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LWT - Food Science and Technology 48 (2012) 69e74

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Mechanical and microstructural properties of milk whey protein/espina corona gum mixed gels María Julia Spotti*, Liliana G. Santiago, Amelia C. Rubiolo, Carlos R. Carrara Grupo de Biocoloides, Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química, Universidad Nacional del Litoral, 1 de Mayo 3250, 3000 Santa Fe, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 August 2011 Received in revised form 14 February 2012 Accepted 20 February 2012

Mechanical and microstructural properties of gels composed of whey protein isolate (WPI) and Espina Corona Gum (ECG) were studied. WPI gels were made varying the protein concentration from 12 to 16 g/ 100 g while mixed gels were obtained at 12 g/100 g protein and varying ECG concentration from 0.12 to 0.60 g/100 g. All gels were obtained by heat treatment (30 min, at 80  C). Mechanical properties were studied by uniaxial compression and stress relaxation test and gelling properties were discussed in terms of pore size, opacity index, colour parameters and microstructure. Stress (maximum stress) and deformability (Henky’s strain) of mixed gels were seen to increase with the increase in WPI and ECG concentration, thus revealing a higher solid character. This behaviour was consistent with a reduced pore size and increased opacity index of the gel matrix. Microstructure of WPI/ECG mixed gels revealed the existence of a biomacromolecule segregative phenomenon which could promote a greater local protein concentration in separated microdomains. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Espina Corona Gum WPI Mixed gels Uniaxial compression Confocal microscopy

1. Introduction Proteins offer an extensive range of desirable functional properties for use in the food industry, as well as pharmaceuticals and cosmetics. Whey proteins are widely used because of their technological applications and nutritional value. They represent 20 g/ 100 g of total milk proteins and are composed of b-lactoglobulin (50 g/100 g), a-lactalbumin (20 g/100 g), serum albumin, and immunoglobulins, among other proteins (Cayot & Lorient, 1997). One of the most important and extensively studied functional properties of whey proteins is their ability to form gels (Bryant & Mc Clement, 2000; Li, Ould Eleya, & Gunasekaran, 2006). For whey proteins (like other globular proteins), partial or complete denaturation is needed as a first step for gelation (Bertrand & Turgeon, 2007; Bryant & Mc Clements, 2000; Li et al., 2006). During denaturation, proteins suffer an unfolding of their threedimensional structure into extended chains without breaking their peptide (covalent) bonds. Proteins can be denatured in different ways, the usual method being heat treatment (Donato, Garnier, Novales, Durand, & Dublier, 2005; Fitzsimons, Mulvihill, & Morris, 2008; Ikeda, 2003; Walsh-O’Grady, Kennedy, Fitzgerald, & Lane, 2001). Nevertheless, other treatments like high pressure,

* Corresponding author. Tel.: þ54 342 4571252x2602. E-mail address: [email protected] (M.J. Spotti). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2012.02.023

mechanical agitation, organic solvents, etc., can be used. Protein gels are, in general, thermally irreversible (unlike many polysaccharide gels that could return to their original state by means of heating). The stability of whey protein gel structures is mainly the result of covalent bonds, particularly disulfide bonds (between cysteine residues) and, to a lesser extent, noncovalent bonding like hydrophobic interactions and hydrogen bonds (Betrand & Turgeon, 2007; Foegeding, Davis, Doucet, & Mc Guffey, 2002). Gelation, as well as other functional properties of proteins, is altered in the presence of polysaccharides (Baeza, Gugliota, & Pilosof, 2003; Baeza & Pilosof, 2001; Bryant & Ms Clements, 2000; Li et al., 2006). Since proteins and polysaccharides are the major components of food systems and they have an essential role in their structure, texture and stability (Perez, Carrara, Carrera, Santiago, & Rodríguez Patino, 2010; Pérez, Wargon, & Pilosof, 2006), it is important to study mixed system behaviour so as to develop desirable properties in food products (Perez, Carrara, Carrera, Rodríguez Patino, & Santiago, 2009; Pérez et al., 2006). The whey milk protein gelation has been studied in the presence of a wide variety of polysaccharides (Bertrand & Turgeon, 2007; Bryant & Mc Clements, 2000; Li et al., 2006). Different types of mixed gels can be obtained according to the relative concentration of each macromolecule, their nature (neutral or ionic) and environmental conditions (temperature, pH and ionic strength) (Spahn, Baeza, Santiago, & Pilosof, 2008). Textural, mechanical and sensory properties of these gels are a consequence of their

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microstructure, and this depends on the type and degree of proteineprotein and proteinepolysaccharide interactions, among other things (van den Berg, Rosenberg, van Boekel, Rosenberg, & van de Velde, 2009). In aqueous mixed systems where different macromolecules are used, the enthalpic interactions between chains of different macromolecules may be more or less favourable than the interactions between chains of the same type, leading to association or segregation, respectively (Bryant & Mc Clements, 2000). Association occurs by electrostatic interaction between macromolecules with opposite electric charges. Segregative interactions are more common, and they take place in mixtures of macromolecules where medium conditions promote thermodynamic incompatibility (Perez et al., 2009; Tolstoguzov, 1997). For gelling macromolecules, phase segregation in aqueous medium can be trapped during the formation of the network, giving a two-phase co-gel with a continuous and a dispersed phase. Phase separation in mixtures of gelling macromolecules can lead to substantial increases in gel strength due to an increase in local effective concentration of both macromolecules. In mixtures comprising a gelling and a non-gelling macromolecule, segregative interactions can also lead to increase in gel strength (Fitzsimons et al., 2008). Phase separation of proteinepolysaccharide systems is strongly affected by the chemical structure of the polysaccharide, especially by its charge, concentration of both macromolecules and medium conditions (Baeza & Pilosof, 2001). Galactomannans are a group of neutral polysaccharides naturally occurring in seeds of some Leguminosae tree, consisting in chains of mannose residues with randomly attached galactose units as side-chains (Tavares, Monteiro, Moreno, & Lopes da Silva, 2005). The galactoseemannose ratio can vary from 1 to 10, depending on the type and source (Azero & Andrade, 2006; de Jong & van de Velde, 2007). Many galactomannans like guar gum, locust bean gum and tara gum, among others, have been used in gelation studies with proteins (Fitzsimons et al., 2008; Tavares & Lopes da Silva, 2003; Tavares et al., 2005). The polysaccharide used for this work, Espina Corona gum (ECG), is a galactomannan obtained by grinding the endosperm of Espina Corona seeds (Gleditsia amorphoides), which grows up in Argentina, Brazil and Paraguay. The galactoseemannose ratio of ECG is 2.5 (Cerezo, 1965) and has a molecular weight of 1390 kDa (Perduca, Santiago, Judis, Rubiolo, & Carrara, 2011). It is used as a food thickener and stabilizer. It is a non-gelling biopolymer; since it cannot form a gel structure by itself. The study of WPI/ECG mixed system is important to characterize and obtain new applications of this South American polysaccharide. The aim of this work was to study the effect of a neutral polysaccharide (espina corona gum, ECG) concentration on the mechanical properties of WPI gels, through uniaxial compression and stress relaxation tests. Confocal scanning laser microscopy and CIE Lab colour parameters of the gel structure were also analyzed. 2. Materials and methods 2.1. Materials Whey Protein Isolate (WPI) (BIPROÔ) was kindly provided by Davisco Foods International Inc. (Minnesota, USA). It is composed of 97.9 g/100 g (dry basis) protein, 0.2 g/100 g fat, 1.9 g/100 g ash and 4.8 g/100 g moisture. The Espina Corona Gum (ECG) (10.04 g/100 g moisture, 1.44 g/100 g ash, 0.27 g/100 g fat, 2.17 g/100 g protein, 0.70 g/100 g crude fibre and 85.38 g/100 g polysaccharide) (Perduca et al., 2011) was obtained from Idea Supply Argentina SA (Chaco, Argentina).

2.2. Preparation of solutions A solution of 20 g/100 g WPI powder was prepared with distilled water, agitated for 2 h at room temperature and cooled overnight for complete hydration. Then, it was diluted to other concentrations (12e16 g/100 g) with distilled water. A suspension of ECG was prepared at 2 g/100 g, kept overnight at 4  C, and then centrifuged at 2000g for 20 min. The supernatant was filtered with an ASTM 40 (420 mm) sieve. Finally, soluble solids (1.77 g/ 100 g) were determined by weight loss at 105  0.1  C in an oven. Mixed solutions were obtained by adding ECG to WPI at 0.12, 0.36 and 0.60 g/100 g, leading to final ratios of 1:0.01, 1:0.03 and 1:0.05. Mixed solutions were left under agitation for 15 min, adjusting the pH at 7 and degassing with vacuum. Sodium azide (0.2 g/100 g) was added as a bactericide in all solutions. 2.3. Preparation of WPI and WPI-ECG heat induced gels WPI and WPIeECG mixed solutions were poured into 100 mm length and 30 mm inner diameter glass cylinders, closing both ends with rubber stoppers. Cylinder inside surface was coated with silicon oil to prevent the gel from sticking. Heat induced gels were obtained by heat treatment at 80  C during 30 min in a water bath. Then, they were stored at 4  C for 24 h before analysis. 2.4. Uniaxial compression Uniaxial compression test was carried out using a universal testing machine (model 3344, INSTRON Corp., Norwood, USA) until fracture using two parallel plates (diameter: 60 mm) lubricated with a thin layer of paraffin oil to minimize friction. The measurements were performed at a crosshead speed of 1 mm/s (de Jong & van de Velde, 2007; Ribeiro, Rodriguez, Sabadini, & Cunha, 2004; Yamamoto & Cunha, 2007) in a room with controlled temperature (22  C). The samples were cylinders 30 mm long and 30 mm in diameter. The true or Hencky stress, sH, can be defined as (Yamamoto & Cunha, 2007):

sH ¼ FðtÞ$HðtÞ=ðH0 $A0 Þ

(1)

Similarly, the Henky strain, εH, was calculated as:

εH ¼ lnðHðtÞ=H0 Þ

(2)

where F(t) and H(t) are the force and the height at a given time t, and Ao and Ho are the initial area and height of the gel, respectively (Yamamoto & Cunha, 2007). The parameters calculated from data compression were: maximum stress (sM), which is the maximum value of sH until rupture, calculated from Eq. (1); maximum strain (εM), which is the maximum value until rupture, calculated with Eq. (2); WF, which is the work of fracture associated with the hardness, calculated as the area under the curve sH vs. εH between 0 and εM; Young’s modulus (E), which is calculated as the slope of the linear and initial region of the curve sH vs. εH (5% strain) (Steffe, 1992); and rupture deformation (Rup. Def (%)), which is the deformation of the gels calculated as follow: (final height  initial height)/initial height  100. 2.4.1. Pore size determination Pore size was calculated taking into account the rubber elasticity theory (Aklonis, Mac Knight, & Shen, 1972) using the following Eq. (3):



x ¼ 3$Kb $T$ r02 =rf2

1=3

=E

(3)

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where: x is the pore size, Kb is the Boltzmann constant, T is the temperature in Kelvin, E is Young’s modulus determined by uniaxial compression, and (r02 =rf2 ) can be regarded as the average deviation of the network chains from the dimensions they would assume if they were isolated and free from all constraints (front factor). For covalent networks it is considered to be 1. 2.5. Stress relaxation Stress relaxation test was conducted in uniaxial compression form using the Instron universal testing machine. Gel samples, cylinders 30 mm in height and 30 mm in diameter, were compressed to 5% of its original height at a crosshead speed of 1 mm/s, and then stress relaxation was recorded for 300 s. After performing this test for different strain levels, the percentage of deformation used (5%) was found to be within the linear viscoelastic region (data not shown). Graphs of Hencky Stress (sH) vs. time were obtained with the relaxation data. The maximum stress relaxation modulus (G0) was calculated as:

G0 ¼ sM =gconst

(4)

where sM is maximum stress calculated with Eq. (1) and gconst is the adimensional strain applied in the test (0.05 in this case) (Steffe, 1992). 2.6. Determination of gel colour and opacity Minolta colorimeter model 508D/8 8 mm (Tokio, Japán) was used for the experience. The CIE Lab system, defined in rectangular coordinates (L*, a*, b*), with a 65 illuminant and 10 observer angle was applied. The following parameters: L* (lightness), a* (þ red, green) and b* (þ yellow, blue) were evaluated for 6 mm thick discs. The opacity index (OI%) was obtained taking L value with a black background (LBb) and then with a white background (LBw). The following equation was then applied:

OI% ¼ LBb =LBw  100

(5)

2.7. Confocal scanning laser microscopy An inverted Nikon Eclipse Model TE-2000-E2 microscope, motorized with optical DIC/Nomarski and infinity corrected optics was used. Protein solutions were non-covalently stained with 10 ml/ gprot of Rhodamine B solution at 1 mg/ml. The required amount of Rhodamine B was mixed with biopolymer solutions before gelation. Then, the coloured solutions were placed in glass cylinders with rubber stoppers. The cylinders were placed in a water bath at 80  C for 30 min, and then at 4  C for at least 24 h before observations. Observations of whey proteins were made by excitation of Rhodamine B at 544 nm, the emission being recorded between 550 and 750 nm. A 40 objective and a zoom of 10 were used in all the samples. Each image was composed into 1024  1024 pixels with a field of 63.65  63.65 mm. 2.8. Statistical analysis All measurements were performed at least in triplicate. Mean values and their corresponding standard error were calculated and presented in graphs as coordinate pairs with their corresponding error bar. For statistical treatment of data, StatGraphics Centurion XV software was used and analysis of variances (ANOVA) was done. When statistical differences were found, Duncan’s test (a ¼ 0.05) was carried out. Analysis and graphic presentations were performed using OriginPro 7.5 SR0 software (OriginLab Corporation, Northampton, USA).

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3. Results and discussion 3.1. Effect of ECG on mechanical properties of WPI gels 3.1.1. Uniaxial compression Mechanical parameters obtained by uniaxial compression are presented in Table 1 and examples of the stressestain curves in Fig. 1. As expected, an increase in mechanical properties can be observed for WPI gels when concentration increases. The maximum stress tolerated by the gels increases linearly (r2 ¼ 0.999) with the increase in protein concentration (Fig. 2) since the higher the concentration, the denser the network structure formed (Foegeding et al., 2002), which allows a better distribution of the applied stress and thereby more resistance to rupture and collapse (Li et al., 2006). For mixed gels, the increment in ECG concentration produces an increase in maximum stress (sM), this being significant from 0.36 g/100 g of ECG onwards. The addition of polysaccharide might cause protein molecules to come closer to each other, probably acting as filler (Tavares & Lopes da Silva, 2003). However, there are others proteinepolysaccharide interactions that could be considered in the gel formation, such as hydrogen and electrostatic bonds between certain groups of polysaccharide and proteins. Electrostatic interactions between WPI and ECG should be underestimated because ECG is a neutral polysaccharide and all the experiences were carried out at pH 7. Similar results were reported in other works, where the interactions of WPI with other galactomannans, such as Locust Bean Gum (de Jong & van de Velde, 2007; Tavares et al., 2005; Tavares & Lopes da Silva, 2003) and Tara Gum and Guar Gum (Fitzsimons et al., 2008; de Jong & van de Velde, 2007; Tavares et al., 2005) have been studied. The same behaviour was also observed for other polysaccharides such as xanthan gum (Bertrand & Turgeon, 2007; Li et al., 2006) and Gellan Gum (de Jong & van de Velde, 2007). Gel maximum Hencky’s strain (εM) decreases with the protein concentration (Table 1); nevertheless, it is not affected by ECG (results without significant differences, P > 0.05). With regard to Young’s modulus (E) and work of fracture (WF), the same trend as that of maximum stress was observed (Fig. 3A and B). A linear increase was found in these parameters with the increment in both protein and ECG concentration, it being more pronounced in the first case, so that the total energy needed to break the gel and the gel elasticity increased with the total concentration of macromolecules. Similar results were found by Fitzsimons et al. (2008) in WPI/guar gum mixed gels. These results suggest that the addition of ECG has a synergistic effect on the mechanical properties of WPI gels, which could be

Table 1 Maximum strain and deformation at rupture calculated by uniaxial compression test, pore size gels calculated by rubber elasticity theory and relaxation modulus calculated by stress relaxation test for WPI and WPI/ECG mixed gels. WPI conc ECG conc Max. strain (g/100 g) (g/100 g) (εH)

Deformation Pore size G0 (kPa) at rupture (%) (x) (nm)

Gels without ECG 12 0 14 0 16 0

0.79  0.01a 0.72  0.01ab 0.65  0.07b

54.7  0.7a 51.3  0.6ab 47.6  3.9b

157  2a 75  3b 63  3c

12.71  1.29a 21.23  4.82b 81.67  10.45c

Gels with ECG 12 0.00 12 0.12 12 0.36 12 0.60

0.79  0.01a 0.80  0.06a.b 0.74  0.02a.b 0.72  0.02b

54.7  0.7a 54.9  2.8a.b 52.3  0.7a.b 51.5  1.0b

157  2d 118  2e 98  3f 87  3 g

12.71  1.29d 13.34  2.92e 26.58  5.04f 38.57  3.83 g

Conc ¼ Concentration. The values with the same letter did not have differences with Duncan’s test with a ¼ 0.05.

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Fig. 3. Work of fracture (WF ) and Young’s modulus (E ) vs (A) WPI concentration and (B) ECG concentration, for WPI and WPI/ECG mixed gels, respectively. Fig. 1. Hencky stress (sH) vs. Hencky strain (εH) for WPI gels (12, 14 and 16 g/100 g) and WPI/ECG mixed gel: (12*: 12 g/100 g WPI-0.6 g/100 g ECG).

explained considering segregative interactions between these macromolecules. The consequence of macromolecules segregation is a local concentration increase of each component, this behaviour being more pronounced when the polysaccharide is neutral (Bertrand & Turgeon, 2007; Li et al., 2006; Perez et al., 2006; Tavares & Lopes da Silva, 2003). Table 1 shows gel pore sizes with and without ECG. Both increments in protein and ECG concentration produce a reduction in pore size of gel networks. Pore sizes are significantly different (P < 0.05) for all concentrations. The decrement in pore size is more affected by protein concentration than by polysaccharide concentration. This behaviour might be explained considering that the gel network is mainly built by disulfide bonds among cysteine residues of b-lactoglobulin molecules (Foegeding, 2005), so that an increase in protein concentration produces more interactions among protein molecules and a subsequent decreased pore size, while the polysaccharide (which is itself unable to gelify) scatters among the protein molecules in the network, which leads to a smaller change in the pore size (Bertrand & Turgeon, 2007). 3.1.2. Stress relaxation In a step strain test, the sample is subjected to an instantaneous strain, the stress required to maintain the deformation being followed as a function of time. A wide range of behaviour may be found in stress relaxation tests. In ideal elastic materials, no relaxation is observed, while ideal viscous substances relax instantaneously. Viscoelastic materials, in turn, relax gradually, and the end point depends on the molecular structure of the material

Fig. 2. Maximum stress (sM) vs. ECG (lower abscissa) and protein concentration (upper abscissa) for WPI gels ( ) and WPI/ECG mixed gels ( ).

being tested (Steffe, 1992). Relaxation curves versus time are shown in Fig. 4 A and B. The curves showed that all gels have viscoelastic behaviour. An increase in protein concentration gave a more solid gel that withstood a significant amount of effort without relaxing its structure in a significant way. The increment in ECG concentration at constant protein concentration had the same effect but to a lesser extent. These results suggest that the increase in both protein and ECG concentration promotes gels with more solid behaviour. It can be seen that the initial stress (stress at time zero) is bigger for gels with higher macromolecule concentration. This can also be seen in Table 1 where the maximum stress relaxation modules are shown. These modules are higher when the protein and polysaccharide concentrations increase. 3.2. Effect of ECG on colour properties of WPI gels The values of the CIE Lab parameters and the Opacity Index (OI%) for WPI and WPI/ECG mixed gels are shown in Table 2. The increase in ECG and protein concentration can be seen to produce a slight change in gel colour. The most affected parameter was b*, which appears as yellow colour when it is positive. This parameter increased with protein concentration for WPI gels. On the other hand, for mixed gels the b* value increased from 25 (0 g/100 g ECG) to 50 (0.12 g/100 g ECG), nevertheless this value decreased for higher ECG concentration. It should be noted that gel transparency can affect the colour parameters of the gels. The opacity index in protein gels increases with protein concentration. In mixed gels, the minimum ECG concentration produced gels with some loss of transparency, but a subsequent increase in ECG concentration does not affect the opacity index. In our case, gels might become more opaque in the presence of a polysaccharide since ECG would interfere with the orderly organization of the filaments of whey proteins, with the resultant loss of transparency. A gel is opaque or transparent depending on the organization of its structure (Bertrand & Turgeon, 2007; Pilosof, 2000). Transparent gels are those which are formed by an association of ordered protein

Fig. 4. Stress relaxation curves (sH vs. time) for (A) WPI (12, 14 and 16 g/100 g WPI) and (B) WPI/ECG mixed gels (12 g/100 g WPI with 0.00, 0.12, 0.36 and 0.60 g/100 g ECG).

M.J. Spotti et al. / LWT - Food Science and Technology 48 (2012) 69e74

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Table 2 Colour parameters (Cielab system) and Opacity index for WPI and WPI/ECG mixed gels. WPI Conc. (g/100 g)

ECG Conc. (g/100 g)

L

a*

b*

OI %

Gels without ECG 12 14 16

0.00 0.00 0.00

636.43  4.06a 639.13  2.87b 661.41  2.80c

0.46  0.05a 0.34  0.04a 0.35  0.04a

69.20  1.40a 79.35  1.01b 73.96  1.01c

33.50  0.70a 43.62  0.36b 41.17  0.36c

Gels with ECG 12 12 12 12

0.00 0.12 0.36 0.60

636.43  6.18a 630.53  0.51a 631.26  1.26a 617.63  1.41b

0.46  0.02a 0.29  0.02b 0.40  0.02a 0.42  0.03a

69.20  2.02a 80.73  0.84b 74.33  0.53c 69.63  0.20a

33.50  0.71a 38.90  0.40b 40.00  0.10b 39.70  0.70b

Conc ¼ Concentration. The values with the same letter did not have differences with Duncan’s test with a ¼ 0.05.

molecules and the thickness of the filaments is very small. This structure of thin filaments is characteristic of globular proteins under conditions of pH greater or less than the isoelectric point and low ionic strength (Foegeding, 2005; Pilosof, 2000; Tavares & Lopes da Silva, 2003). On the other hand, opaque gels are not ordered and are particulate. This kind of gel is formed when the fluctuations in macromolecule density approach macroscopic size and effectively scatter light (Oakenfull, Pearce, & Burley, 1997). Such networks are characterized by regions of high macromolecule concentration separated by regions almost devoid of them. 3.3. Effect of ECG on microstructure of WPI gels Microstructure of protein and mixed gels by confocal scanning laser microscopy can be observed in Fig. 5. Labelling the whey protein with Rhodamine B allowed protein matrix identification in the mixture by confocal microscopy, since the ECG chains are not fluorescent. Whey proteins appeared grey in the picture owing to Rhodamine B, while the dark areas corresponded to zones devoid of protein, which contained the ECG. No observable differences among WPI gels could be seen (Fig. 5 A, B and C). Besides, gel pores could not be distinguished. These results were consistent

with the study of pore size given by the theory of rubber elasticity discussed in Section 3.1.1. In these gels, pore sizes were all below 200 nm, which is the limit resolution of optical microscopes, including confocal microscopes. In mixed gels, at the lowest ECG concentration the polysaccharide can be seen like little droplets dispersed in a protein network (Fig. 5D). As ECG concentration increases, droplet sizes increase too (Fig. 5E and F), because more places are occupied by polysaccharide molecules. These images show phase separation, where one phase (the continuous phase) contains mainly whey proteins and the other one is enriched with the polysaccharide. This behaviour might be promoted by thermodynamic incompatibility between both macromolecules. In mixed systems of macromolecules the entropy of mixing is less than the segregation enthalpy and often the mixture separates spontaneously into two separate phases, each enriched with a particular macromolecule and devoid of the other one (Fitzsimons et al., 2008). When the gelation process takes place, this phase segregation forms the gel structure with little areas (microdomains) with increased protein concentration (Li et al., 2006). The same results were observed by Tavares et al. (2005) for different polysaccharides studied (Locust Bean gum, Guar gum and Tara gum). Similar images of WPI and Gellan gum, Locust

Fig. 5. CLSM micrographs for all studied gels: (A) 12 g/100 g WPI, (B) 14 g/100 g WPI, (C) 16 g/100 g WPI, (D) 12 g/100 g WPIe0.12 g/100 g ECG, (E) 12 g/100 g WPIe0.36 g/100 g, (F) 12 g/100 g WPIe0.60 g/100 g ECG.

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Bean gum, pectin and k-carrageenan were obtained in several studies of cold set gels (van den Berg et al., 2009; van den Berg, van Vliet, van der Linden, van Boekel, & van de Velde, 2007). The images of protein and mixed gels microstructure are consistent with the results discussed in all previous items, where the phase segregation is responsible for the synergistic effect of the ECG on mechanical and optical properties of WPI gels. 4. Conclusions Mechanical properties, colour and microstructural characteristic of WPI gels were strongly affected by protein concentration and by ECG (even in low concentrations). In general, an increase in mechanical properties and higher gel solid character were observed when macromolecule concentration (WPI and ECG concentration) was increased. Besides, a reduction in pore size was found with macromolecule concentration increment. On the other hand, colour parameters, except for the opacity index, which increased with ECG concentration, were not affected by changes in macromolecule concentration. Microstructure observed by confocal scanning laser microscopy showed mixed gels with phase separation between macromolecules under the studied conditions. This phase separation can explain the improvement in gelling properties of WPI/ECG gels, due to higher protein concentration in local areas (microdomains). These results show/showed that it is possible to manipulate several properties of WPI gels varying both protein and ECG concentration. Acknowledgements Authors would like to thank the financial support of CAIþD PI57283 project and to Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina (CONICET) for the postgraduate fellowship awarded to María Julia Spotti. References Aklonis, J., Mac Knight, W., & Shen, M. (1972). Rubber elasticity. In J. Aklonis, W. Mac Knight, & M. Shen (Eds.), Introduction to macromolecule viscoelasticity (pp. 105e140). New York: Wiley-Interscience. Azero, E., & Andrade, C. (2006). Characterization of Proposis juliflora seed gum and the effects of its addition to k- carrageenan systems. Journal of the Brazilian Chemical Society, 17(5), 844e850. Baeza, R., Gugliota, L., & Pilosof, A. (2003). Gelation of b-lactoglobulin in the presence of propylene glycol alginate: kinetics and gel properties. Colloids and Surfaces B: Biointerfaces, 31(1e4), 81e93. Baeza, R., & Pilosof, A. (2001). Mixed biopolymer gels systems of b-lactoglobulin and non gelling gum. In E. Dickinson, & R. Miller (Eds.), Food colloids-fundamentals of formulation (pp. 392e403). Cambridge, UK: The Royal Society of Chemistry. Bertrand, M. E., & Turgeon, S. L. (2007). Improved gelling properties of whey protein isolate by addition of xanthan gum. Food Hydrocolloids, 21(2), 159e166. Bryant, C. M., & Mc Clements, D. J. (2000). Influence of xanthan gum on physical characteristic of heat-denatured whey protein solutions and gels. Foods Hydrocolloids, 14(4), 383e390. Cayot, P., & Lorient, D. (1997). Structure-function relationships of whey proteins. In S. Darmodaran, & A. Parraf (Eds.), Food proteins and their applications (pp. 225e256). New York: Marcel Dekker. Cerezo, A. (1965). The constitution of a galactomannan from the seed of Gleditsia amorphoides. Journal of Organic Chemistry, 30(3), 924e927.

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