Characteristics of Pressure-induced Gels of β-Lactoglobulin at Various Times after Pressure Release

Lebensm.-Wiss. u.-Technol., 31, 10–19 (1998)

Characteristics of Pressure-induced Gels of β-Lactoglobulin at Various Times after Pressure Release Eliane M. Dumay, Monica T. Kalichevsky and J. Claude Cheftel* Unite´ de Biochimie et Technologie Alimentaires, Centre de Genie ´ Biologique et Sciences des Aliments, Universite´ Montpellier II, F-34095 Montpellier Cedex 05 (France) (Received January 6, 1997; accepted May 6, 1997) Aggregation and gelation of aqueous solutions of a β-lactoglobulin (β-Lg) isolate (pH 7.0; 100 to 140 g/kg protein) were induced by pressure application and release (P-gels; 450 MPa, 25 °C, 15 min), or by heating (T-gels; 87 °C, 45 min). Pressure-induced aggregation led to porous gels prone to exudation in contrast to heat-induced gels which displayed a finely stranded network with high water retention. Pore size and strand thickness were greater for P-gels than for T-gels by one or two orders of magnitude. The matrix of the strands of P-gels consisted of highly packed particles 10 to 20 nm in diameter, as estimated by SEM, suggesting a random aggregation model with equally attractive sites per β-Lg particles (or primary aggregates). P-gels displayed a lower rigidity than T-gels. Moreover, P-gels could be totally dispersed and solubilized by homogenizing in water immediately after pressure release. Thus, pressure treatment at 450 MPa induced weaker intermolecular or interparticular forces than heating at 87 °C for 45 min. In contrast to T-gels, P-gels of β-Lg underwent mechanical and protein solubility changes when stored at 4 °C following pressure release, clearly indicating a time-dependent strengthening of protein–protein interactions. It appears that primary aggregates of β-Lg further aggregated during storage through hydrophobic interactions and disulfide bonds. Increasing the protein concentration of the initial solutions from 100 to 140 g/kg, and therefore the probability of protein–protein interactions, increased pore size and strand thickness of P-gels, with a marked trend to phase separation and protein microparticulation. Adding sucrose to the initial solutions decreased pore size and strand thickness and lessened the solid behaviour of P-gels, probably by reducing the number of protein–protein interactions induced by pressure.

©1998 Academic Press Limited Keywords: high pressure; β-lactoglobulin; gelation

Introduction Heat-induced gels prepared from β-lactoglobulin (β-Lg) isolates have been extensively studied in the last decade, since β-Lg isolates have become available with a reasonable level of purity and specific functional properties (1, 2), namely gelling (1, 3) and microparticulation ability (4, 5). With the growing interest in high hydrostatic pressure applied to the food field (6, 7), pressure-induced gelation has become attractive in view of improved food sanitation and development of new textures. It was possible to obtain gels from fresh egg white and yolk (8) or soy protein (9) upon pressurisation at 300 to 600 MPa and 25 °C for 10 to 30 min. Van Camp and Huyghebaert (10, 11) studied pressure-induced gels of whey protein concentrate near neutral pH: higher protein concentrations, higher pressure levels (between 200 and 400 MPa and 20 to 30 °C for 30 min) induced stronger gels; pressure-induced gels were shown to have weaker networks than heatinduced gels (80 °C for 30 min) prepared from the same *To whom correspondence should be addressed.

whey protein solutions (10, 11). Zasypkin et al. (12) formed gels with a sponge-like texture prone to exudation by pressurization of β-Lg isolate solutions (pH 7.0; ≥ 70 g/kg protein) at 450 MPa and 25 °C for 30 min. The rigidity of pressure-induced gels increased with the concentration of β-Lg isolate but remained lower than that of heat-induced gels of the same protein concentration (12). Elasticity was much higher for heat- than for pressure-induced gels of the same protein concentration (12). Previous studies on the pressure-induced unfolding and aggregation of β-Lg at pH 7.0 were carried out at 25 or 50 g protein/kg of solution (13–15) and in the presence of 0 to 50 g/kg sucrose as a baroprotective agent (13). Results indicated that processing at 450 MPa and 25 °C for 15 min induced: (i) a 50% decrease in the enthalpy of thermal denaturation, i.e. a partial loss of β-Lg structure; (ii) the formation of β-Lg aggregates, which increased in size with protein concentration and time after pressure release. These aggregates were partly dissociated into oligomers (di- to hexamers) by sodium dodecylsulfate and completely dissociated into β-Lg monomers by β-mercaptoethanol; (iii) some reversibil-

0023-6438/98/010010 + 10 $25.00/0/fs970290

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ity in β-Lg unfolding and aggregation was observed only at the lowest protein concentration (25 g/kg protein). Recent data (16) indicated that the reactivity of the free SH group of β-Lg increased with pressure up to 150 MPa. It is likely that the partial unfolding of β-Lg under pressure was followed by aggregation through SH/S–S interchange reactions (primary aggregates) and hydrophobic interactions (higher molecular weight aggregates). Adding sucrose (0 to 50 g/kg) to aqueous solutions of β-Lg (25 g/kg protein; pH 7.0) decreased pressure-induced unfolding and the subsequent aggregation of β-Lg (13). The present study deals with the microstructure of pressure-induced gels (450 MPa and 25 °C for 15 min) of β-Lg isolate at pH 7.0 in the presence or absence of added sucrose. The rigidity and protein solubility of these gels were determined as a function of time after pressure release to investigate the mechanisms of pressure-induced aggregation and gelation of β-Lg. Temperature-induced gels (T-gels) prepared from the same β-Lg solutions were also studied as a basis for comparison.

Materials and Methods β-lactoglobulin isolate The β-lactoglobulin (β-Lg) isolate from sweet whey (batch 650; Besnier-Bridel, Retiers, ´ France) was the same as in previous studies (12–15). It contained 58 g/kg moisture, and per kg of dry solids, 5.6 g nonprotein nitrogen (NPN), 859 ± 4 g protein [(total N – NPN) 3 6.38)], 915 ± 24 g protein as measured by the bicinchoninic acid procedure (see below), 50 g ash, 0.4 g calcium, < 10 g fat, and ca. 40 g lactose. The β-Lg isolated contained 890 g native β-Lg and 20 g native α-lactalbumin per kg soluble protein [(total N – NPN) 3 6.38], as determined by gel permeation chromatography at pH 6.0. The high solubility of the β-Lg isolate in the pH range 3.0 to 7.0 (ø totally soluble at pH 7.0) and the high enthalpy of thermal denaturation (14.1 J/g of protein) indicated a highly native state (13).

Chemicals All chemicals were of analytical grade. Sucrose came from Carlo Erba (art. no. 365157, lot no. 03890100 Milan, Italy). Pure β-Lg (three times crystallized, L-0130 lot no. 51117210), sodium dodecylsulfate (SDS L-4509-lot no. 85110449), Trizma base (T-1503, lot no. 45115734) bicinchoninic acid solution (B-9643), copper II sulfate pentahydrate solution (C-2884) and iodoace˙ tamide (art. no. I-6125, lot no. 103115004) came from Sigma (St-Louis, MO, U.S.A.). Dithiothreitol (DTT) was from Merck (art. no. 11474, lot no. 19250674 Darmstadt, Germany).

Preparation of β-Lg isolate solutions before high pressure processing Solutions of β-Lg isolate were prepared in degassed deionized water at 100, 120 and 140 g/kg of protein [calculated as (total N – NPN) 3 6.38]. Sucrose was then added at a concentration of 50, 100 and 150 g/kg, and the spontaneous pH (6.8 to 6.9) was adjusted to 7.0 by addition of 0.5 mol/L NaOH. The solutions were gently stirred for 60 min with a magnetic rod (avoiding foam formation), then centrifuged at 1200 3 g at 20 °C for 5 min (Sorvall RC5B, GSA rotor) for clarification and elimination of air bubbles that could hinder subsequent mechanical and microscopic gel characterization. The supernatants were assayed for protein concentration by measuring their absorbance at 280 nm and by the bicinchoninic acid procedure (UV2 spectrometer, Unicam, Argenteuil, France), and for dry solid content by oven-drying at 104 °C for 24 h. The small pellet obtained by centrifugation was neglected since the protein and dry solid contents of the solutions were not significantly modified by the centrifugation step.

Preparation of pressure-induced or heat-induced gels To prepare pressure-induced gels (P-gels), β-Lg isolate solutions were placed in polyvinylidene chloride tubings (diameter 26 mm, Krehalon, Eygalieres, ` France) sealed with three knots at both ends, equilibrated at 25 ± 0.5 °C for 30 min, then subjected to high pressure in water as already described (13), using a 1 L vessel (ACB, Nantes, France). The pressure was raised to 450 MPa in 3 min 45 s, held at 450 ± 1 MPa and 25 ± 1 °C for 15 min, then released in 1 min 20 s. Heat-induced gels (T-gels) were prepared from similar solutions of β-Lg isolate at pH 7.0. Solutions were placed in glass tubes (i.d. = 21 mm; height 46 mm) with two silicone stoppers, and gelled by heating in a water-bath at 87 °C for 45 min. The gels were then cooled in tap water. Pand T-gels were stored at 25 °C for the first 2 h and at 4 °C for longer periods in unopened tubings or glass tubes before analysis. An equilibration period of at least 1 h at 25 ± 0.5 °C was applied before mechanical evaluation.

Scanning electron microscopy (SEM) Small pieces of gel (6 3 1.5 3 1 mm) were cut with a razor blade and fixed at 4 °C in potassium phosphate buffer (50 mmol/L, pH 7.0) containing 40 mL/L glutaraldehyde for 16 h. Gels pieces were then rinsed with phosphate buffer, dehydrated in a graded series of ethanol/water solutions (from 0.5 to 1 L/L ethanol) with a residence time of at least 2 h in each solution, then critical-point dried with carbon dioxide (Bal-Tec CPD 030, Balzers Technique, Balzers, Lichtenstein). Dried samples were fractured in air at ambient temperature, mounted on aluminium stubs with silver lacquer, ˚ using a sputtering coated with gold (80 to 100 A) apparatus (Bal-Tec SCD 050, Balzers Technique) and

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examined with a field effect scanning electron microscope (Jeol JSM 6300F, Tokyo) at an acceleration voltage of 10 kV and a magnification of 3 500 to 40,000. This resulted in a three-dimensional evaluation of gel dimensions. In some cases, ethanol-dehydrated samples were fractured in liquid nitrogen before critical-point drying so as to obtain flat fractures and a better two-dimensional evaluation of network sizes. For each experiment, samples from at least two different high pressure runs were examined by SEM.

Measurement of mechanical characteristics The mechanical characteristics of gels were determined with a Stevens LFRA Texture Analyzer (St. Albans, U.K.) using a flat cylindrical compression probe (diameter 51 mm) at a displacement speed of 0.2 mm/s. Gel cylinders (diameter 21 mm and height 20 ± 0.5 mm for T-gels; diameter 20 to 26 mm and height 20 ± 0.5 mm for P-gels) were prepared with a razor blade. The diameter of P-gels varied due to syneresis taking place after pressure release. The surface area (A, cm2) of each gel was therefore measured before mechanical evaluation. The interfaces between gel samples and probe or anvil were not lubricated. Gel rigidity (F0/A, g/cm2) was defined as the force F0 per cm2 measured at 10% compression (gel strain of 2 mm). For each gel sample, results are the average ( ± sX) from measurements on four to five different pieces of gel.

Determination of the exudation and of the protein solubility of P-gels The solubility of the protein constituents of P-gels was determined within 40 to 60 min of pressure release and after storage at 4 °C for 24 or 45 ± 0.2 h in unopened tubing. The nonincorporated liquid (NIL or liquid exudate) and the remaining gel fraction of P-gels were carefully separated and weighted. Two grams of the remaining gel fraction were sliced into small pieces with a razor blade, and homogenized with an Ultraturrax (T25 tool, IKA, Staufen, Germany) for 1 min at 8000 rpm plus 1 min at 9500 rpm, in 48 g of each one of the three following solutions: (i) deionized water; (ii) deionized water plus 5 g/L sodium dodecylsulfate (SDS); (iii) deionized water plus 5 g/L SDS and 10 mmol/L dithiothreitol (DTT). During homogenization, the temperature of the mixture remained below 25 °C. When necessary, the pH of the gel dispersions was readjusted to 7.0. After gentle magnetic stirring for 1 h at ø 25 °C, the gel dispersions (5 to 6 g/L protein) were centrifuged at 12,000 3 g for 20 min (Sorvall RC5B, SS-34 rotor) to remove insoluble protein. The protein concentration was determined in the supernatants by the bicinchoninic acid (BCA) procedure (17) as modified by Hill and Straka (18) to minimize interference from thiol groups and reducing sugars: 50 µL of supernatant (containing 10 to 100 µg protein) was incubated at 37 °C for 15 min with 50 µL of 0.1 mol/L iodoacetamide in 0.1 mol/L Tris-HCl buffer, pH 8.0.

Two millilitres of BCA reagent (one part of C-2884 solution + 50 parts of B-9643 solution) were then added and thoroughly mixed with a vortex. The resulting mixture was incubated at 37 °C for 30 min, then cooled in cold water for 5 min to stop colour development. Absorbance of the protein–BCA mixture was measured at 562 nm (Unicam UV2 spectrometer) in triplicate and corrected for the absorbance of the same BCA solution without protein. Calibration curves were made with crystallized β-Lg solubilized in water, in water plus 5 g/L SDS or in water plus 5 g/L SDS and 10 mmol/L DTT. The protein concentration of initial β-Lg solutions used to prepare the gels, and the protein concentration of NIL from P-gels were also evaluated with the same BCA procedure. The protein concentration of the remaining gel fraction of P-gels was calculated as the difference between the total protein contained in the tubing and the protein contained in NIL. The solubility of the protein constituents of P-gel was taken as: 100 3 (soluble protein in the remaining gel fraction/total protein in the same remaining gel fraction). The determination of dry solid content in NIL or gels was carried out in triplicate by oven-drying at 104 °C for 24 h. Solubility data are the means of three independent high pressure runs followed by gel solubilization.

Results and Discussion Water retention and mechanical behaviour of pressure-induced gels at various times after pressure release While aqueous pH 7.0 solutions of β-Lg isolate containing 25 to 50 g/kg remained clear after processing at 450 MPa and 25 °C for 15 min (13), opaque and white gels were obtained when solutions containing 100 to 140 g/kg protein were processed under the same conditions. Pressure-induced gels (P-gels) consisted of a central gelled cylinder surrounded by nonincorporated liquid (NIL) or exudate; they displayed a sponge-like behaviour, losing water when compressed by hand, and reabsorbing this water when the strain was released. In contrast, T-gels were homogeneously gelled, looked like cooked egg white and displayed complete water retention both after heating and under manual compression. Since P-gels underwent progressive syneresis and exudation after pressure release, their liquid loss (NIL), texture and solubility of protein constituents were measured as a function of storage time at 4 °C. Results of P- and T-gels prepared from solutions containing 120 g/kg protein ± 100 g/kg sucrose are shown in Table 1 and Fig. 1. P-gels ( ± sucrose) displayed increasing exudation with storage time (Table 1), but less so when sucrose was present, probably due to osmotic effects. NIL contained 60 to 70 g/kg protein in the case of β-Lg isolate alone and 70 to 80 g/kg in the presence of sucrose. Protein lost in NIL (g protein/kg of initial total protein) significantly increased (P ≤ 0.001) with storage time (Table 1), and was lower in the presence than in the absence of

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sucrose (significant only at 1 h; P ≤ 0.05). In parallel to their exudation behaviour, P-gels of β-Lg ( ± sucrose) displayed increasing rigidity as a function of storage time after pressure release (Table 1). In spite of higher dry solid contents, P-gels containing sucrose displayed equal or lower rigidity than P-gels of β-Lg alone (Table 1). This less solid behaviour of P-gels containing sucrose may be attributed to weaker interactions between protein aggregates. In contrast, T-gels (120 g/kg protein ± 100 g/kg sucrose) displayed no or negligible exudation (not shown). The rigidity of T-gels increased during the first hour following the end of heat treatment and gel formation (results not shown), a period corresponding to cooling and final setting of the gel network. After 1 h, T-gel rigidity remained equal to 140 ± 6 or 155 ± 6 g/cm2 in the absence of presence of sucrose, respectively. T-gel rigidity was slightly (but significantly; P ≤ 0.05) higher in the presence than absence of sucrose. Overall, P-gels of β-Lg isolate ( ± sucrose) displayed lower rigidity than the corresponding T-gels. This may be attributed to weaker interactions between protein aggregates in P- than in T-gels and confirms (i) previous results obtained with the same β-Lg isolate in the absence of sucrose and for longer pressurisation times (450 MPa, 25 °C, 30 min) (12), and (ii) the characteristics of P-gels obtained from a whey protein concentrate (10, 11).

Solubility of the protein constituents of pressure-induced gels at various times after pressure release Within the first hour after pressure release, the P-gel fractions obtained from solutions containing 120 g/kg protein ± 100 g/kg sucrose were easily dispersed and completely ‘solubilized’ (no sedimentation at 12,000 3 g for 20 min) by homogenizing in deionized

water at pH 7.0 (24 mL water per g of gelled fraction) (Fig. 1). These soluble aggregates indicate that at this stage, only weak interactions between β-Lg molecules or aggregates contribute to gel formation. Solubility in water of the protein constituents of P-gels (no sucrose) decreased markedly with gel ageing, and did not exceed 48 or 41% after 24 or 45 h, respectively (Fig. 1a). This indicates a marked strengthening of the network of P-gels upon storage at 4 °C after pressure release. Such strengthening implies a greater number of stronger intermolecular protein–protein interactions. It is therefore likely that the increasing rigidity of P-gels during storage (Table 1) is due to chemical changes in the gel network in addition to the progressive raise in dry solids resulting from exudation. These chemical changes may even be responsible for gel syneresis and exudation. The solubility of protein constituents was also determined by homogenizing P-gel fractions in 5 g/L solutions of SDS with or without 10 mmol/L DTT. Twenty four hours after pressure release, protein solubility of P-gels (no sucrose) was 88% in SDS and ø 100% in SDS plus DTT (Fig. 1a). This suggests that (i) water-insoluble aggregates (large molecular weight) formed within the first 24 h of pressure release are predominantly stabilized through hydrophobic interactions, while (ii) some intermolecular S–S bonds, probably formed through SH/S–S interchange reactions (14, 15) also contribute to the gel network. The protein solubility of P-gels (no sucrose) 45 h after pressure release was only 56% in SDS, and still close to 100% in SDS plus DTT (as compared to 41% in water) (Fig. 1a). This indicates that 45 h after pressure release, the gel network is now predominantly stabilized by intermolecular S–S bonds. Similar measurements were carried out on P-gel fractions containing sucrose (Fig. 1b). The protein solubility in water 24 or 45 h after pressure release

Table 1 Characteristics* of the gelled fraction and the nonincorporated liquid (NIL) of pressure-induced gels (450 MPa and 25°C for 15 min) from β-Lg isolated in water at pH 7.0 with or without sucrose as a function of time after pressure release Time after pressure release (h)

Gelled fraction

Rigidity (g/cm2)

g of gelled fraction/kg of initial solutiona

g of dry solids/kg of gelled fractionb

g of protein/kg of gelled fractionc

Nonincorporated liquid g of NIL/kg of initial solutiona

g of g of protein in protein/kg of NIL/kg of initial NILc total proteinc

Without sucrosed 1 27.4 (3.7) 24 76.6 (5.8)*** 45 88.3 (10.8)***

694 (13) 541 (20)*** 523 (8)***

171 (3) 188 (4)** 193 (7)**

156 (5) 178 (1)*** 178 (1)***

306 (13) 459 (20)*** 477 (8)***

60.5 (3.3) 69.0 (3.1)* 70.0 (4.5)*

145 (12) 234 (7)*** 270 (26)***

With 100 g/kg sucrosee 1 28.5 (3.1) 24 66.5 (4.8)***■ 45 69.2 (5.8)***■

794 (14)■ ■■ 634 (23)***■ ■■ 590 (4)***■ ■■

265 (3)■ ■■ 271 (2)*■ ■■ 272 (1)*■ ■■

144 (4)■ ■ 159 (7)*■ ■ 162 (7)**■

206 (14)■ ■■ 366 (23)***■ ■■ 410 (4)***■ ■■

70.0 (3.2)■ ■ 80.0 (8.4)■ 80.0 (7.2)*

112 (8)■ ■ 217 (25)*** 254 (26)***

*Means (±sx) from 5–6a or 3–4b, c independent pressure treatments. cProtein concentration measured by the BCA procedure. The initial solutions of β-Lg isolate contained 127.8 g/kg protein as measured by the BCA procedure, which was equivalent to 120 g/kg protein as (MAT-NPN)×6.38). The dry solid content of initial β-Lg solutions was 140d or 241e g/kg. Significant differences relative to the corresponding gels 1 h after pressure release (same sucrose concentration) for *P ≤ 0.05, **P ≤ 0.01 or ***P ≤0.001. Significant differences relative to the corresponding gels without sucrose (same time after pressure release) for ■ P ≤0.05, ■ ■ P ≤0.01 or ■ ■ ■ P ≤0.001

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decreased significantly less (P ≤ 0.05) than for P-gels without sucrose. This supports the previously mentioned hypothesis that sucrose decreases the probability and extent of pressure-induced protein–protein interactions. SDS completely solubilized the waterinsoluble aggregates formed in P-gels 24 h after pressure release, and almost completely solubilized (at 91%) those present 45 h after pressure release. These aggregates are thus predominantly stabilized by hydrophobic interactions, and the presence of sucrose reduces both the proportion of S–S bonds and the rigidity of P-gels. In contrast, T-gels (120 g/kg protein, pH 7.0) displayed very low protein solubility in water, slightly decreasing with storage time from 8.5% (sX = 0.08) at 1 h to 6.8% (sX = 0.09) 45 h after gel formation. These results are in agreement with previous ones (19) and emphasize the irreversibility of thermal aggregation and gelation. With 5 g/L SDS, the protein solubility of T-gels increased only to 17% (sX = 0.12) or 14% (sX = 0.07) 1 or 45 h after gel formation, respectively. SDS was therefore insufficient to dissociate thermally-induced aggregates. With SDS plus DTT, protein constituents of T-gels 1 to 45 h old were totally soluble. This underlines the role of S–S bonds in the thermal aggregation and gelation of 120

(a)

100 80

Solubility of protein constituents (%)

60 40 20 0 120

Water

SDS

SDS + DTT

Water

SDS

SDS + DTT

(b)

100 80 60 40 20 0

Fig. 1 Solubility of protein constituents of P-gels prepared from β-Lg isolate solutions in water at pH 7.0, containing 120 g/kg protein and (a) no sucrose or (b) 100 g/kg sucrose. Solubilization of gelled fractions by homogenizing in either water, water plus 5 g/L sodium dodecylsulfate (SDS) or water plus 5 g/L SDS and 10 mmol/L dithiothreitol (DTT), within 1 h ( ) of pressure release, or after 24 ( ) or 45 h (`). Means of three independent pressurisation experiments at 450 MPa and 25 °C for 15 min followed by the solubilization procedure

β-Lg. The protein solubility of T-gels was not significantly modified (P ≤ 0.05) when these contained 100 g/kg sucrose (results not shown). Thus sucrose had much less effect on heat- than on pressure-induced gelation of β-Lg.

Ultrastructure of pressure-induced gels after pressure release P-gels were fixed for microscopy 24 ± 1 h after pressure release (Figs 2–4). Two structural levels were studied: the overall network at a low level of magnification (Figs 2 and 3) and the stranded or particulated structure at a higher level (Fig. 4). At a low level of magnification ( 3 500 to 3000) and in the studied range of protein and sucrose concentrations (100 to 140 g/kg protein ± 100 g/kg sucrose in the initial solutions), SEM examination of P-gels fractured in air at ambient temperature and after critical pointdrying revealed a three dimensional and coral-type network with large interstitial pores and large strands (or ‘pillars’), the size of which varied with the protein and sucrose concentrations (Fig. 2a–e). P-gel networks were apparently organized in a tetra- or pentahaedral mode, i.e. with four to five pillars emerging from the junction zones (see Fig. 2a,b). Such a microstructure is similar to that of filtration membranes (20). The high porosity of P-gels must be responsible for the marked exudation. In the cases of the smallest size pores, as for P-gels induced from solutions containing 120 g/kg protein plus 100 g/kg sucrose (Fig. 2d), it was possible to observe fine strands of aggregates threaded between the small size pillars, probably corresponding to soluble protein still present in the pore liquid. In contrast, T-gels prepared from the same solutions revealed no or nonsignificant exudation, and displayed by SEM at low magnification a filled matrix with a smooth surface (Fig. 2f). Micrographs from T-gels containing 100 (not shown) or 120 g/kg protein (Fig. 2f (left)) were similar. Rougher surfaces were observed for T-gels with 140 g/kg protein (Fig. 2f (right)). The presence of additional 100 g/kg sucrose (not shown) in T-gels did not change the SEM appearance at low magnification. Pore size and pillar thickness were more easily determined after fracturing P-gels in liquid nitrogen following ethanol dehydration and before critical pointdrying, so as to obtain flat surfaces and two-dimensional SEM pictures (Fig. 3). When the protein concentration of initial solutions increased from 100 to 140 g/kg, junction zones became less numerous per surface unit of gel sample, and the size of pores, pillars and sections through the junction zones increased (Fig. 3a–c). Pore size was 1 to 7 µm, 3 to 20 µm or 7 to 50 µm for initial solutions containing 100, 120 or 140 g/kg protein, respectively. Pillar thickness was 0.2 to 2 µm, 0.8 to 5 µm or 3 to 20 µm for the same P-gels, respectively. It was verified on a semi-log scale that the size parameters of the gel network appeared to be exponentially proportional to the protein concentration of the initial solutions. It is likely that a higher

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probability of protein–protein interactions was responsible for the increased porosity (in terms of pore size and pillar thickness) of the pressure-induced network. When prepared from solutions containing 140 g/kg of protein, P-gels displayed an expanded matrix containing numerous spheric areas (0.5 to 2 µm in diameter) visible through fractured pillars and junction zones, plus round particles 1 to 4 µm in size located outside of the pillars (Fig. 2c). When the initial solutions (120 g/kg protein) contained sucrose, the number of junction zones in P-gels increased, and the pore size and pillar thickness decreased (Fig. 3d–f). Pore size was about 2 to 13 µm, 1 to 7 µm or 0.5 to 4 µm for initial solutions containing 50, 100 or 150 g/kg sucrose, respectively. Pillar thickness was about 0.5 to 4 µm, 0.3 to 2 µm or 0.2 to 1 µm for the same P-gels, respectively. It was verified on a semi-log scale that the size parameters of the gel network decreased exponentially with increasing sucrose con-

centration of the initial solution. Adding sucrose was equivalent to reducing protein concentration, 100 g/kg sucrose in 120 or 140 g/kg protein solutions giving gel microstructures similar to those observed for 100 or 120 g/kg protein solutions without sucrose, respectively (Figs 2b,e and 3a,e). It is likely that sucrose acts by reducing the probability of protein–protein interactions. At greater magnification levels ( 3 10,000 to 40,000; Fig. 4a–h), the density of the matrix of P-gel was clearly visible through fractured pillars and junction zones. For initial solutions of 100 g/kg protein, or 120 g/kg protein plus 50 to 150 g/kg sucrose, the gel matrix was dense and continuous, and consisted in highly packed β-Lg particles of small size (10 to 20 nm) (Fig. 4a–f). As a basis for comparison, the dimension of the β-Lg dimer has been estimated to be 3.58 nm 3 6.93 nm (21). The external surface of pillars appeared to be rougher than their internal side (Fig. 4a–d), especially in the case of

Fig. 2 Three-dimensional structure of (a–e) P-gels (450 MPa, 25 °C, 15 min) and (f) T-gels (87 °C; 45 min) from β-Lg isolate in water at pH 7.0. SEM observation at low magnification and after fracturing samples in air at ambient temperature after critical point-drying. P-gels prepared from solutions containing (a) 100, (b) 120 or (c) 140 g/kg protein without sucrose, and (d) containing 120 g/kg protein plus 100 g/kg sucrose or (e) 140 g/kg protein plus 100 g/kg sucrose. T-gels prepared from solutions containing (f(left)) 120 or (f(right)) 140 g/kg protein without sucrose. P-gels pillars (1) and junction zones (2); spheric areas visible through fractured pillars and junction zones (3); microparticles located outside pillars (4); fine strands of aggregates in pore lumen (5)

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small pillars, possibly because some NIL protein was precipitated during sample fixation or dehydration. By comparison, T-gels of β-Lg prepared from solutions of 100 (not shown) or 120 g/kg protein displayed finely stranded aggregates (Fig. 5a), as already reported (22). The size of individual aggregates ranged between 10 and 40 nm and the thickness of strands between 50 and 100 nm (Fig. 5a). Such a finely stranded network explains the high water retention of T-gels of β-Lg. Increasing the protein concentration to 140 g/kg led to a coarser network and to larger strands (Fig. 5b), because of the greater probability of protein–protein interactions and faster random aggregation. The presence of 100 g/kg sucrose did not induce changes in T-gel microstructure (Fig. 5c,d), except for a small strandthinning effect for T-gels at 140 g/kg protein (Fig. 5d). In the case of globular proteins, unfolding is often required before gelation, and a critical balance between attractive and repulsive forces is needed for gel network formation and stabilization (3, 23)). Consider-

ing that pressure processing of the present β-Lg isolate in water at pH 7.0 (450 MPa, 25 °C, 15 min) induced unfolding of only about half of the native structure (13), it may be assumed that pressure-induced gelation results from predominantly random aggregation reactions between attractive sites rather than from latticelike organization as described for T-gels after extensive unfolding (3, 23). In the case of pressure-induced gelation, partial unfolding plus random aggregation lead to phase-separated gels with large pore size, thus reducing the ability of the network to immobilize water via capillary forces. Phase separation has indeed been considered as a possible gelation mechanism (23). All P-gel samples were prepared for SEM examination 24 h after pressure release but also, for most of them, within the first 30 min after pressure release or 3 to 5 d afterwards (samples being kept at 4 °C in unopened tubings). Three-dimensional SEM examination (results not shown) did not reveal any marked difference in the size parameters of the gel network observed after the

Fig. 3 Two-dimensional structure of P-gels (450 MPa, 25 °C, 15 min) from β-Lg isolate in water at pH 7.0. SEM observation at low magnification and after fracturing ethanol-dehydrated samples in liquid nitrogen before critical point-drying. Gels prepared from solutions containing (a) 100, (b) 120 or (c) 140 g/kg protein without sucrose, or 120 g/kg protein plus (d) 50, (e) 100 or (f) 150 g/kg sucrose

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different storage times, but finer evaluation would have required image analysis on a large number of twodimensional gel micrographs. The spheric areas (0.5 to 2 µm) located inside the large size pillars of P-gels induced at 120 to 140 g/kg protein appear to have a lesser density that the surrounding matrix (Fig. 4e–h) and contain finely stranded particles of 10 to 50 nm (Fig. 4f). These structures may be explained by microphase separation between diluted and concentrated liquid phases, or between highly

unfolded plus aggregated and less denatured or more soluble β-Lg molecules. During the phase inversion step of gelation (β-Lg aggregates initially dispersed in the aqueous phase being converted into a continuous phase), droplets of diluted aqueous phase may have remained entrapped inside the gel matrix, thus forming a kind of W/W emulsion. At the highest protein concentrations, pressure probably induced phase separation because of very fast and excessive aggregation with respect to unfolding, leading

Fig. 4 Three-dimensional structure of P-gels (450 MPa, 25 °C, 15 min) from β-Lg isolate in water at pH 7.0, as observed by SEM at high magnification. P-gels prepared from solutions containing (a,b) 100 g/kg protein without sucrose, (c,d) 120 g/kg protein plus 100 g/kg sucrose, (e,f) 120 g/kg protein without sucrose, or (g,h) 140 g/kg protein without sucrose. Samples fractured in air at ambient temperature after critical point-drying (a,c,d,e,g,h), or fractured in liquid nitrogen before critical point-drying (b,f). Dense matrix of P-gels (1); rough external surface of pillars (2); spheric areas visible through fractured pillars and junction zones (3); microparticles located outside pillars (4)

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to protein precipitation as microparticles (Figs 2c and 4g). Such microparticles resemble those observed in T-gels of β-Lg alone prepared near the isoelectric point (Fig. 5e) (22).

Conclusions For each of the two aggregation modes, heat or pressure, hydrophobic contacts and S–S bonds appear to represent the main protein–protein interactions (13–15, 19). However, the pressure-induced and heatinduced gels of β-Lg alone in water at pH 7.0, differed by: (i) the size parameters of the gel network; (ii) the organization of β-Lg aggregates; and (iii) the physicochemical stability and evolution of the gels after pressure release or heating. Previous results (13) indicated that processing at 450 MPa and 25 °C for 15 min induced only partial unfolding of β-Lg. The energy corresponding to this compression is much smaller than that of the usual heat gelling treatment at 87 °C for 45 min (24). However, the energy applied is not the sole parameter to take into account. Extensive protein aggregation may result from

decreased protein–water interactions between the protein network and the surrounding aqueous media due to volume and equilibrium changes in free and bound water during pressure build up and release (24). The disruption of hydrophobic and electrostatic interactions that cause protein dissociation and unfolding at relatively low pressure levels is probably triggered by the conversion of free water into more compact water bound to the additional protein surface. However, at higher pressure levels ( ≥ 150 MPa), protein aggregation (as a result of unfolding) could be enhanced due to the higher compressibility and smaller volume of free water as compared to that of protein-bound water. This may explain the compactness of the matrix of pressureinduced gels and the phase separation mechanisms. A weakening of hydrophobic interactions during the last phase of pressure release could be responsible for the high solubility of P-gels in water immediately after pressure release. Upon storage at atmospheric pressure, interactions between primary aggregates appear to build up progressively in P-gels, causing the observed increase in rigidity and decrease in protein solubility. At atmospheric pressure, sucrose is known to stabilize native protein structures through a preferential exclu-

Fig. 5 Three-dimensional structure of T-gels (87 °C, 45 min) from β-Lg isolate in water, as observed by SEM at high magnification. T-gels prepared from solutions at pH 7.0 and containing (a) 120 or (b) 140 g/kg protein without sucrose, or (c) 120 or (d) 140 g/kg protein plus 100 g/kg sucrose. (e) T-gels prepared from solutions at pH 5.4 and containing 120 g/kg protein without sucrose. Samples fractured in air at ambient temperature after critical point-drying

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sion of sugar from the protein domain, which affects the surface tension of water (i.e. the work necessary to increase the surface area between the protein and the solvent) and minimizes the protein–solvent interface (25). The observed effects of sucrose under pressure in the present study could result from a protective effect against protein unfolding at intermediate pressure levels and against subsequent aggregation. Such baroprotective effects of sucrose on β-Lg have already been reported at lower protein concentrations (13).

11 12 13

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Acknowledgements 15

This study was funded in part by Research Contract AIR 1-CT 92-0296 from the European Communities (Agricultural and Agroindustry Programme, DG XII, Brussels). M.T.K. has benefited from a post-doctoral grant given by the French Ministry of Higher Education and Research, Paris.

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