peak, as a function of the ratio of ANS: protein species. The protein concentrations used gave an absorbance of 0.1–0.15 A.U. at 390 nm, with an ANS: protein ratio of 10. The results show that regardless of the molecular mass used in the calculations, the serum aggregates exhibited higher fluorescence intensity, and, hence, a higher surface hydrophobicity than standard b-Lg or micellar casein (NPPC). This clearly indicates that the heat-induced serum k-casein/whey protein aggregates had quite a high hydrophobicity. The two aggregate dispersions of different concentrations gave quite similar results, except at the high ANS: protein ratio where deviations were observed. This trend suggested that both the estimations of the molecular mass of the serum k-casein/whey protein aggregates used in this study were satisfactory. As suggested by the large range of elution times found for the FPLC aggregate peak (Fig. 1), the size distribution of these serum k-casein/whey protein aggregates is probably very large and may therefore include the two molecular masses used in this study to convert the concentrations of the two aggregate samples, dispersions 1 and 2, from mM to g L1. The very low fluorescence intensity found for native micellar casein (NPPC) could be explained by the very low ANS: protein ratio used in this study. Other authors generally used ratios much higher than 105 mol mol1 to 350 dispersion 1

Fluorescence intensity (arbitrary units)

312

300

Heat-induced milk serum aggregates

250 200

dispersion 2

150

standard native β-lactoglobulin

100 50 micellar cas ein

0 13

3.5. Surface hydrophobicity Analysis of the emission spectra (not shown) of the ANS-protein mixtures of NPPC and aggregates revealed a slight hypsochromic shift of the maximum peak from 480 nm (expected maximum wavelength for emission fluorescence) to 468–470 nm. Gatti, Risso, and Pires (1995) observed a similar phenomenon for micelle suspensions and attributed this shift to a low-polarity

5 7 ANS: protein ratio (mol:mol)

9

Fig. 6. Surface hydrophobicity, given as the maximum fluorescence intensity in the range 465 to 485 nm due to the binding of 8-anilino-1naphthalene sulphonic acid (ANS) by: ( -shaped) native phosphocaseinate (NPPC) as standard milk casein micelles, (K-shaped) standard native b-lactoglobulin, and purified heat-induced milk serum aggregates (m-shaped as dispersion 1, using a molecular mass of 3  103 kDa to calculate molar concentrations; and ~-shaped as dispersion 2, using a molecular mass of 2  104 kDa). See Section 2.15 for experimental details.

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study changes in surface hydrophobicity of casein micelles in milk (e.g., Bonomi, Iametti, Pagliari, & Peri, 1988; Gatti et al., 1995; Zbikowska, Szerszunowicz, & Smyk, 2004). Hence, further investigation using a large range of ANS:NPPC ratios (up to 106 mol mol1) indicated that micellar casein started to show significant fluorescence intensity at an ANS:NPPC ratio of 104 mol mol1. Quenching of the fluorescence signal occurred at 2  105 mol mol1 (not shown). Quenching of the fluorescence signal for the heat-induced serum aggregates occurred at an ANS: aggregate ratio of 2  103 mol mol1. However, the intensities measured at ANS: particle ratios up to 2  103 mol mol1 were higher for the aggregates than for NPPC (data not shown). Relkin (1998) found that the increased binding of ANS by pure b-Lg on increasing the heat-treatment was related to the heat-induced irreversible unfolding of the protein. The change in surface hydrophobicity of heated whey proteins following sulfhydryl-mediated aggregation is, however, rather unclear. In the study of Relkin (1998), the surface hydrophobicity of heated b-Lg increased with temperature in the range 60–85 1C. However, Carbonaro, Bonomi, Iametti, and Carnovale (1996) reported that the surface hydrophobicity of whey protein extracts isolated from milk decreased on increasing the temperature from 75 to 80 1C. This shows that the degree of aggregation may affect the surface hydrophobicity. Consequently, the slight increase of the size of the serum aggregates on isolation and/or reconstitution (Fig. 1) may have affected the results presented in this study. In the current study, the surface hydrophobicity of kcasein/whey protein aggregates produced during high heat treatment (90 1C for 10 min) appeared to be high at neutral pH, despite the surface charge of the aggregates. The solubility of protein particles as a function of pH is a balance between the stabilising forces (ionic interactions with the aqueous solvent) and destabilising forces (surface hydrophobicity, charge screening). The changes in turbidity of a suspension of serum k-casein/whey protein aggregates as a function of pH (Fig. 5a) indicate that these aggregates precipitate at pH 4 and 5, i.e., before reaching their Ip, possibly as a consequence of their surface hydrophobicity and/or other destabilising factors. The net effect of the various surface properties of the heat-induced serum aggregates in milk and the changes in these properties with pH should help in clarifying the behaviour of these aggregates during acid gelation of skim milk.

4. Conclusions Isolation of the heat-induced milk protein aggregates allowed extensive characterisation of their structure

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and surface properties. They appeared to consist of almost spherical particles with a diameter of 25–70 nm. They were essentially composed of k-casein and denatured whey proteins linked together through disulfide bonds. The isoelectric point of the aggregates was 4.5 in standard milk ultrafiltrate. The surface hydrophobicity of the aggregates was significantly larger than that of standard b-Lg and micellar casein. Our results suggested that the heat-induced k-casein/whey protein aggregates may precipitate at high pH on acidification, due to a specific balance of stabilising/ destabilising forces.

Acknowledgements The authors wish to acknowledge Pierre Schuck and Jacques Fauquant for material preparation, Saı¨ d Bouhallab, Thomas Croguennec and Christelle Lopez for advice and discussion and Franc- ois Ordronneau for surface hydrophobicity measurements. We also wish to thank Taco Nicolai at the Universite´ du Maine (Le Mans, France) for MADLS analysis and Daniel Thomas at the Universite´ de Rennes I (Rennes, France) for TEM analysis. Finally, we thank John Hannon for carefully reading the manuscript.

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