Effect of dynamic high pressure on whey protein aggregation: A comparison with the effect of continuous short-time thermal treatments

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FOOD

HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 1014–1032 www.elsevier.com/locate/foodhyd

Effect of dynamic high pressure on whey protein aggregation: A comparison with the effect of continuous short-time thermal treatments Alvar Gra´cia-Julia´, Malika Rene´, Marianela Corte´s-Mun˜oz, Lae¨titia Picart, Toma´s Lo´pez-Pedemonte, Dominique Chevalier, Eliane Dumay Equipe de Biochimie et Technologie Alimentaires, UMR 1208, De´partement Agro-Ressources et Proce´de´s Biologiques et Industriels, Universite´ Montpellier II, F-34095 Montpellier Cedex 05, France Received 21 November 2006; accepted 21 May 2007

Abstract Dispersions of whey protein isolate containing 6% or 10% (w/w) protein at pH 6.5 were processed using a 15 L/h homogeniser with a high-pressure (HP) valve immediately followed by cooling heat exchangers. The effect of dynamic high pressure or ‘‘ultra-high pressure homogenisation’’ (UHPH) between 100 and 300 MPa was investigated on protein solubility, physical characteristics of dispersions (viscosity, protein voluminosity, L*, a*, b* colour parameters) and size distribution of protein aggregates. Temperatures T1 and T2 were determined at the inlet and outlet of the HP valve, as they varied during UHPH. UHPH did not induce protein aggregation below 225 MPa. Photon correlation spectroscopy (PCS) revealed protein aggregation at 250–300 MPa for both dispersions, corresponding to T2 values of 66–74.6 1C and 68–76.5 1C at 6% and 10% protein, respectively. PCS (in particle number frequency) indicated a main population of aggregates at 7, 26 or 50 nm, at, respectively, 250, 275 or 300 MPa and 6% protein, while a monomodal distribution at 26 nm was observed at the 3 pressure levels and 10% protein, resulting in controlled protein aggregation without gelation. The effects of mechanical forces predominated over those of short-time heating, since more insoluble protein was found after UHPH of the 6% protein dispersion at 275–300 MPa (T2 ¼ 71–74.6 1C) than after control assays of continuous heating (4 s) at 71–7470.5 1C and atmospheric pressure. Atomic force microscopy confirmed protein aggregation at X250 MPa. Polyacrylamide gel electrophoresis under dissociating and reducing or non-reducing conditions suggested that UHPH-induced aggregation occurred mainly through hydrophobic interactions. r 2007 Elsevier Ltd. All rights reserved. Keywords: Dynamic high pressure; Ultra-high-pressure homogenisation; Whey proteins; Protein aggregation; Novel technologies; Photon correlation spectroscopy

1. Introduction Protein ingredients, such as whey protein isolates (WPI, containing X90% protein, d.b.) or whey protein concentrates (WPC, containing 60–85% protein, d.b.) are widely used in the food industry due to their high nutritional quality, desirable sensory characteristics and high technofunctional potentiality (De Wit, 1998; Huffman & Harper, 1999). b-Lactoglobulin (b-Lg), the major protein in whey (about 50%) is a globular protein existing as 36.7 kDa dimers in aqueous solution at close to neutral pH Corresponding author. Tel.: +33 467 143 351; fax: +33 467 143 352.

E-mail address: [email protected] (E. Dumay). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.05.017

(McKenzie, 1971). b-Lg monomer has two intermolecular disulphide bridges (Cys 66–160 and Cys 106–119), and one free thiol group (Cys 121) (Hambling, McAlpine, & Sawyer, 1992). Heat-induced unfolding and aggregation of b-Lg alone or in the presence of other whey proteins and caseins have been widely studied in the past years (De la Fuente, Singh, & Hemar, 2002; Livney, Corredig, & Dalgleish, 2003). Denaturation/aggregation phenomena depend on physical and biochemical parameters such as heating temperature and holding time, heating rate, protein concentration, presence of other proteins or hydrocolloids, pH, ionic strength and mineral content (Durand, Gimel, & Nicolai, 2002; Hoffmann, Roefs, Verheul, van Mil, & de Kruif, 1996; Laligant, Dumay, Valencia, Cuq, & Cheftel,

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1991; Verheul, Roefs, & de Kruif, 1998). Most of the recent studies have been carried out with purified proteins to propose models/mechanisms of heat-induced aggregation reactions (Croguennec, O’Kennedy, & Mehra, 2004; Galani & Apenten, 1999; Hoffmann & van Mil, 1997; Manderson, Hardman, & Creamer, 1998; Prabakaran & Damodaran, 1997; Qi, Brownlow, Holt, & Sellers, 1995; Schokker, Singh, Pinder, Norris, & Creamer, 1999). Some studies deal with industrially prepared whey protein products or skim milk to evaluate heat-induced effects on protein native state after thermal processing and drying at an industrial scale (De la Fuente, Hemar, Tamehana, Munro, & Singh, 2002; De Wit, 1990, 1998; Oldfield, Singh, Taylor, & Pearce, 1998; Oldfield, Taylor, & Singh, 2005). Initial steps of heat-induced denaturation at neutral pH involve the dissociation of b-Lg native dimers into native/modified monomers at a critical temperature close to 60 1C, accompanied by sulphydryl group exposure and thiol/disulphide exchanges (Croguennec, Bouhallab, Molle´, O’Kennedy, & Mehra, 2003; Iametti, de Gregori, Vecchio, & Bonomi, 1996; Sawyer, 1968; Shimada & Cheftel, 1988, 1989), and then irreversible formation of polymers. Hydrophobic-driven associations are also involved in aggregation mechanisms, depending on heating temperature and leading to larger aggregates (Cairoli, Iametti, & Bonomi, 1994; Galani & Apenten,1999). a-Lactalbumin (a-La; 20% of bovine whey proteins) has a highly compact globular structure with four disulphide bonds and no free thiol group. Although a-La is quite susceptible to denaturation at 65 1C, its high degree of renaturation is responsible for its apparent high heat resistance (Dalgleish, Senaratne, & Franc- ois, 1997). Schokker, Singh, and Creamer (2000) have proposed a heat-induced polymerisation mechanism for a-La and b-Lg mixtures via SH/S–S exchanges. Havea, Singh, and Creamer (2001) suggested that thiol exposure in unfolded b-Lg or bovine serum albumin (BSA) catalysed the formation of a-La (homoand hetero-) polymers when the proteins were heated together at 75 1C. Effects of isostatic (also hydrostatic) high pressure (HP) have been studied these past 15 years with a view of potential applications in the dairy industry (Cheftel & Dumay, 1996; Huppertz, Kelly, & Fox, 2002; Lo´pezFandin˜o, 2006; Trujillo, Capellas, Saldo, Gervilla, & Guamis, 2002). Isostatic high pressure also promoted b-Lg aggregation through both thiol-disulphide interchanges and hydrophobic interactions (Funtenberger, Dumay, & Cheftel, 1995, 1997). However, studies carried out at high b-Lg concentration (FTIR or gel studies) indicated that pressure and temperature induced different protein/protein interactions and aggregation mechanisms (Dumay, Kalichevsky, & Cheftel, 1998; Panick, Malessa, & Winter, 1999). Dynamic high pressure (or ultra-high pressure homogenisation, UHPH) is a novel technology actually studied in food and pharmaceutical areas, with a view to assess inactivation of microorganisms, enzymes or viruses (Brinez, Roig-Sagues, Herrero, & Lopez, 2006;

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Diels, De Taeye, & Michiels, 2005; Hayes, Fox, & Kelly, 2005; Moroni, Jean, Autret, & Fliss, 2002; Picart et al., 2006; Vachon, Kheadr, Giasson, Paquin, & Fliss, 2002), as well as to fragment particles, oil droplets or fat globules in dispersions and emulsions (Floury, Desrumaux, & Lardie`res, 2000; Keck & Muller, 2006). Recent progress in HP technology and the design of new homogenisation valves (ceramic seats and needles) able to withstand pressures up to 350 MPa have indeed opened new opportunities to homogenisation processing. By comparison, classical homogenisation, the standard industrial process used in the dairy, pharmaceutical and cosmetic industries to reduce particle sizes operates with an up-stream pressure of about 20–60 MPa. Studies of UHPH processing for food applications, namely for producing milk, dairy products and vegetable milks have been recently supported by national or European research projects. This technology effectively allows a more efficient reduction in fat globules/oil droplets (than classical homogenisation) with a concomitant reduction of microbial load (Brinez et al., 2006; Hayes & Kelly, 2003; Thiebaud, Dumay, Picart, Guiraud, & Cheftel, 2003). Isostatic and dynamic HP are different from a technological standpoint. During UHPH, a fluid under pressure is forced through a small orifice of some micrometres width, the HP valve gap (Floury, Bellettre, Legrand, & Desrumaux, 2004). The resulting strong pressure gradient between the inlet and outlet of the HP valve generates intense shear forces, simultaneously to cavitation, impact and short-life heating phenomena. The level of protein denaturation/aggregation may effectively depend on the intensity of mechanical forces and/or temperature. As a consequence, UHPH may induce changes in protein (techno-) functionality. This is why the UHP homogeniser used in the present study has been specially instrumented with thermocouples and pressure gauges to follow the changes in pressure and temperature during processing and measure the increase in fluid temperature generated by passing through the HP valve. Few data are yet available concerning whey protein denaturation/aggregation through dynamic HP up to 300–350 MPa. Homogenisation up to 140–200 MPa without fluid pre-heating did not seem to induce whey protein denaturation (Hayes & Kelly, 2003; Paquin, Lacasse, Subirade, & Turgeon, 2003; Sandra & Dalgleish, 2005; Subirade, Loupil, Allain, & Paquin, 1998). Bouaouina, Desrumaux, Loisel, and Legrand (2006) showed that UHPH treatment up to 300 MPa induced disruption of large powder protein particles present in a WPI dispersion without affecting the bulk protein solubility, but enhancing foaming ability and foam stability of the processed proteins. In the present study, the effect of UHPH up to 300 MPa was investigated on WPI dispersions (6% or 10%, w/w, protein, pH 6.5) by determination of protein solubility, physical characteristics of the processed dispersions, protein electrophoresis behaviour and size determination of protein aggregates by photon correlation spectroscopy. The results were compared with those obtained by

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continuous short-time (4 s) thermal treatments at various temperatures, with a view to assess the relative effects of heat and of mechanical forces in UHPH treatments.

HP valve

2. Material and methods 2.1. Whey protein dispersions A single batch of WPI from Lactalis (Prolacta 90, lot 196 P9, Retiers, France) was used. The WPI has been industrially prepared in mild conditions by microfiltration of the milk, and then by ultrafiltration and spray drying of the microfiltrate. It contained 96.2 g dry solids per 100 g powder, and on dry basis (w/w), 1.04% non-protein nitrogen (NPN), 89.1% protein [(total N–NPN)  6.38], 1.1% ash (including 0.27% calcium, 0.43% potassium, 0.16% phosphorus, 0.10% sodium, 0.05% magnesium and 0.04% chloride) and 1.6% lactose, as given by the producer. The WPI contained 68.5% b-Lg, 21.5% a-La, 5.2% immunoglobulins, 2.5% bovine serum albumin (BSA) and 1.3% lactoferrin per 100 g soluble protein, as indicated by the producer. WPI dispersions containing 6% or 10% (w/w) protein were prepared using deionised water (Millipores), by gentle magnetic stirring at room temperature for 2 h, avoiding foam formation. The spontaneous pH values of the WPI dispersions were 6.5970.03 at 6%, and 6.4770.03 at 10% (w/w) protein. A mean value of 6.5 is taken in the following text. 2.2. Reagents All chemicals were of analytical grade. Tris[hydroxymethyl]aminomethene (Tris, T-1503), bicinchoninic acid solution (B-9643), copper sulphate (III) pentahydrate solution (C-2284), iodoacetamide (I-6125), acrylamide (A8887), N,N0 -methylenebisacryl-amide (M7279) and ammonium persulphate (A3678) came from Sigma (St. Louis, MO). Sodium mono- and di-phosphate (480226; 480087) came from Carlo Erba (Milano, Italy). b-Mercapthoethanol (MSH, 805740) and tetramethylethylenediamine (TEMED, 10732) came from Merck (Darmstadt, Germany). 2.3. Ultra-high-pressure homogeniser The present study was carried out with an UHP homogeniser (model FPG7400H, Stansted Fluid Power Ltd., Essex, UK), specially instrumented with T thermocouples of low inertia, pressure gauges and Bourdon manometers to follow the fluid history during processing. The sole HP valve (first stage) with ceramic needle and seat (350 MPa maximum) was used in this study. The highpressure generating system consisted of a single intensifier, operating in a pulsating mode as already described (Picart et al., 2006). During the processing, temperature and pressure were measured at the inlet (T1, P1) and at the outlet (T2, P2) of the HP valve, and after rapid cooling downstream of the HP valve (T3) as indicated by Fig. 1.

Cold water at 10°C

Food liquid

T1 / P1

Cooling unit

11 s

9s

Cold water at 10°C

LP valve

Tin

Intensifier

1016

T2 / P2

Cooling unit T3

T4

Treated food liquid

Cold water at 10°C

HP pump

Fig. 1. Schematic representation of the ultra-high-pressure homogeniser with the high-pressure (HP) and low-pressure (LP) valves. Cooling devices (heat exchanger with circulating water at 10 1C) were installed around the intensifier, at the immediate outlet of the HP valve (thus avoiding fluid overheating) and after the LP valve. The HP valve was also cooled by circulating water at 10 1C in an external jacket to avoid changes in the valve geometrical dimensions during processing. Temperature (T1, T2, T3 and T4 of the fluid) and pressure (P1, P2) were measured at different positions of the processing pathway as indicated.

Rapid cooling by heat exchanger with circulating water at 10 1C, located at the immediate outlet of the HP valve, avoided fluid overheating due to the conversion of kinetic energy into heat at the pressure drop. With this cooling device, the residence time of the fluid at temperature T2 in the present HP homogeniser is o1 s (i.e. 215 ms for the fluid travelling at T2 through the HP valve plus an additional time 5550 ms needed to cool the fluid from T2 to T3 in the cooling device) for a single-pass homogenisation (Picart et al., 2006). The initial temperature of protein dispersions in the feeding tank (Tin ¼ 2471 1C) and the temperature at the homogeniser outlet (T4 ¼ 13.570.5 1C) were measured with thermistors. WPI dispersions were processed between 100 and 300 MPa (P1). The coefficients of variation (CV%) in (P1) values were p3% over 5–30 min of processing. The pulsed flow rate ranged between 16.0 L/h at 100 MPa and 13.0 L/h at 300 MPa. Three independent UHPHs were carried out on different days. Two controls were considered in the present study: the non-processed WPI dispersion (control A) and the WPI dispersion run through the homogeniser without applying high pressure (control B). Samples were collected in sterile containers after the maximal residence time of a particle through the whole homogeniser (i.e. 160 s of operating or 8 pulsating cycles), and kept at 4 1C before analysis. 2.4. Continuous short-time thermal treatments WPI dispersions (6% or 10%, w/w) were submitted to continuous short-time thermal treatment (STTT) in view of assessing the relative effects of heat and of mechanical forces in UHPH treatment. A STTT of 4 s (4.170.3 s) was chosen to be compared with the short heating (o1 s) of the fluid travelling through the homogeniser. Continuous

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STTTs were obtained by circulating the WPI dispersion through a stainless-steel tubing (2.10 mm inner Ø; 3.18 mm outer Ø) immersed in a water bath previously equilibrated at various temperatures, from 59 to 8370.5 1C. The inlet temperature (Tin) of the dispersions was 2470.5 1C. The temperature of heated dispersions was checked at the tubing outlet, and was lower than the water bath temperature with a maximum difference of 1 1C. Samples were collected in sterile containers cooled with ice, and kept at 4 1C before analysis. At least two independent runs were carried out on different days for each studied STTT. 2.5. Protein solubility index The solubility of protein components was measured 1.5–2.5 h after processing (UHPH or STTT). Control and processed WPI dispersions (6% or 10%, w/w, protein) were diluted with deionised water to a final protein concentration of 1% (w/w). Solubility measurements were carried out by gentle magnetic stirring for 45 min at 2071 1C of 100 g dispersion at 1% (w/w) protein and pH 6.62 (spontaneous pH of the diluted dispersion), or after pH adjustment to 4.7 with 0.1 M HCl. The 1% (w/w) protein dispersions were then centrifuged in 50 mL tubes (Nalgene, Rochester, USA) at 12,000g and 20 1C for 15 min (Sorvall RC5B, rotor SS34) to eliminate insoluble constituents. Protein concentration was assessed in the non-centrifuged dispersions (total proteins) and in the supernatants after centrifugation (soluble proteins). Protein determination was carried out by the bicinchoninic acid (BCA) procedure modified to minimise interferences from thiol groups and reducing sugars as previously published (Dumay et al., 1998): 100 mL of supernatant (containing 10–100 mg proteins) was incubated at 37 1C for 15 min with 50 mL of 0.2 M iodoacetamide in 0.1 mM Tris–HCl buffer, pH 8. BCA reagent (2 mL) was then added. After 30 min incubation at 37 1C, the absorbance of protein–BCA mixtures was measured in quadruplicate at 562 nm (Unicam UV2 spectrometer, Thermo Optek, Montigny-le-Bretonneux, France), and corrected for the absorbance of the same BCA solution without protein. Calibration curves were performed with purified bovine b-Lg (non-crystallised, L-2506, Sigma) in 50 mM pH 7.0 sodium phosphate buffer. The solubility of protein constituents, also called protein solubility index (PSI), was taken as 100  (soluble protein present in the supernatant/total protein in an equal volume of the noncentrifuged dispersion). The protein loss upon centrifugation, corresponding to insoluble aggregates, was taken as 100  (total proteinsoluble protein)/(total protein). PSI was measured for at least two independent experiments (UHPH or STTT). 2.6. Particle size distribution by photon correlation spectroscopy The particle size distribution of control or processed (UHPH or STTT) samples was measured at 2570.5 1C by

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photon correlation spectroscopy (PCS), using a Zetasizer 3000 HS (MALVERN Instruments Ltd., Malvern, UK) with a 5 mW He–Ne laser operating at 632.8 nm. Initial dispersions were diluted to 0.1% (v/v) protein with a 50 mM pH 6.5 sodium phosphate buffer previously filtered through a 0.45 mm cellulose acetate membrane (Sartorius, Goettingen, Germany). Such dilution was necessary to avoid multiple diffusion phenomena during PCS measurements. The 0.1% (v/v) protein samples were then filtered through a 0.45 mm cellulose acetate membrane to check the absence of dust coming from the WPI powder, and to eliminate possible air bubbles by air expansion at the filter outlet. Most of the samples have been analysed both filtered and non-filtered. No significant difference could be seen between the size distribution curves of filtered and non-filtered samples, indicating the absence of disturbing particles and no loss of protein aggregates in the studied size range due to the filtration step. Experimental data were assessed by Contin’s algorithm designed to obtain size distribution curves in light intensity frequency for polydispersed samples, when the polydispersity index ranged between 0.08 and 0.5 (Malvern Zetasizer Handbook, Principles of operation, 1996). The polydispersity index is taken as the deviation of the autocorrelation function G(t) to the linearity on a semilogarithmic scale [log G(t) ¼ a+bt+ct2 where a, b and c are the polynomial coefficients and t the delay time; the polydispersity index equals 2c/b2] (Malvern software). In the present study, most of the sample polydispersity indices ranged between 0.28 and 0.5. For each sample, 6 PCS measurements of 90 s were carried out at a scattering angle of 901. PCS parameters were set as follows: the particle size ranged between 0.5 and 1000 nm; delay times were set at 1.6 and 4 ms for the fast and slow correlators, respectively; the first analytical point of the correlator was selected automatically; the number of points was set up to 60; the logarithmic spacing between points was 1.2; the point weighting was quartic. A number distribution was calculated from the intensity distribution, according to Mie’s theory. For this calculation, the dispersant viscosity was taken as 0.89 mPa s at 25 1C and the refractive index as 1.33. The dispersed particle characteristics were taken as for milk proteins (Regnault, Thiebaud, Dumay, & Cheftel, 2004): 0.004 and 1.36 for the imaginary and the real refractive indices, respectively. PCS measurements were performed 1.5–4 h after processing (UHPH or STTT). Size distributions were represented as intensity or number fraction (%) curves and called size distribution in light intensity frequency or size distribution in particle number frequency. It should be recalled that, according to Rayleigh’s law, in the case of particles much smaller than the measuring wavelength (632.8 nm), light scattering intensity is proportional to the sixth power of particle diameter (d 6). In the case of particles with dimensions close to the light wavelength, light scattering intensity, according to Mie’s theory, is proportional to d a.

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The exponent a decreases from 6 to 3.5 when the particle diameter increases from 50 to 1000 (Finsy & De Jaeger, 1991). In spite of this correction, the distribution curves in light intensity frequency reflect the influence of largest particles. In contrast, the distribution in number frequency is sensitive to particles of small size. Cumulated intensity or particle number fractions were equal to 100. For each independent experiment carried out on different days, a mean distribution curve was calculated from six measurements per sample. The corresponding mean diameters (arithmetical mean) were calculated from the intensity or number fraction (%) curves. 2.7. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis without a dissociating or denaturating agent (native PAGE) was performed, with a running 50 mM sodium phosphate buffer (PBS) at pH 7.0, using minigels (80  80 mm) prepared at pH 7.0 with 120 g acrylamide/L in 50 mM PBS for the separating gel, or 35 g acrylamide/L in 25 mM PBS for the stacking gel. Ammonium persulphate and TEMED were added at 1.8 and 2.8 mM, respectively. Protein samples were diluted in the 25 mM pH 7.0 PBS in the presence of glycerol (150 mL/L). Five microliters of samples containing 3.8 mg of protein/mL were pippeted on the plates. Electrophoresis was carried out 24 h after processing at a constant power of 12 W and 1871 1C for 4 h. Electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed under both reducing and non-reducing conditions, using a linear gradient at 80–200 g acrylamide/L in a pH 8.7 Tris–HCl buffer as a separating gel (Funtenberger et al., 1997). The stacking gel contained 60 g acrylamide/L in a pH 6.8 Tris–HCl buffer. Protein samples were prepared 4 h after processing by dilution in a pH 6.8 Tris–HCl buffer in the presence of 20 g SDS/L and 150 mL glycerol/L, with or without 210 mM MSH. After 4 min of heating at 100 1C, 8 mL of the mixtures containing 3.5 mg protein/mL were pipetted on the plates. Electrophoresis was carried out in a 25 mM Tris–glycine buffer, pH 8.3, containing 1 g/L SDS, at a constant power of 30 W for 6 h. During electrophoresis, temperature was controlled by circulating water at 1871 1C. Electrophoresis gels were stained with R-250 Coomassie blue and then washed as already described (Funtenberger et al., 1997). For protein identification and molecular weight (MW) determination, purified proteins (Sigma, St. Louis, MO) were used: b-Lg (L-2506); a-La (L-5385); BSA (A2153); MW SDS-6 H and SDS-7 kits. 2.8. Viscosity measurement Flow times of protein dispersions were measured using an Ubbelohde-type glass capillary tube (0.58 mm inner Ø, Prolabo, France) submerged vertically in a water bath maintained at 2070.1 1C. Measurements were carried out

at least in triplicate 1.5–4 h after processing. Viscosity (mPa s) was calculated from Eq. (1): Zd ¼

Zs t d r d , ts r s

(1)

where Zs and Zd are the respective viscosity of the solvent (deionised water) and the WPI dispersion; ts and td, their respective flow time (s); and rs and rd, their respective density (kg m3). Protein voluminosity was calculated according to Boulet, Britten, and Lamarche (1998) using Lee’s Eq. (2), relating the volume fraction (Fv; mL/mL) to the relative viscosity (Zd/Zs): Zd =Zs ¼ 1 þ 2:5Fv þ 7:031Fv2 þ 37:371Fv3 .

(2)

The voluminosity is of equivalent spheres. 2.9. Colour measurement Colour measurements of the control or processed samples were carried out in triplicate, 1.5–4 h after processing using a Luci 100 colorimeter (Dr. Lange, Du¨sseldorf, Germany) equipped with an integrating sphere (illuminant D65; observer at 101). Colour measurements were performed against a white background. The CIE 1976 L*, a* and b* colour system was used to evaluate the lightness, the red–green and the yellow–blue components, respectively. 2.10. Atomic force microscopy Atomic force microscopy (AFM) of protein samples was carried out using a Dimension 3100 microscope equipped with the Nanoscope IIIa electronic device (Digital Instruments-Veeco, Santa Barbara, USA) at the ‘‘Near Field Microscopy’’ Laboratory of Montpellier II University as previously described (Regnault et al., 2004). A 3–4 mL droplet of protein dispersion (untreated or UHPH-processed samples examined 1–2 h after processing) was placed on a previously cleaned mica disc (MG 40093, Metafix, F80500 Montdidier, France) and dried in ambient air. Nanoprobes cantilevers made of silicon (Type PPP-FM, Digital Instruments) with a spring constant of 0.5–9.5 N/m and a resonance frequency of 45–115 kHz were used. Topographical images were generated from vertical movements of the cantilever tip during scanning with a preset cantilever deflection (tapping mode). The oscillation amplitude was 50–70 nm. Images were treated with the Digital Nanoscope Software (Version 5.30r3, Digital Instruments) for 3-D representation. 2.11. Statistical analysis Statistical analyses of experimental data were carried out using Fisher’s and Student’s tests.

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3. Results and discussion 3.1. Effect of dynamic high pressure on whey protein solubility

90

T2 = 74.6°C

T2 = 76.5°C

80

T2 70

60

Temperature (°C)

Protein Solubility Index (%)

for p ¼ 0.05) increase in PSI at pH 4.7 (Fig. 2). It was possible that a better dispersion and hydration of WPI powder particles was achieved at 100–200 MPa. This trend was not observed at 10% (w/w) protein, after similar processing conditions (Fig. 2). A highly significant (pp0.01) decrease in PSI at pH 4.7 was observed above 250 MPa for the dispersion at 6% (w/w) protein, as compared with the untreated dispersion at pH 4.7 (Fig. 2). This decrease in soluble protein was accompanied by an opalescence of the dispersion at the homogeniser outlet, and by a change in sample colour from yellow–gold to milky white, suggesting the formation of protein aggregates able to scatter light. Comparing both dispersions (6% and 10%, w/w, protein) processed at 275–300 MPa, significantly (p ¼ 0.05) higher PSI values were found at the higher protein concentration (10%) (Fig. 2), instead of slightly higher (by 2–3 1C) T2 values measured at the outlet of the HP valve (Fig. 3). For the processed dispersion at 10% (w/w) protein, pH 4.7 insoluble protein effectively reached 1371% and 2171% of total initial protein constituents, at 275 and 300 MPa, respectively, instead of 2472% and 2871% for the 6% (w/w) protein dispersion at the two respective pressure levels (means from two independent experiments7standard deviation). Similar results dealing with the effects of dynamic high pressure on protein solubility for various initial protein concentrations and up to 300 MPa are not yet available. Paquin et al. (2003) reported protein solubility measured at pH 6.0 and 4.6 after processing at 150 MPa (Tin ¼ 22 1C), pH 6.0, WPI dispersions of 14% protein only: such processing with an AvestinTM ceramic valve did not induce higher losses of soluble protein as compared with that of untreated samples at both pH values. In the present study, the slightly higher temperatures T2 noticed at 10% (w/w)

∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

∗∗ ∗∗∗

∗

80

T2 = 71°C

∗

∗∗

∗

100

T2 = 73°C

PSI values equalled 100.672.4% for the untreated (control) dispersions as measured after dilution to 1% (w/w) protein and pH 6.62, and reached 93.572.1% after pH adjustment to 4.7 (means and standard deviations for 4 independent dispersions prepared on different days) (Fig. 2). Such PSI values reflect the highly native state of the WPI, industrially prepared using mild processes. No significant difference (pp0.05) could be observed between the PSI values of both A and B control dispersions, as compared at pH 6.62 or at pH 4.7. PSI at pH 6.62 did not reveal any significant difference (pp0.05) between the untreated and UHPH-processed samples, for both dispersions at 6% or 10% (w/w) protein (results not shown), indicating that all protein constituents remained soluble after centrifugation at neutral pH. These first results agree with those obtained by Bouaouina et al. (2006) after UHPH up to 300 MPa (Tin ¼ 20 1C; similar HP valve than in the present study): no change in protein solubility was observed upon centrifugation at pH 6.7 of processed 3% WPI dispersions. In contrast, PSI determined at pH 4.7 in the present study revealed the processing-induced effect through the precipitation of denatured/aggregated whey proteins at pH close to the isoelectric pH (Fig. 2). UHPH of the 6% (w/w) protein dispersion at 100–200 MPa induced a slight (even not always significant

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40 20

60 50

T3

40 30

T1

0 Control B

100

150

200

225

250

275

300

Pressure (MPa) Fig. 2. Protein solubility index at pH 4.7 (PSI, %) of WPI dispersions containing (w/w) 6% (’, ) or 10% ( , &) protein, after ultra-highpressure homogenisation (UHPH) at 100–300 MPa (Tin ¼ 24 1C). For each protein concentration, two sets of independent experiments were performed. For each independent experiment, means and standard deviation of four measurements are shown. PSI values expressed as 100  (soluble protein present in the supernatant of centrifugation/total protein in an equal volume of the non-centrifuged dispersion). Significant differences for *p ¼ 0.05, **p ¼ 0.01 or ***p ¼ 0.001, as compared with the corresponding untreated (control) dispersion at pH 4.7.

20 10 0 0

50

100

150

200

250

300

350

Pressure (MPa) Fig. 3. Temperature measured before the HP valve (T1, E, ), at the HP valve outlet (T2, –, ) and after immediate cooling at the HP valve outlet (T3, K, ), during UHPH at 100–300 MPa (Tin ¼ 24 1C). WPI dispersions containing (w/w) 6% (E, K, –) or 10% ( , , ) protein. Means of three independent experiments are shown. The standard deviations are included in the symbols.

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protein could result from higher frictional stress in the HP valve (followed by heat dissipation), in relation with the higher viscosity of the initial dispersion. Viscosity (Zd at 20 1C) was effectively significantly (p ¼ 0.01) higher at 10% (2.44 mPa s) than at 6% (1.63 mPa s) protein (CV p1.6%) (Tables 1 and 2). The influence of fluid viscosity during UHPH has recently been underlined by Diels, Callewaert, Wuytack, Masschalch, and Michiels (2005). The disruption mechanisms in the HP valve are not yet completely elucidated. It appears therefore that an increase of the initial fluid viscosity decreases the prevalence of turbulence, impact with solid surfaces and cavitation that could contribute to disruption of particles such as microorganism cells at the outlet of the gap valve (Diels, Callewaert, Wuytack, Masschalch, & Michiels, 2005). On the contrary, increase of the initial fluid viscosity could lead to more extensional stress and viscous shear effects able to favour disruption of cells, oil droplets or large macromolecules during the fluid travelling through the narrow gap of the HP valve (Floury, Desrumaux, Axelos, & Legrand, 2002). 3.2. Effect of continuous short-time thermal treatment on whey protein solubility Continuous STTTs (4 s) of protein dispersions were investigated with a view to assess the relative effects of heat and mechanical forces during UHPH. PSI values at pH 4.7 are presented in Fig. 4, as a function of fluid temperature at

the tubing outlet during STTT. A clear and significant (pp0.05) decrease in protein solubility was observed above 69 1C for the dispersion at 10% (w/w) protein, and above 74 1C at 6% protein. At 10% (w/w) protein, short-time heating at X77 1C induced a partial protein gelation in the tubing, explaining the lack of reported results at these temperatures. Considering the temperatures measured at the tubing outlet during continuous STTT and the temperatures T2 measured at the HP valve outlet during UHPH, in the case of the 6% (w/w) protein dispersion, it appears that processing at 275 MPa (T2 ¼ 71 1C) or 300 MPa (T2 ¼ 74.6 1C) induced significantly (p ¼ 0.01) lower values of PSI than STTT at 71 or 74 1C, respectively (Figs. 2 and 4): X20% of insoluble protein was observed after UHPH at 275–300 MPa, while p13% was observed after continuous STTT at 71 1C or 74.6 1C. It must be recalled that the residence time of the fluid at temperature T2 in the present HP homogeniser is 51 s and close to 215 ms for a single-pass homogenisation (Picart et al., 2006). Since the loss of soluble protein was significantly higher for UHPH than for STTT (4 s), there must be a combined effect (additional or synergistic effect) of mechanical forces and short-life heating phenomena taking place in the HP valve. The efficient cooling device installed after the HP valve in the present homogeniser avoided fluid over-heating and limited the exposure time (51 s) of protein constituents to temperature prone to denaturation, thus permitting one to reveal effect of mechanical forces as

Table 1 Physical characteristics (viscosity, voluminosity and CIE L*, a*, b* colour parameters) of the 10% (w/w) WPI dispersions processed by UHPH or STTT1 UHPH Pressure (MPa)

Viscosity (mPa s)

Voluminosity (mL/g)

L*

a*

b*

Control A2 Control B2 100 150 200 225 250 275 300

2.4470.01a 2.4570.04a,b 2.4470.03a,b 2.4570.02a,b 2.4570.01a,b 2.4670.01b 2.5370.02c 2.6970.03d 2.8170.02e

2.2770.01a 2.2870.03a,b 2.2870.03a,b 2.2870.02a,b 2.2870.01a,b 2.2970.01b 2.3570.01c 2.4770.02d 2.5570.01e

28.8770.25a 28.6570.94a 29.0272.42a,b 29.7070.67a 30.5371.81a,c 31.8970.16b,c 33.6970.09d 33.9970.01e 34.8970.02f

0.4170.03a 0.4370.01a 0.4570.03a 0.4470.02a 0.4870,05a 0.6470.00b 0.9370.03c 1.2370.02d 1.5470.01e

3.3970.34a 2.9770.09a,b 2.6070.34b 3.0270.17a,b 3.0770.46a,b 2.0370.10c 0.4470.10d 0.3170.01d 1.1370.01e

STTT Temperature (1C)

Viscosity (mPa s)

Voluminosity (mL/g)

L*

a*

b*

Control3 58 61 66 69 73 77

2.4470.02a,b 2.4670.01a 2.4270.01b 2.4970.01c 2.4970.01c 2.5570.01d 2.5670.02d

2.2770.02a,b 2.2970.01a 2.2570.01b 2.3270.01c 2.3170.01c 2.3670.01d 2.3770.02d

31.4470.01a 31.8770.13b 32.2670.21b,c 32.5470.04c 33.5570.07d 36.6170.03e 53.4670.03f

0.4470.04a 0.5470.01b 0.5670.03b 0.6470.03c 0.7870.01d 1.4170.01e 4.2170.01f

3.6770.33a 3.2370.12a 3.3570.27a 2.2370.06b 0.2370.04c 3.2170.04d 4.9570.02e

Values followed by the same letter within a column are not significantly different at pp0.05. For determination of physical characteristics see the text. 1 Measurements carried out 1.5–4 h after UHPH or STTT. Mean values (7standard deviation) for at least 3 measurements. 2 Untreated dispersion (control A) or dispersion run through the homogeniser without applied high pressure (control B). 3 Control dispersion at 24 1C.

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Table 2 Physical characteristics (viscosity, voluminosity and CIE L*, a*, b* colour parameters) of the 6% (w/w) WPI dispersions processed by UHPH or STTT1 UHPH Pressure (MPa)

Viscosity (mPa s)

2

Voluminosity (mL/g)

a

a

1.6370.01 1.6370.01a 1.6470.01a 1.6470.01a 1.6670.02a 1.6470.02a 1.6670.02a 1.6970.02b 1.7370.02b

Control A Control B2 100 150 200 225 250 275 300

L*

a* a

2.3770.03 2.3970.03a 2.4070.02a 2.4070.02a 2.4470.05a 2.4270.04a 2.4470.05a 2.5270.05b 2.6070.03b

32.0870.11 31.8770.06b 31.7870.28a,b 31.0771.59a,b 32.1970.77a,b 32.0470.08a,b 32.4870.10c 34.5070.06d 36.2970.05e

b* a

0.4570.04 0.4670.01a 0.4570.03a 0.4170.02a 0.3470.02b 0.5370.03a 0.5870.03c 0.8170.02d 1.1570.02e

1.5170.27a 1.8570.11a 1.4870.09a 1.2970.14a 0.5370.22b 1.4470.32a 0.5970.31b 1.3270.11c 3.0670.05d

STTT Temperature (1C)

Viscosity (mPa s)

Voluminosity (mL/g)

L*

a*

b*

Control3 58 61 67 71 74 76 78 82

1.6170.01a 1.5970.01b 1.5970.01b 1.6070.01a,b 1.6070.01a,b 1.5970.01b 1.6070.02a,b 1.6270.01a,c 1.6370.01c

2.3370.03a 2.2770.02b 2.2770.03b 2.3070.01a,b 2.3070.01a,b 2.2870.01a,b 2.3170.05a,b 2.3770.03a,c 2.3970.01c

31.9870.18a 32.7170.06b 32.2270.05a 32.1471.13a,b 33.7470.37c 35.0770.27d 39.1570.56e 44.3470.24f 60.5270.12g

0.4670.02a 0.3870.01b 0.5170.01c 0.4870.06a,b,c 0.5370.06a,c 0.7170.03d 1.4570.21e 2.6370.07f 4.5870.01g

1.5070.25a 0.3070.02b 1.1970.14a 0.0070.13c 0.9570.12d 2.4670.12e 6.8170.03f 8.9370.13g 7.5070.17h

Values followed by the same letter within a column are not significantly different at pp0.05. For determination of physical characteristics see the text. 1 Measurements carried out 1.5–4 h after UHPH or STTT. Mean values (7standard deviation) for at least 3 measurements. 2 Untreated dispersion (control A) or dispersion run through the homogeniser without applied high pressure (control B). 3 Control dispersion at 24 1C.

∗∗ ∗∗∗

Protein Solubility Index (%)

∗∗∗

∗∗ ∗∗∗ ∗∗∗

∗

∗ ∗∗∗∗

∗∗ ∗∗

∗∗

∗∗

∗∗ ∗∗

∗

100 80

compared with heating phenomena. These results agree with previous data (Picart et al., 2006) showing higher inactivation of milk endogenous alkaline phosphatase after UHPH than STTT for similar processing conditions than in the present study.

60

3.3. Effect of dynamic high pressure on protein aggregate sizes

40 20 0 24

58

61

66 67 69 71 73 74 Temperature (°C)

76 77 78

82

Fig. 4. Protein solubility index at pH 4.7 (PSI, %) of WPI dispersions containing (w/w) 6% (’, ) or 10% ( , &) protein, after continuous short-time (4 s) thermal treatment (STTT) at various temperatures between 58 and 82 1C (temperatures measured at the tubing outlet). Tin ¼ 24 1C. For each protein concentration, two sets of independent experiments were performed. For each independent experiment, means and standard deviation of four measurements are shown. Protein solubility indices expressed as for Fig. 2. Significant differences for *p ¼ 0.05, **p ¼ 0.01 or ***p ¼ 0.001, as compared with the corresponding untreated dispersion (control at 24 1C) at pH 4.7.

Fig. 5a–d shows the size distribution curves in light intensity frequency and in particle number frequency obtained for non-processed WPI dispersions, initially prepared at 6% or 10% (w/w) protein, and then diluted to 0.1% (v/v) for PCS measurements (control A), and the size distribution curves for the same dispersion after running it through the homogeniser without applying high pressure (control B). The size distribution curves in intensity (Fig. 5a, c) (sensitive to particles of large sizes) showed a first population with a maximum located at 10 nm (peak 1), a second population with a maximum at 70 nm (peak 2), plus a minor population at 300–500 nm (peak 3). The distribution curves of the two controls A and B were similar without any significant difference (pp0.05) between the corresponding mean diameters in intensity or

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50

50

a

30 20

30 20 10

0

0

30 20

d

Control A Control B 100 MPa 150 MPa 200 MPa 225 MPa 250 MPa 275 MPa 300 MPa

10 0

e 40 30 20

Control A 58°C 61°C 67°C 71°C 74°C 76°C 78°C 82°C

30 20 10 0

f 30 20 10

0

0

g 30 20

Control A 58°C 61°C 67°C 71°C 74°C 76°C 78°C 82°C

40

10

40

Control A Control B 100 MPa 150 MPa 200 MPa 225 MPa 250 MPa 275 MPa 300 MPa

40

Size distribution in number frequency (%)

c

Control A Control B 100 MPa 150 MPa 200 MPa 225 MPa 250 MPa 275 MPa 300 MPa

40

10

40

Size distribution in intensity frequency (%)

b Control A Control B 100 MPa 150 MPa 200 MPa 225 MPa 250 MPa 275 MPa 300 MPa

40

h

Control 58°C 61°C 63°C 66°C 69°C 73°C 77°C

Control 58°C 61°C 63°C 66°C 69°C 73°C 77°C

40 30 20

10

10

0

0 1

10

100

1000

Diameter (nm)

1

10

100

1000

Diameter (nm)

Fig. 5. Particle size distribution curves of WPI dispersions after UHPH at 100–300 MPa for Tin ¼ 24 1C (a–d), or STTT for 4 s at various temperatures between 58 and 82 1C at the tubing outlet (e–h). WPI dispersions containing (w/w) 6% (a, b, e, f) or 10% (c, d, g, h) protein at pH 6.5. Size distribution curves in light intensity frequency (a, c, e, g) or in particle number frequency (b, d, f, h). Measurements carried out by photon correlation spectroscopy at 25 1C. Mean curves from six PCS measurements are shown.

in number, indicating that running the sample close to atmospheric pressure through the HP valve gap of few micrometres did not induce any particular effect on protein particle size. The size distribution curves in particle number frequency (sensitive to particles of small size) showed a

main population between 2 and 10 nm, with a peak maximum at 3.6 nm and a mean diameter of 4.37 0.1 nm, for both control samples A and B, and indicated that 90% of the hydrated protein particles are p7 nm in diameter. From hydrodynamic or X-ray crystallographic

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measurements, the b-Lg dimer is approximately an ellipsoid with a length of 6.9 nm and a width of 3.6 nm and composed of two spheres of 1.8 nm radius in contact with one another (Mulvihill & Donovan, 1987). Nuclear magnetic resonance (NMR) studies indicated a Stokes radius of 1.9 nm for b-Lg monomers and 3.8 nm for b-Lg octamers (Pessen, Purcell, & Farrell, 1985). Using smallangle X-ray scattering (SAXS), Panick et al. (1999) have calculated diameters of 3.9–4.2 nm for a solution of 1% purified b-Lg at pH 7.0 and 20 1C, with maximal dimensions around 11 nm. Using solution X-ray scattering, Kataoka, Kuwajima, Tokunaga, and Goto (1997) have determined a radius of gyration of 1.72 nm for holo-alactalbumin at pH 8.0. Taking into account that PCS indicates hydrodynamic diameters, i.e., the diameter of the protein particle itself plus the width of the hydration shell, a size range of 2–7 nm calculated by PCS in the present study for most of WPI native proteins (68.5% b-Lg plus 21.5% a-La) is in agreement with the expected values for such globular proteins. Using dynamic light scattering (DLS) at 901, Roefs and de Kruif (1994) reported a particle diameter of 10.4 nm for a WPI isolate (87% b-Lg; pH 6.8), as calculated by the cumulant fit (i.e. assuming a monodisperse distribution). Using DLS and a calculation method similar to Contin’s algorithm, Gimel, Durand, and Nicolai (1994) have found a diameter of 5.870.2 nm for purified b-Lg at pH 7.0. For b-Lg isolated from WPC at pH 7.0, Sharma, Haque, and Wilson (1996) have calculated a particle diameter of 11 nm by the cumulant fit, and reported that 60% of the percentile size distribution given by Contin’s analysis corresponded to the 1–9 nm range, while 40% corresponded to a 10–99 nm range. These DLS results are in accordance with the peak (1) and (2) size ranges given by the distribution curves in light intensity frequency (Contin’s analysis) in the present PCS study. As already underlined, the intensity distribution curves primarily reflect large particles. In constrast, the distribution in particle number frequency is sensitive to the particles of small size. This explains the different representations given by both kinds of size distribution, in light intensity or in particle number frequency, and the usefulness of the two representations. Fig. 5a–d shows the size distribution curves of samples processed by UHPH, compared with continuous STTT (Fig. 5e–h). PCS revealed few changes after UHPH at 100, 150 or 200 MPa. Two main populations are shown by the size distribution curves in intensity, which mainly corresponded to peaks (1) and (2) of the untreated sample (Fig. 5a). There was also a reduction of peak (3) intensity and a global shift of the intensity curves towards smaller particle sizes. This shift towards lower sizes clearly appeared on the size distribution in particle number frequency of the 6% (w/w) protein dispersion (Fig. 5b), and could explain the slightly higher PSI values found at these pressure levels (Fig. 2). This apparent reduction in particle sizes could result from a dispersive effect of UHPH on some protein agglomerates present in the WPI

1023

dispersion (peak 3, Fig. 5a). The effect of low pressure (100–200 MPa) did not affect the size distribution of 10% (w/w) sample (Fig. 5d) in accordance with solubility results (Fig. 2). At 225 MPa, the size distribution in intensity frequency showed a decrease in peak (1) and a concomitant increase in peak (2), accompanied with a shift towards larger sizes for both dispersions at 6% and 10% (w/w) protein (Fig. 5a, c). This slight positive shift by a few nm (Fig. 5d) suggests that some irreversible structural changes (unfolding and hydration of protein particles) could start at this pressure level. UHPH at 250, 275 and 300 MPa induced marked shifts towards larger sizes as shown by the size distribution curves in both intensity and particle number frequency, indicating clear aggregation phenomena above 225 MPa at both protein concentrations (Fig. 5a–d). Aggregation phenomena are also clearly indicated by an increase in the mean diameters calculated from the distribution curves in intensity or in particle number frequency (Fig. 6a, b). Bimodal distributions with two maxima at 7 and 26 nm were observed at 6% (w/w) protein and 250 MPa (size distribution in particle number frequency; Fig. 5b), suggesting that aggregation phenomena occurred firstly with the formation of small size aggregates of twice the initial particle sizes, followed by the formation of larger aggregates of 10–20-fold the initial particle sizes. Aggregation phenomena were amplified at the highest pressure levels. At 6% (w/w) protein, the largest aggregates reached 300–400 nm as indicated by the size distribution curve in intensity frequency (Fig. 5a), but most of the aggregates did not exceed 100–200 nm as indicated by the size distribution curve in particle number frequency (Fig. 5b). On the contrary, at 10% (w/w) protein, a monomodal distribution was observed as soon as 250 MPa was reached (Fig. 5d), with a maximum at 26 nm and most of the particle sizes under 100 nm, in spite of a marked shift towards higher sizes shown by the distribution curves in intensity (Fig. 5c). This fact could be explained by a possible equilibrium between aggregate formation and disruption occurring at 250–300 MPa and 10% (w/w) protein. It is likely that most of the biggest aggregates induced by UHPH were broken by mechanical forces, but still a few of them remained, giving the highest sizes on the distribution curves in intensity. This trends is also visible in Fig. 6. Despite the higher mean diameter in intensity (Fig. 6a), the presence of more small size aggregates led to smaller mean diameter in particle number frequency (Fig. 6b) for the 10% (w/w) sample than for the 6% (w/w) sample. High protein concentration increases the probability of particle collisions during the fluid travelling through the narrow gap of the HP valve (i.e. in a laminar flow; Floury et al., 2004) and at the valve gap outlet where cavitation phenomena, turbulence and impact take place. All these mechanical forces could favour both (i) particle nucleation/aggregation and (ii) aggregate disruption. As already suggested, it is likely that higher frictional effects took place in the HP valve at 250–300 MPa and 10% (w/w)

ARTICLE IN PRESS A. Gra´cia-Julia´ et al. / Food Hydrocolloids 22 (2008) 1014–1032

1024 350

350

a

250

250

200

200

150

150

100

100

50 0

b

100

c

300

Mean diameter (nm)

Mean diameter (nm)

300

50 0

80

80

60

60

40

40

20

20

0

d

100

0 0

50

100

150

200

250

300

350

Pressure (MPa)

20

30

40

50

60

70

80

90

Temperature (°C)

Fig. 6. Mean diameters of protein particles in WPI dispersions processed by UHPH at 100–300 MPa for Tin ¼ 24 1C (a, b), or by STTT for 4 s at various temperatures between 58 and 82 1C at the tubing outlet (c, d). Mean diameters calculated from the PCS distribution curves in light intensity frequency (a, c) or in particle number frequency (b, d). WPI dispersions containing (w/w) 6% (K) or 10% (m) protein at pH 6.5. Means of two independent experiments plus deviation to the mean are shown.

protein (compared with 6%), probably in relation with the higher dispersion viscosity at the entrance of the HP valve. The initial viscosity of untreated dispersions was indeed 2.44 mPa s for the 10% (w/w) protein sample instead of 1.63 mPa s for the 6% sample (significant difference for p ¼ 0.01) (Tables 1 and 2, controls). At the pressure drop across the HP valve and in a laminar flow, the frictional loss coefficient effectively depends on viscosity (Stevenson & Chen, 1997). Increase in the initial viscosity of dispersions could lead to higher frictional effects, extensional stress and aggregate disruption, thus limiting aggregate maximal size to 100 nm (Figs. 5d and 6b). Further studies are needed to thorough the effects of high shear rates on particle aggregation/disruption at a high protein concentration. Shear rate reaches effectively high values in such UHPH valves (shear rates b107 s1; Floury et al., 2002). The present results therefore point out the possibility to use high shear forces for controlling aggregate size. For the present UHPH conditions, 225 MPa appears as a transition pressure for protein aggregation. AFM images display the topography of multilayer samples. Even if we cannot observe independent particles as it could be possible with highly diluted samples, the present images indicate a different topography for the control sample and the sample processed at 200 MPa (Fig. 7a, b) on the one hand, and the samples processed at

250–275 MPa (Fig. 7c, d) on the other hand. AFM 3-D representation displayed quite homogeneous layers of elementary protein particles for the untreated sample and the sample processed at 200 MPa (Fig. 7a, b). AFM examination indicated that these elementary protein particles were 5–8 nm in diameter, in accordance with PCS results (given in particle number frequency). In contrast, protein particles were roughly agglomerated at X250 MPa (Fig. 7c, d). Section analyses of AFM windows (Fig. 7a0 , d0 ) indicate the organisation of protein particles in the dried samples. In the case of the untreated sample and the sample processed at 200 MPa (Fig. 7a0 , b0 ), the elementary particles of 5–8 nm (small size peaks) were regularly arranged into 10–40 nm heaps homogeneously distributed on the mica sheet. At X250 MPa, section analyses (Fig. 7c0 , d0 ) indicated that the elementary particles (small size peaks) were gathered into large size conglomerates of 60–170 nm width, probably due to stronger particle–particle interactions than for the other two samples. These topographical data agree with the presence of protein aggregates observed by PCS at X250 MPa (but not at 200 MPa). These preliminary results need to be completed by AFM examination of more diluted samples according to Ikeda and Morris (2002), for example, to characterise in depth aggregate assemblage, surface and shape.

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Fig. 7. Atomic force micrographs (a–d) of WPI dispersions containing 10% (w/w) protein at pH 6.5 and corresponding section analyses (a0 –d0 ) along the up-down diagonal of the AFM windows. Untreated sample (a, a0 ). UHPH-processed samples at 200 MPa (b, b0 ), 250 MPa (c, c0 ) or 275 MPa (d, d0 ) for Tin ¼ 24 1C. The spherical yellow coloured particles represent protein, while the brown areas represent the mica holder. (a–d) Bar ¼ 0.2 mm in the direction of X-axis and Z-axis, 28 nm in the direction of Y-axis. (a0 –d0 ) Bar ¼ 0.25 mm in the X-axis.

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3.4. Effect of continuous short-time thermal treatment on protein aggregate size Fig. 5e–h shows the size distribution curves in light intensity and in particle number frequency obtained for the WPI dispersions at 6% or 10% (w/w) protein after continuous STTT for 4 s. When the temperature increased from 58 to 77 1C, the size distribution curves in intensity showed a progressive decrease of peak (1) with a concomitant increase of peak (2) and the disappearance of peak (3) (Fig. 5e, g). The peak (2) maximum was progressively shifted towards larger sizes, from 70–80 nm (control) up to 200 nm for samples heated at highest temperature, with the maximum aggregate size reaching 300 nm for 6% (w/w) samples. The present results are in accordance with those of Sharma et al. (1996) who, using DLS, observed a sharp decrease in the small size particles (1–9 nm) with a concomitant increase in aggregates of larger sizes (100–500 nm) after heating 5% (w/v) b-Lg (pH 7.0) at 70 1C for 5 min. The distribution in particle number frequency (Fig. 5f, h) also indicated a progressive increase in particle size with temperature increase. Moderate heating of protein samples up to 66–67 1C for 4 s induced a shift of the peak maximum from 3.6 nm (control) to 6–7 nm (heated samples), i.e. twice the initial diameters at both protein concentrations. This size increase could be caused firstly by a progressive and irreversible (after cooling) unfolding/ hydration of b-Lg, most of the hydrodynamic radii being o10 nm until 63 1C. Cairoli et al. (1994) have effectively shown by in situ intrinsic fluorescence studies of purified b-Lg solution (0.34%, w/v, pH 6.8) that irreversible changes occurred above the temperature threshold of 60 1C, with a rapid exposure of tryptophan residues in the dead time measurement of 2–5 s. In the present study, a beginning of aggregation at 66–67 1C (Fig. 5f, h) is not excluded, as the observed particle size of 6–7 nm (peak maximum) could correspond to b-Lg octamers (Panick et al., 1999). Then, clear aggregation took place from 69 1C to higher temperatures with a clear dependence of aggregate size upon temperature. Fig. 5f displays bimodal curves at 71–74 1C, suggesting two main populations of aggregates with a first maximum at 10 nm and a second one near 50 nm (maximal sizes of 20 and 200 nm, respectively). At X74 1C, the 50 nm peak increased at the expense of the 10 nm peak with a progressive shift of the peak maximum towards 100 nm (Fig. 5f) and of the maximal particle size towards 300 nm (Fig. 5e) at the highest temperature. Such an influence of temperature on aggregate growth has already been reported by Le Bon, Nicolai, and Durand (1999) for b-Lg isolate dispersions prepared at various concentrations and pH 7.0, and heated between 55 and 87 1C for 10 min to longer heating times. Kazmierski and Corredig (2003) also observed a temperature-dependent aggregation for 10% WPI dispersions at pH 7.0. In this latter study, higher extents of insoluble protein were observed after heating at 75 or 85 1C than at 65 1C for

various heating times (5–120 min). Furthermore, larger aggregates (of higher MWs) formed at 85 1C than at 65 1C in accordance with the present results. It is effectively known that thermal reactivity of b-Lg (related to the reactivity of b-Lg free sulphydryl group) occurred at a faster rate at the highest temperature (Hambling et al., 1992; Qi et al., 1995; Le Bon et al., 1999). Aggregation can be interpreted as an unfolding first-order step followed by second-order aggregation steps (Verheul et al., 1998). b-Lg unfolding is fast at high temperatures and therefore no more rate limiting for the whole aggregation process, favouring the formation of large aggregate sizes even after short heating time (Cairoli et al., 1994) as in the present study. This could explain why a main population of large aggregates is observed at 78 and 82 1C instead of both small and large aggregates at lower temperatures (Fig. 5f). Higher aggregate sizes were reached after short-time heating of the dispersion at 10%, w/w, protein (compared with 6%), as shown by the mean diameters of both distributions in intensity and number frequency (Fig. 6c, d). These results suggested a dependence of aggregate sizes upon protein concentration in accordance with already published data with purified b-Lg (Hoffmann et al., 1996; Verheul et al., 1998; Le Bon et al., 1999) or WPI (Kazmierski and Corredig, 2003). Both the size and the amount of aggregates increase with increasing protein concentration (Hoffmann et al., 1996). Indeed, heating of dispersions at high protein concentration increases the rate of aggregation propagation (but not the rate of the unfolding step). This probably explains the wider particle distributions with a predominance of large aggregates observed at 10% (w/w) protein as compared with 6% at 76–78 1C (Fig. 5f, h). Comparing processed samples at 275–300 MPa with short-time (4 s) heated samples at temperatures similar to T2 (i.e. 71–74 1C for the 6% (w/w) protein dispersions or 73–77 1C for the 10%), it appears that aggregates of highest sizes (50 nm) were more numerous after UHPH than shorttime heating, at 6% but no more at 10% (w/w) protein (Figs. 5 and 6). These results underline the role of frictional effects developed by dynamic high pressure to limit aggregate growth at high protein concentration. 3.5. Physical characteristics of processed protein dispersions Table 1 gives some physical characteristics of the 10% (w/w) protein dispersion processed by UHPH or STTT. Significant (p ¼ 0.05) increase in viscosity, voluminosity and lightness (whiteness), and significant (p ¼ 0.05) decrease in a* and b* values were noticed at X225 MPa, as compared with the untreated dispersion (control A). The level of 225 MPa also appeared as a pressure threshold for aggregation as revealed by PCS. This increase in viscosity and fluid whiteness resulting from UHPH-induced aggregation at 250–300 MPa could be of interest in product development. Voluminosity values calculated in the present study were close to those published by Boulet et al. (1998)

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for a WPI dispersion at 4% (w/v) protein, pH 6.8 and p0.1 M ionic strength. Taking into account the protein composition of WPI (Section 2.1) and the corresponding protein molecular weights, it was possible to calculate a total number of particles (N) per mL of dispersion using dispersion density and Avogadro number. This leads to an apparent mean particle diameter of 5.35 nm by dividing the voluminosity value of untreated sample per N, assuming spherical spheres. This value agrees with the particle size of untreated sample, as determined by PCS in particle number frequency. STTT for 4 s induced clear and significant (p ¼ 0.05) changes in viscosity, voluminosity and lightness from 66 1C and above, as compared with the untreated dispersion at 10% (w/w) protein (control, Table 1). Comparing processed samples at 275–300 MPa with short-time (4 s) heated samples at 73–77 1C, it appears that heating induced significantly (pp0.05) lower viscosity and voluminosity values, but marked changes in colour parameters, suggesting different aggregate characteristics such as particle density (inversely correlated to voluminosity) and size in accordance with PCS results (Figs. 5d, h and 6a–d). Considering all the data obtained for both UHPH and STTT samples at 10% (w/w) protein, it indeed appears that lightness increased with the mean particle size in number according to a polynomial relation (lightness ¼ 7  105S38  103S2+0.33S+ 29.52, where S is the mean particle size in number; R2 ¼ 0970). The significantly (p ¼ 0.05) higher increase in voluminosity (by 12.3%) observed at 300 MPa, as compared with the 4.4% increase after STTT at 77 1C for 4 s, suggested aggregate assemblies of lesser density, i.e. more permeable to the aqueous solvent and able to develop higher viscosity in the former case. Significantly lower viscosity values were measured at 6% (Table 2) than at 10% (w/w) protein (Table 1). Table 2 also indicates slightly higher voluminosity and lightness values, and lower b* values (yellow component) as expected for a lower WPI powder content in the 6% samples. Processing induced the same trends in physical characteristics at both protein concentrations, but with much lesser intensity at 6% than at 10% (w/w) protein. Lesser increases in viscosity and voluminosity values (as compared with control samples) were effectively observed at 6% than at 10% (w/w) protein after UHPH, with a pressure threshold at 250 MPa (Table 2) instead of 225 MPa (Table 1) for significant changes. STTT of the 6% (w/w) protein dispersion induced significant (pp0.05) increase in viscosity and voluminosity only at the highest temperatures (X78 1C, Table 2), and significant (pp0.05) increase in lightness from 71 1C and above (Table 1) instead of 66 1C in the case of the 10% (w/w) protein sample (Table 1). 3.6. Electrophoresis characterisation of protein aggregates induced by dynamic high pressure Native PAGE under non-dissociating and non-reducing conditions allows proteins to separate based on size, shape

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and charge. The main proteins present in untreated WPI dispersion were b-Lg (A and B variants) and a-La, followed by BSA and 3–4 other minor protein species (Fig. 8a–d). SDS-PAGE patterns of WPI samples plus identification of MW with protein markers (Fig. 9) confirmed the presence of the main proteins as b-Lg (band 6) and a-La (band 7), and revealed that the minor protein species could be immunoglobulins (band 1), lactoferrin (band 2) and BSA (band 3). Traces of b-Lg di-/trimers (band 4, 33–40 kDa) and traces of caseins (band 5; 29–33 kDa) were also visible on SDSPAGE patterns as weak bands. These results are in agreement with the composition given by the producer and with previous studies (Patel, Singh, Havea, Considine, & Creamer, 2005). Identification of caseins traces was supported by the disappearance of peak (5) from SDSPAGE pattern of pH 4.7 WPI-soluble fraction (results not shown). The traces of b-Lg di-/trimers (peak 4) disappeared from SDS-PAGE+MSH (results not shown), indicating that they were linked through disulphide bonds, due to oxidative phenomena. However, the low level of b-Lg di-/ trimers in the untreated dispersion underlines the highly native state of the industrial WPI. Native PAGE patterns did not reveal any difference between the untreated (controls) and the processed dispersions up to 200 MPa at both pH values (6.5 and 4.7) and for both 6% and 10% (w/w) protein dispersions (see Fig. 8a for the 6% protein sample). This result suggested that UHPH did not induce structural changes (MW and/or charge) in proteins up to 200 MPa in accordance with solubility (Fig. 2) and PCS data (Figs. 5a–d and 6a, b) obtained in the present study, or with previous studies carried out at p200 MPa without fluid pre-heating. Subirade et al. (1998) effectively did not observe b-Lg denaturation/aggregation after homogenisation of b-Lg isolate dispersion at 140 MPa (Tinp25 1C). No b-Lg denaturation was reported after HP homogenisation of reconstituted skim milk up to 186 MPa (Tin ¼ 25 1C; Sandra & Dalgleish, 2005) or raw whole milk up to 200 MPa (Tin ¼ 10 1C; Hayes & Kelly, 2003) in accordance with the present results. At X250 MPa, a strong band (X) appeared at the top of the stacking gel on native PAGE patterns for both protein concentrations (see Fig. 8b for the 6% protein sample), corresponding to protein constituents not entering the gel. The intensity of band X increased at the highest pressure levels (275–300 MPa). Besides, a band located at the borderline between the stacking and separating gels appeared stronger at this pressure range (Fig. 8b). Not detected on the native PAGE patterns of pH 4.7-soluble fractions (as compared with control sample, Fig. 8b), these bands corresponded mainly to the insoluble protein aggregates quantified by PSI at pH 4.7. Whatever the pressure level, SDS-PAGE patterns of WPI dispersions processed by UHPH were similar to SDSPAGE patterns of the untreated sample, for both 6% and 10% (w/w) protein samples. Fig. 9 shows SDS-PAGE patterns for the 6% (w/w) protein dispersion processed at

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Fig. 8. Native polyacrylamide gel electrophoresis (native PAGE) patterns of proteins from WPI dispersions processed by UHPH (a, b) or STTT (c, d). WPI dispersions at 6% (a–c) or 10% (w/w) protein (d). Native PAGE patterns of proteins from dispersions prepared at pH 6.5 (total proteins) or after pH adjustment to 4.7 followed by centrifugation (pH 4.7 protein soluble fractions). (a) and (b) Native PAGE patterns of control or UHPH-processed dispersion at 6% (w/w) protein. Purified proteins from Sigma: bovine serum albumine (BSA) (a1, b1); a-lactalbumin (a-La) (a2, b2); b-lactoglobulin (b–Lg) (a3, a12, b3). Control samples (a4, 5 and b4, 5). UHPH-processed samples at 100 MPa (a6, 7), 200 MPa (a8, 9), 225 MPa (a10, 11), 250 MPa (b6, 7), 275 MPa (b8, 9) or 300 MPa (b10, 11). Total proteins from non-centrifuged dispersions at pH 6.5 (lanes 4, 6, 8, 10) and proteins from pH 4.7-soluble fractions (lanes 5, 7, 9, 11). (c) Native PAGE patterns of proteins from control (c 2, 3) or STTT-processed dispersion (c4–11) at 6% (w/w) protein. Purified proteins from Sigma: b-Lg (c1), mixture of BSA and a-la (c12). STTT-processed dispersion at 82 1C (c4, 5), 78 1C (c6, 7), 76 1C (c8, 9) or 74 1C (c10, 11) for 4 s. Total proteins from noncentrifuged samples at pH 6.5 (lanes 2, 4, 6, 8, 10) and proteins from pH 4.7-soluble fractions (lanes 3, 5, 7, 9, 11). (d) Native PAGE patterns of proteins from control (d3) or STTT-processed dispersion (d4–10) at 10% (w/w) protein. Purified proteins from Sigma: b-Lg (d1), a-la (d2), mixture of b-Lg and a-La (d11) and BSA (d12). STTT-processed samples at 58 1C (d4), 61 1C (d5), 63 1C (d6), 66 1C (d7), 69 1C (d8), 73 1C (d9) or 77 1C (d10) for 4 s. Total proteins from non-centrifuged dispersions at pH 6.5. Migration was from the top to the bottom. For all processed samples, Tin was 24 1C.

200–300 MPa. No aggregate bands could be seen at the protein deposit location or in the stacking gel for the UHPH-treated samples as compared with both control (A and B) samples. SDS-PAGE in the presence of MSH also

gave similar electrophoresis patterns for the untreated and UHPH-processed samples at both protein concentrations (results not shown). These results allowed us to conclude that protein aggregates induced by UHPH at X250 MPa

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Fig. 9. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns of proteins from WPI dispersion at 6% (w/w) protein. Untreated WPI (control A, lanes 1, 10). Mixture of BSA and a-La from Sigma (lane 2). Dispersion run through the UHP homogeniser without pressure (control B, lane 3). UHPH-processed dispersion at 200 MPa (lane 4), 225 MPa (lane 5), 250 MPa (lane 6), 275 MPa (lane 7) or 300 MPa (lane 8). MW protein markers from Sigma (lane 9). STTT-processed dispersion at 71 1C (lane 11), 74 1C (lane 12), 76 1C (lane 13), 78 1C (lane 14) or 82 1C (lane 15) for 4 s. b-Lg from Sigma (lane 16). St ¼ stacking gel, Sp ¼ separating gel. Migration was from the top to the bottom. MW protein markers from Sigma (lane 9): BSA (66 kDa); ovalbumin (OVA, 45 kDa); glyceraldehyde-3-phosphate dehydrogenase (GPD, 36 kDa); bovine erythrocyte carbonic anhydrase (CA, 29 kDa); bovine pancreas trypsinogen (TRY, 24 kDa); soybean trypsin inhibitor (TI, 20.1 kDa); a-La (14,4 kDa). WPI proteins: (1) immunoglobulins (Igs, X110 kDa); (2) lactoferrin (Lf, 78 kDa); (3) BSA (66 kDa), (4) b-Lg di-/trimers (33–40 kDa); (5) caseins (29–32 kDa); (6) b-Lg monomers (18.4 kDa); (7) a-La (14.4 kDa). Heated-induced aggregates not entering the stacking gel are marked as Z and mainly visible in lanes 12–15. For all processed samples, Tin was 24 1C.

were dissociated by SDS and therefore cross-linked mainly via hydrophobic bonds. Bouaouina et al. (2006) processed WPI dispersions (3%, w/w, protein; pH 6.7) by UHPH between 50 and 300 MPa (Tin ¼ 20 1C). Similar to our present study, they did not observe any significant changes between UHPH-treated and untreated samples in the band intensity of a-La, b-Lg and BSA on SDS-PAGE under dissociating/non-reducing or reducing conditions. In the present study, no intermediate constituents (corresponding to low MW polymers of b-Lg) appeared behind the b-Lg bands (A and B variants) as already observed by native and SDS-PAGE after isostatic HP treatment of b-Lg isolate (2.5%, w/w, protein; pH 7.0) at 350–450 MPa and 25 1C for 15–30 min (Funtenberger et al., 1995, 1997). Comparing both processes, isostatic and dynamic HP, it appears that during UHPH (dynamic HP) samples are subjected to high pressure for a very short holding time followed by a rapid pressure drop. Indeed, the effect of holding protein samples at high pressure during the short 11 s corresponding to the pressure built up to 200–300 MPa in 2–3 s in the homogeniser intensifier, followed by fluid discharge through the HP valve, cannot be easily compared to the published data obtained with isostatic (and hydrostatic) HP equipments. In isostatic HP experiments, the pressure is built up to X300 MPa in 1–2 min (in pilot-scale equipments) or more (in

spectroscopy studies) and the usual holding time under pressure varies from 3 to 40 min. Such studies have shown that: (i) bovine a-La is pressure-resistant up to 400 MPa (Hinrichs & Rademacher, 2005; Huppertz, Fox, & Kelly, 2004; Tanaka & Kunugi, 1996); (ii) bovine b-Lg starts to unfold at 130 MPa with a clear aggregation process taking place under 230–350 MPa at 20 1C and a 2-fold increase in b-Lg particle diameter from 4.2 nm (at 0.1 MPa) to 7 nm (at X250 MPa) (small angle X-ray scattering; Panick et al., 1999). Comparing these data with the present results, it is clear that protein aggregation (and probably aggregation of b-Lg, the major and most pressure sensitive of whey proteins) took place in the same range of pressure levels for both isostatic and dynamic HP treatments. But the possible effect on protein conformation induced by the rapid pressure drop itself in dynamic HP processing is not exactly known. 3.7. Electrophoresis characterisation of protein aggregates induced by continuous short-time thermal treatment Native PAGE patterns of short-time (4 s) heated samples revealed a band Y at the top of the stacking gel at both protein concentrations (Fig. 8c, d). This band Y progressively increased in intensity with temperature (Fig. 8d) and was the strongest from 69 1C and above at 10% (w/w)

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protein (Fig. 8d), and from 74 1C and above at 6% (Fig. 8c). Besides, at the highest temperatures (77–82 1C; Fig. 8c, d) the intensity of the high MW bands, BSA and b-Lg slightly decreased, and a band located at the borderline of the stacking and separating gels appeared stronger. These new bands that did not enter the stacking/ separating gels disappeared from the native PAGE patterns of pH 4.7-soluble fractions (Fig. 8c, lanes 5, 7, 9) and corresponded to the heat-induced insoluble aggregates quantified by PSI. In contrast to what was observed for UHPH samples, SDS-PAGE revealed some protein constituents remaining at the top and in the stacking gel (band Z, Fig. 9) corresponding to residual aggregates not dissociated by SDS. These aggregates disappeared from SDS-PAGE patterns in the presence of MSH (data not shown), indicating that they were linked through disulphide bonds. In the present study, STTT did not induce the formation of heterogeneous a-La and/or b-Lg low MW polymers typically observed after heating whey protein dispersions at close to neutral pH and low protein concentration (o1%, w/w) (Laligant et al., 1991; Hoffmann & van Mil, 1997; Schokker et al., 1999). The quasi-absence of low MW intermediates and the formation of large aggregates not able to migrate into the separating gel have already been reported by Dalgleish et al. (1997) and Havea, Singh, and Creamer (2001) with mixtures of a-La and b-Lg (10%, w/w, total protein; neutral pH values) heated at 75 1C for 1–10 min, which is in accordance with the present results. More recently, Kazmierski and Corredig (2003) also did not observe intermediate-sized aggregates after heating of 10% WPI dispersions (w/v) at pH 7.0 and 65, 75 or 85 1C for various periods of time (X5 min). The presence of heat-induced large aggregates, in parallel to the absence of intermediate-sized aggregates in the present study, could be attributed to (i) the fast heating rate and (ii) the high protein concentration (6–10%), both favouring aggregation propagation; and (iii) the low a-La/(a-La+b-Lg) ratio ( ¼ 0.24) in the present WPI. Dalgleish et al. (1997) have effectively observed that (i) b-Lg polymerisation was favoured in the first 3–4 min of heating leading to high MW aggregates when the a-La/(a-La+b-Lg) ratio equalled 0.30 and (ii) intermediate-sized aggregates were formed only when approximately equal amounts of the two proteins were present in the heated mixture. 4. Conclusion Few studies deal with protein denaturation/aggregation through dynamic high pressure. Bouaouina et al. (2006) studied the effects of high pressure (up to 300 MPa) on the physico-chemical properties and functionalities of 3% (w/w) WPI at pH 6.7. They found that dynamic high pressure did not affect the conformation (DSC studies) or the solubility of the proteins at neutral pH. It was suggested that dynamic high pressure dissociated large protein aggregates,

leading to unmasking of the buried hydrophobic groups without affecting protein solubility. The present study clearly indicated that UHPH of 6% or 10% (w/w) WPI dispersions (Tin ¼ 24 1C; pH 6.5) followed by a rapid cooling immediately after the HP valve to minimise the heating effect induced protein aggregation above 225 MPa. Protein aggregation resulted from a combined effect of mechanical forces and short-life heating phenomena. Protein aggregates remain ‘‘soluble’’ upon centrifugation at pH 6.5 but not at pH 4.7. Solubility index carried out at pH 4.7 revealed 13–28% loss of soluble protein constituents after UHPH processing at 275–300 MPa. These levels of insoluble protein are relatively low due to the short-time processing and the efficient cooling of the fluid immediately after the HP valve. These levels of aggregation were yet sufficient to significantly change physico-chemical characteristics of dispersion especially at 10% (w/w) protein (increase in viscosity, protein voluminosity and dispersion lightness). Higher extents of protein denaturation could be obtained by combining UHPH and heating in a continuous processing using higher inlet temperature and/or longer residence of the fluid at the outlet temperature (Datta, Hayes, Deeth, & Kelly, 2005). The present results suggest different mechanisms for UHPH- and STTT-induced aggregation, with a predominance of hydrophobic interactions without formation of intermolecular disulphide bond in the case of dynamic high pressure, in contrast to heating. The higher voluminosity values found for UHPH-induced aggregates as compared with heat-induced aggregates agree with a scheme of protein assemblies more permeable to aqueous solvent and with elementary units not strongly tightened by disulphide bonds. Among PCS results, the most interesting is that the UHPH applied at sufficiently high protein concentration can lead to protein aggregates of less than 100 nm, which is in the size domain of nanoparticles. UHPH thus permits one to control the aggregate size below an upper limit. This finding could open new applications in the field of binding ligand to proteins or in the field of techno-functional properties especially for the dairy industry. Such small size aggregates could effectively be used to carry molecules of interest with transport proteins. In another hand, controlled size aggregation may be desired to develop protein ingredients with improved techno-functional properties (viscous and gelling properties for example), thus limiting the use of additives in formulated foods. Acknowledgements This study was funded in part by the FP6 CRAFT European Project UHPH 512626. M. Rene´ benefited from a doctoral grant given by the Ministe`re de la Jeunesse, de l’Education Nationale et de la Recherche, Paris. M. Cortes benefited from a doctoral grant given by University of Costa Rica and the Regional

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French Cooperation for Central America in San Jose´, Costa Rica. Tomas Lopez benefited from a grant given by ‘‘Stages outside Catalunya BE 2004’’, Catalunya Agency for management of university and research support.

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