Low-Methoxyl Pectin

Biomacromolecules 2005, 6, 374-385

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Heat-Induced Gelation of Bovine Serum Albumin/Low-Methoxyl Pectin Systems and the Effect of Calcium Ions Laurence Donato, Catherine Garnier,* Bruno Novales, Sylvie Durand, and Jean-Louis Doublier Unite´ de Physico-Chimie des Macromole´ cules, INRA, Rue de la Ge´ raudie` re, BP 71627, 44316 Nantes Cedex 03, France Received August 12, 2004

Influence of low-methoxyl pectin (LM pectin) and calcium ions (3 mM) on mechanical behavior and microstructure of bovine serum albumin (BSA) gels (pH 6.8, in 0.1 M NaCl) was evaluated. Protein and LM pectin concentrations were fixed at 2, 4, and 8 wt % and 0.21, 0.43, and 0.85 wt %, respectively. Rheological measurements and confocal laser scanning microscopy coupled with texture image analysis by use of the co-occurrence method were performed. Heat treatment of BSA/LM pectin mixtures induced protein gelation and a phase separation process between the two biopolymers, which was kinetically trapped. Calcium ions induced pectin gelation and modified BSA gel properties. Depending on biopolymer concentrations, a balance between pectin and/or protein gel contribution on final gel strength exists. The microstructures of the mixed systems in the presence of calcium can be interpreted as interpenetrated structures. Texture image analysis allowed one to classify more precisely the different microstructures observed in relation with mechanical properties. Introduction Globular proteins and polysaccharides are two major components of many food products and are often used to control the structure, texture, and stability of the products.1 Textural and structural properties of these two types of biopolymers depend on their capacity to form organized structures.2 There is therefore a need for the understanding of protein-polysaccharide interactions, to produce materials for the improvement of conventional foods and for the development of novel formulated foods.3 Biopolymer mixtures frequently lead to phase separation. Either segregative phase separation is observed, resulting in an unmixing system of two phases, each being enriched in one of the biopolymers, or an associative phase separation leading to complex formation between the two biopolymers can occur.4 The thermodynamic behavior of protein/polysaccharide aqueous mixed systems has been widely investigated and it has been shown that segregative phase separation often occurs through thermodynamic incompatibility or depletion-flocculation mechanisms.5 Attention has been recently focused on the gelation of mixed aqueous solutions of protein and polysaccharide that lead to multicomponent biopolymer gels.3 When one or both biopolymers gel, a kinetic competition between gelation and phase separation takes place. By changing the relative rates of these processes, a wide range of microstructures and a large variety of textures become accessible.6 The present study aimed to evaluate the competition between phase separation and gelation process into a model system composed of a globular protein, bovine serum * To whom correspondence should be addressed. Phone: 33-(0)2-4067-50-45. Fax: 33-(0)2-40-67-50-43. E-mail: [email protected].

albumin (BSA), and an anionic polysaccharide, low-methoxyl pectin (LM pectin). BSA is one of the most widely studied proteins and is the most abundant protein in plasma that contributes to colloid osmotic blood pressure. BSA primary structure is composed of 582 amino acid residues7-10 with an average molecular weight of 66 300 Da. Its isoelectric point is around 5.2.11 At pH 5-7, it contains 17 intrachain disulfide bridges and one sulfhydryl group. This protein is not uniformly charged within the primary structure, but the distribution on the ternary structure seems fairly uniform.12 Under heat treatment, BSA undergoes two structural modifications. The first stage that occurs up to 65 °C is a reversible denaturation of the protein. Then unfolding of the hydrophobic zones and exposition of the cysteine residues takes place, giving easy access to the formation of hydrophobic interactions and disulfide bridges resulting in aggregates. Since disulfide bridges are covalent bonds, this stage is irreversible. A further aggregation step can lead to gel formation under appropriate conditions of concentration, pH, and ionic strength.13 LM pectins are complex polysaccharides that belong to cell walls of plant materials. The main chain of LM pectins is a polygalacturonic acid partially esterified by methoxyl groups interrupted by insertion of rhamnose residues. Side chains composed mainly of neutral sugars can be linked to either the galacturonic or rhamnose groups.14 LM pectins, with a degree of methoxylation below 50%, show a high affinity for calcium ions. This interaction leads to the formation of cross-links between LM pectin chains via calcium ions, causing gel formation. In practice, LM pectins are gelled by use of soluble calcium salts, which can be naturally present in fruit or milk or added as a dilute solution.15

10.1021/bm040061f CCC: $30.25 © 2005 American Chemical Society Published on Web 10/29/2004

Influence of LM Pectin and Calcium Ions on BSA Gels

Native BSA/LM pectin mixtures formed at 20 °C at pH higher than the isoelectric point of the protein stays homogeneous and translucent even after centrifugation. These biopolymers are therefore considered compatible.16,17 Similar observations were reported for β-lactoglobulin (β-Lg)/LM pectin mixtures17-19 and for BSA/sodium alginate.20 Different hypotheses have been proposed to explain this compatibility. The two biopolymers may coexist in one phase although some domains of one polymer are excluded by those of the other, causing inhomogeneities of very small size in the solution but no visible phase separation. Another explanation is that the presence of local electrostatic interactions between positively charged patches on the protein molecule, even above the isoelectric point, and the anionic polysaccharide could explain this thermodynamic behavior.19,20 The coexistence of these two phenomena was also considered. Semenova et al.17 suggested that BSA/LM pectin compatibility is not of electrostatic origin, since no phase separation was noticed when pH or ionic strength was varied. Results obtained by dynamic light scattering and microcalorimety suggested that compatibility between the biopolymers would be controlled by an increase in mixing entropy, which would result from dehydration of biopolymer macromolecules during contact formation. Influence of heat-induced globular protein gelation on properties of anionic polysaccharide/globular protein mixtures was the subject of many studies.6,18,20-23 Medium conditions, nature of heat treatment, and intrinsic properties of biopolymers play key roles in the gelling and unmixing kinetics of these systems. However, few studies dealt with the use of both biopolymers as gelling ingredients in a composite system. Recently, Gilsenan et al.24-26 studied, in associative and segregative phase separation conditions, mixtures of LM pectin with a nonglobular protein (gelatin) in the presence and absence of calcium. From the mechanical properties analysis, the protein/polysaccharide interactions were explained depending on medium conditions. Very few studies can be found on polysaccharide/globular protein systems when both biopolymers were gelled. β-Lactoglobulin/sodium polypectate mixtures were studied by Ndi et al.,27 and whey protein/LM pectin in the presence of CaCl2 was investigated by Bernal et al.28 and more recently by Beaulieu et al.29 According to our knowledge, no study was performed on BSA/LM pectin mixed gelled systems at pH > pHi. Results showed a variety of mechanical and structural properties depending on the nature of biopolymers and experimental conditions. Several hypotheses have been proposed on the types of interaction that occur during heating and cooling of the biopolymer mixtures. A competition between the biopolymers for the binding of calcium and water was proposed,29 and a temperature dependence of calcium binding for the different biopolymers was assumed.27 A similar approach on BSA/calcium alginate systems suggested the existence of electrostatic interactions between the biopolymers.30 For unravelling the type of interactions that takes place in such complex systems, the objective of the present investigation was first to understand the way the heat-induced gelation of BSA/LM pectin system took place in 0.1 M NaCl

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at pH 6.8. In a second step, the influence of the presence of calcium ions on the structural and mechanical properties of the mixed systems was considered. Ultrastructural information on the organization of biopolymers was obtained by confocal laser scanning microscopy (CLSM), whereas viscoelastic properties of the mixed gels were studied by means of oscillatory shear measurements. Materials and Methods Materials. The LM pectin sample, kindly given by Degussa Food Ingredients (Baupte, France), had a degree of esterification of 28.1% and contained 70.6% galacturonic acid. Pectin powder was purified by washing the powder with acidic ethanol in order to eliminate the counterions and to obtain the polysaccharide in an acidic form. LM pectin solutions were prepared by dissolving LM pectin powder in deionized water. pH was slowly adjusted to 6.8 with 0.1 M NaOH and its value was verified after 1 night at 4 °C. LM pectin solutions were filtered on 0.45 µm membrane filters to eliminate aggregates. Concentration of LM pectin solution up to 4.5 wt % at pH 6.8 were obtained by dialysis against dextran T40 (25 wt %) during 72 h. BSA, obtained by cold fractionation without heating (98-99 wt % high purity, lot 85640), was purchased from ICN Biomedicals (Aurora, OH). The protein powder was defatted with n-pentane. The major ions quantified by the manufacturer in the sample were sodium (22 µmol/g) and chlorure (34 µmol/g). Insoluble matter was eliminated by centrifugation (16000 g, 20 min). Solutions of BSA (8, 16, and 32 wt %) were prepared by adding BSA powder to a 0.2 M NaCl solution under gentle magnetic stirring at 4 °C overnight. pH was adjusted at 6.8 with 1 M NaOH. Calcium content of both biopolymer powders was determined by adsorption spectroscopy. Results were 2.4 µmol/g for defatted BSA powder and 34.1 µmol/g for purified LM pectin powder. Sodium azide (0.02 wt %) was added to biopolymer solutions to prevent bacterial contamination. Preparation of Biopolymer Mixtures. Protein and LM pectin solutions were mixed at room temperature to obtain final concentrations of 2, 4, and 8 wt % BSA and 0.21, 0.43, and 0.85 wt % LM pectin in 0.1 M NaCl. It was verified that the pH after mixing was still 6.8. Blends were then stirred at 50 °C before addition of a warm CaCl2 solution in 0.1 M NaCl. Final CaCl2 concentration in the systems was fixed at 3 mM. Dynamic Oscillatory Measurements. Time sweep oscillatory measurements were performed at a frequency of 1 rad/ s, for a strain amplitude of 1%, by use of a controlled-strain rheometer (AR2000, TA Instruments) equipped with a Peltier temperature controller and with a cone-plane device (40 mm diameter, 4° angle, 102 µm gap). Warm protein/LM pectin mixtures were poured at 50 °C in the rheometer. The temperature was increased from 50 to 80 °C at 6 °C/min, kept at 80 °C for 30 min, and decreased from 80 to 20 °C. Temperature was then maintained for 1 h at 20 °C. A strain sweep test was performed to check that measurements have been performed within the linearity limits of the viscoelastic behavior.

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Confocal Laser Scanning Microscope Observations. CLSM was used in the fluorescence mode. Observations were made with a Carl Zeiss LSM 410 Axiovert (Le Pecq, France), equipped with three lasers and four wavelengths available (364, 488, 543, and 633 nm). As LM pectin and protein do not exhibit intrinsic fluorescence at these wavelengths, the biopolymers were labeled with fluorochromes showing different excitation and emission spectra. Covalent labeling of LM pectin was carried out by adding fluoresceinamine (FA) dissolved in dimethyl sulfoxide (DMSO) (0.03 g of FA/g of LM pectin) to a LM pectin solution (0.66 wt %) prepared in water. Acetaldehyde (30 µL) and 30 µL of cyclohexane were then added to the solution as catalysts. The reaction was allowed to perform for 5 h at ambient temperature under magnetic stirring. The polysaccharide was then precipitated with ethanol and ether. Precipitate was dissolved in water containing sodium azide (0.02 wt %) and dialyzed against water to eliminate unreacted dye. Labeled LM pectin was then obtained by freeze-drying. Proteins were stained by adding rhodamine B isothyocyanate (RITC) (2.5 mg of RITC/g of BSA) to the BSA solution under magnetic stirring during 1 h. Mixtures of labeled polymers were prepared as described above and poured between a concave slide and a coverslip and then hermetically sealed. The same heat treatment as for rheological measurement was applied by use of a thermostated stage (Linkam PE 60). It was checked in parallel (results not shown) that labeled biopolymers exhibited the same rheological behavior as unlabeled ones. Observation of LM pectins was made by excitation of FA at 488 nm, the emission being recorded between 510 and 525 nm. Observation of BSA was made by excitation of RITC at 543 nm, the emission being recorded between 575 and 640 nm. These conditions ensure good spectral discrimination between the two dyes. For each system, 10 images were taken after 1 h at 20 °C at the end of heat treatment. Each image was composed into 512 × 512 pixels with gray levels ranging from 0 (black) to 255 (white). Texture Image Analysis. A method of texture image analysis, the gray level spatial interdependence method (also referred to as the co-occurrence method) was applied on the images taken by CLSM. Texture image analysis is commonly used for segmentation and classification.31 A textured area in an image is characterized by a nonuniform or varying spatial distribution of gray level intensity. The variation may be regular or random. It is obvious that for a coarse texture, the distribution changes slightly with distance, whereas for a fine texture, the distribution changes rapidly with distance.32 The co-occurrence method is based on the observation of gray level values of pixels separated by a given distance in a given direction. The result is a two-dimensional histogram describing the probability that pairs of gray values occur in a given spatial relationship. For this study, only adjacent pixels were considered. The number of times that a pixel of gray level i was found adjacent to a pixel of gray level j was computed in each direction. The frequencies of occurrences (Pi,j) were arranged into square tables called the cooccurrence matrixes, whose size is equal to the number of gray level classes. From each co-occurrence matrix, 10 parameters were extracted and named according to Haralick:

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energy, entropy, contrast, correlation, inverse difference moment, maximum probability, average x lines, average y lines, standard deviation of x lines, standard deviation of y lines. Each parameter provides information on the structure of the system (homogeneity, complexity, presence of aggregates, pores, ...). For instance, energy is a measure of the homogeneity of the image: higher values are obtained for uniform images where more pixels have the same gray level values. Entropy measures the complexity within an image, higher values corresponding to larger differences in the range of gray level values. In addition, contrast feature measures the local variations in gray level values: a high contrast value describes the presence of small objects within the image. For a particlelike image with large areas of similar gray level intensities, the correlation feature will be much higher than for an image with small areas of same gray level intensity, indicative of smaller particles. The co-occurrence parameters were then analyzed by principal component analysis (PCA). By this method, synthetic variables called principal components were calculated from original variables. The principal components are uncorrelated and describe the main variations observed among the different systems. Similarity maps can be drawn by plotting the scores of two chosen principal components. In these similarity maps, two points close together correspond to images of similar textures.34 It is possible to explain the differences observed on the maps by associating each of the original parameters with the axes by plotting their scores on the map. To check a possible day effect, images were recorded on different days for varying systems. The parameters extracted from these images were treated by PCA and compared. No differences were observed on the similarity maps. It has also been checked that the optical settings (contrast, brightness) of the microscope did not affect the results of image analysis. Results and Discussion BSA/LM Pectin Systems without Calcium: (A) Effect of LM Pectin on Heat-Induced Gelation of 2 wt % BSA. Variations of the storage modulus (G′) as a function of time for 2 wt % BSA and BSA/LM pectin mixtures are shown in Figure 1. For all samples, a rapid increase of G′ during the plateau at 80 °C and a smoother increase during the cooling step were observed. After 1 h at 20 °C, G′ did not increase significantly and the system was stable. Thus, addition of LM pectin to BSA did not change the overall G′ evolution during the heat treatment. However, addition of 0.21 wt % polysaccharide led to a slightly stronger gel than the pure protein gel. This behavior has also been noticed in the presence of 0.3 wt % LM pectin (not shown). When increasing the LM pectin concentration (0.43 and 0.85 wt %), the G′ evolution was the same as for BSA only all along the thermal process, but weaker gels were obtained. This result was confirmed for LM pectin concentration increasing up to 1.3 wt % (not shown). The corresponding microstructures obtained for BSA and BSA/LM pectin mixtures during the thermal process are shown in Figure 2. For BSA only, the microstructure did not change during thermal treatment (column a). Fluores-


Figure 1. Variation of G′ during the thermal process (dashed line ) temperature profile) for 2 wt % BSA (b) and 2 wt % BSA/LM pectin blends containing 0.21 (]), 0.43 (4), and 0.85 (0) wt % LM pectin, in 0.1 M NaCl, pH 6.8.

cence was regularly distributed in the medium. As gelation has been shown by rheology, it could be concluded that the protein network cannot be evidenced at this scale of observation. For BSA/LM pectin mixtures, the systems remained homogeneous until the beginning of the step at 80 °C. In the micrographs taken after 15 min at 80 °C, inhomogeneities in the distribution of fluorescence were observed: bright zones corresponding to zones enriched in proteins and dark zones corresponding to zones devoid of BSA became visible. The structure was kept during the cooling step. When pectin concentration increased, dark zones appeared larger, and connectivity between protein aggregates decreased. To complete these observations, and to ensure that dark zones were occupied by LM pectin, observations on mixtures containing both RITC-BSA and FA-LM pectin were performed. The fluorescence of each polymer was arbitrarily coded: green for LM pectin fluorescence, red for BSA fluorescence. Figure 3 shows the final microstructures obtained for 2 wt % BSA/0.21 and 0.43 wt % LM pectin mixtures when the two micrographs were superimposed. No zones devoid of polymers were shown, proteins and LM pectin being located in two different phases. These observations confirmed that heat-induced BSA gelation was modified by the presence of LM pectin due to a phase separation process while heating, leading to a modification of the structure of the protein network. Moreover, rheological results showed that addition of LM pectin to BSA solution modified the viscoelastic properties of the final gel. For the system containing a low amount of LM pectin (0.21 wt %), the phase separation process was limited, leading to an increase in the local protein concentration. In these conditions, phase separation resulted in an increase in gel strength and a denser network. The same effect has been shown by Beaulieu et al.29 on whey protein gel (8 wt %) but with a higher LM pectin concentration (1 wt %) at pH 6, in water. For BSA/LM pectin systems at higher LM pectin concentrations, phase separation leads to less connected protein aggregates, and gels weaker than the 2 wt % pure


protein gel were obtained. Indeed, it is well-known that phase separation is enhanced when biopolymer concentration is increased.5 By assuming that protein aggregates behave as spherical particles, phase separation could be the result of a depletion-flocculation mechanism of the BSA aggregates, caused by the presence of the LM pectin chains in the medium. This hypothesis has been already considered by Tuinier et al.35 in the case of aggregated whey protein colloid particle mixed with an exocellular polysaccharide from a lactic acid bacterium. Tanaka36 introduced the concept of viscoelastic phase separation to explain such behaviors. Mixtures of protein and polysaccharide can be considered as dynamically asymmetric mixtures, because of the size difference in component molecules and of the sol-gel transition, which induces slow dynamics. Phase separation leads in that case to an interaction network (a transient gel) of slow particles (the protein aggregates) if the attractive interactions between them are strong enough. This last point was studied by Wolthers et al.37 in viscoelastic experiments on a dispersion of colloidal spheres. They showed that depletion-flocculation mechanism could induced the formation of a colloidal network for which the frequency dependence depends on the interaction between colloidal particles. This phase separation behavior was also described by Bourriot et al.38 on micellar casein/galactomannan mixtures by CLSM observations and rheological measurements. A continuous weak network mostly composed of aggregated micelles was formed resulting from the depletion-flocculation mechanism of the colloidal protein by the polysaccharide chains. (B) Influence of Protein Concentration. For pure protein gels containing 4 and 8 wt % BSA and for mixed gels (results not shown), G′ profiles were similar to those for systems containing 2 wt % BSA described above. Figure 4 shows the variation of final G′ modulus at the end of the thermal treatment for the different BSA gels and for BSA/LM pectin systems. It is clearly seen that the higher the protein concentration, the higher the G′ values, for pure protein gels as well as for the mixtures. For all protein concentrations, increasing pectin concentration caused a decrease of the final storage modulus, except for systems containing up to 0.21 wt % LM pectin. It may be assumed that this pectin concentration is too low to promote a phase separation weakening the protein network. The corresponding microstructures visualized by CLSM are shown in Figure 5. For BSA only, whatever the protein concentration, images of the same type as previously described were obtained. It was difficult to observe the effect of protein concentration. When 0.21 wt % LM pectin was added to BSA, dark zones containing LM pectin seemed bigger when protein concentration increased, but the change of the connectivity between protein aggregates was hardly visible. For 0.43 and 0.85 wt % LM pectin in the mixtures, increasing the protein concentration seemed to lead to smaller dark zones and an increased connectivity between protein aggregates. However, it was quite difficult to differentiate easily all these images just visually. Texture image analysis was then performed on these micrographs as will be described later. These results could be correlated with

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Figure 2. CLSM micrographs for 2 wt % RITC-BSA (a) and 2 wt % RITC-BSA/LM pectin blends containing 0.21 (b), 0.43 (c), and 0.85 (d) wt % pectin during the thermal process in 0.1 M NaCl, pH 6.8. Protein appears in bright. Scale bar is 25 µm.

rheological measurement: as BSA is the only gelling agent, the protein concentration is likely to govern the mechanical properties of the final mixed gel. Addition of LM pectin to protein has the same effect as observed previously for 2 wt % protein on the final gel strength, whatever the protein concentration. BSA/LM Pectin Systems in the Presence of Calcium Ions: (A) Effect of Calcium Ions on Gelation of LM Pectin. Variations of G′ for LM pectin in the presence of 3 mM CaCl2 during the thermal process are shown in Figure 6. Whatever the polysaccharide concentration, no modulus was measured at 80 °C. G′ began to increase during the

cooling step for a temperature below 60 °C and continued to increase as a function of time at 20 °C. As already known, increasing LM pectin concentration for a constant calcium concentration increased gel strength due to more numerous linkages between LM pectin chains via calcium ions.39 It should be noticed that moduli of LM pectin gels did not reach a plateau after 1 h at 20 °C, showing a slow reorganization of LM pectin gel. (B) Effect of Calcium Ions on BSA Gelation. The variations of G′ during the thermal treatment for BSA gels (2, 4, and 8 wt %) in the presence or absence of CaCl2 (3 mM) are shown in Figure 7. Similar G′ profiles were obtained


Figure 3. Double localization CLSM micrographs of 2 wt % RITCBSA/0.21 (a) and 0.43 (b) wt % FA-LM pectin systems taken after 1 h at 20 °C after heat treatment (80 °C for 30 min), in 0.1 M NaCl, pH 6.8. Protein appears in red and pectin in green. Scale bar is 25 µm.

Figure 4. G′ modulus of BSA/LM pectin mixtures (measured after 1 h at 20 °C after the thermal process) as a function of LM pectin concentration for 2 (b), 4 (9), and 8 (2) wt % BSA in 0.1 M NaCl, pH 6.8.

and addition of calcium led to an increase in the gel strength, whatever the protein concentration. It is noticeable that, for


BSA gels, G′ ranged from 102 to 104 Pa whereas pectin gels’ moduli were lower than 102 Pa. The microstructure of 2, 4, and 8 wt % BSA gels with calcium is shown in Figure 8. These pictures should be compared with the first row of Figure 5 (in the absence of CaCl2). Addition of calcium in the system resulted in the appearance of inhomogeneities in the fluorescence distribution, better evidenced at low protein concentration. Dark zones corresponding to zones devoid of proteins were observed and could be assimilated as pores of the BSA network containing the solvent. This coarsening of the protein network due to calcium ions can be paralleled with the increase in gel strength observed by rheology. To discriminate all images, texture image analysis was carried out on images taken at the end of the heat treatment, for BSA gels at different concentrations in the presence or the absence of calcium ions. The similarity map of the first two principal components obtained after PCA of the cooccurrence parameters is shown in Figure 9. On this map, a representative image of the set of 10 images taken for each system is shown at the average position of that group of images. Each image is characterized by parameters that give information on the structure of the system (homogeneity, complexity, presence of aggregates, pores, ...). Two images close together correspond to images of similar texture. In Figure 9, systems with and without calcium were clearly separated according to the first axis. The images corresponding to systems with calcium were found in the left part of the map whereas images of systems without calcium were located in the right part of the map. The first axis separated two groups of parameters. The parameters associated with systems without calcium were energy, maximum probability, and inverse difference moment. These parameters are indicative of homogeneous images. In opposition, the systems with calcium were described by parameters (entropy, contrast, correlation, and standard deviation) related to complexity of the image and the presence of small objects (assimilated as the pores of BSA network). The average final G′ moduli are indicated in parentheses above each picture ((10% variation). It can be seen that the classification obtained for the pictures could be related to rigidity of the gels. For each protein concentration, G′ values increased from the right to the left of the first axis, that is, with the presence of calcium ions in the protein gels. Axis 2 differentiates systems as a function of BSA concentration: the higher the BSA concentration, the more homogeneous the image (the parameters related to these images being the average, the energy, and the maximum probability) and the higher the G′ values. Image analysis appears then to be an objective method to differentiate pictures of similar appearances (pictures a and c, for instance). This suggests that the increased rigidity of BSA gels upon adding calcium is related to modifications of their microstructure. These results could be explained by an affinity between BSA and calcium, as suggested by Powell Baker and Saroff,40 or by an increase in the reactivity of sulfidryl groups and thus hydrophobic interactions, as was reported for β-Lg.41 However, calcium could also have a “salting-in” effect on

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Figure 5. CLSM micrographs of RITC-BSA and RITC-BSA/LM pectin blends in 0.1 M NaCl, pH 6.8, after 1 h at 20 °C after the heat treatment. Protein appears in bright. Scale bar is 25 µm.

Figure 6. Variations of G′ during the thermal process (dashed line ) temperature profile) for 0.21 (]), 0.43 (0), and 0.85 (4) wt % LM pectin in the presence of 3 mM CaCl2 in 0.1 M NaCl, pH 6.8.

Figure 7. Variations of G′ during the thermal process (dashed line ) temperature profile) for 2 (O, b), 4 (], [), and 8 (4, 2) wt % BSA gels without calcium (open symbols) and with (solid symbols) 3 mM CaCl2, in 0.1 M NaCl, pH 6.8.

BSA by reducing electrostatic repulsion between proteins, then increasing protein aggregation, resulting in a denser and stronger network.42

(C) Effect of the Presence of Calcium Ions on BSA/ LM Pectin Mixed Gels. The kinetics of gel formation of 2 wt % BSA/LM pectin mixtures in the presence of 3 mM


Figure 8. CLSM micrographs of 2 (a), 4 (b), and 8 (c) wt % BSA gels in the presence of 3 mM CaCl2, pH 6.8, in 0.1 M NaCl taken after 1 h at 20 °C after the heat treatment (80 °C for 30 min). Protein appears in bright. Scale bar is 25 µm.

CaCl2 are illustrated in Figure 10 and compared to gel formation of 2 wt % BSA only. The profiles for the mixtures differed dramatically from that of BSA alone. Whatever the LM pectin concentration in the system, only a slight increase of G′ (G′ < 3 Pa) was observed during the step at 80 °C. A sharp G′ increase occurred during the cooling step below 60 °C, followed by a leveling at 20 °C. This result suggests that protein aggregation at 80 °C was hindered by the presence of both LM pectin and calcium. The final G′ moduli for BSA/LM pectin gels were much lower than the G′ of the BSA gel and of the same order of magnitude of the corresponding LM pectin gel (see Figure 6). As LM pectin


gelation occurred on cooling, it can be supposed that LM pectin gelation governed the gelation of the mixed systems. The corresponding microscopic observations of BSA and BSA/LM pectin gels in the presence of calcium ions are displayed in the first column of Figure 11. By comparison with the first column of Figure 5, it appears clearly that addition of calcium to BSA/LM pectin system also induced a phase separation process, the final microstructure of the mixtures showing dense fluorescent particles concentrated in proteins and dark zones containing LM pectin gel. However, phase separation was more pronounced in the presence of calcium at the lowest pectin concentration studied (0.21 wt %) and the protein aggregates seemed slightly different in the presence of both calcium and polysaccharide. When LM pectin concentration increased, darker zones seemed to be larger. Observations of the microstructure during the kinetics (results not shown) revealed that the final microstructure was already obtained at 80 °C, after 15 min for LM pectin e 0.43 wt % and between 15 and 30 min at 80 °C for 0.85 wt % LM pectin. It can be supposed that protein aggregation is delayed by the presence of a high amount of LM pectin. Moreover, from rheological results, it has been shown that the heating step resulted in a slight increase of G′. During the cooling step, the apparent microstructure did not change. The final gel microstructure could then be interpreted as an interpenetrated structure,43 protein aggregates forming a very weak gel visible by microscopic observations, interpenetrated by the LM pectin network deduced from rheological results. To determine the effect of protein concentration, the BSA content in the mixed systems in the presence of calcium ions was increased (4 and 8 wt %). When 0.21 wt % LM pectin was present in the systems, the same evolution of the viscoelastic moduli as that with BSA only was observed, but lower moduli were obtained. Contrary to what was observed with 2 wt % BSA concentration, G′ increased strongly during the heat treatment and a slight increase occurred during the cooling step to reach a plateau at 20 °C (results not shown). For a higher concentration in LM pectin (0.43 and 0.85 wt %), similar G′ profiles were obtained as with 2 wt % BSA/LM pectin mixtures, with only a slight G′ increase at 80 °C. Figure 12 summarizes the effect of increasing protein concentration on the final G′ moduli of BSA only, BSA/LM pectin mixtures, and pectin gels in the presence of calcium ions. For BSA only and mixed systems, increasing protein concentration increased gel strength. Influence of calcium ions and increase of BSA concentration (4 and 8 wt %) on final G′ modulus of the systems could be evaluated by comparison with Figure 4. When 0.21 wt % LM pectin was added, G′ values obtained with or without calcium were close to each other. This suggests that protein gelation governs the gelation of the system. On the contrary, for 0.43 wt % pectin, the systems containing calcium were weaker than those without calcium. For 0.85 wt % LM pectin, the mixed system containing 4 wt % BSA were more rigid in the presence of calcium, whereas the inverse effect was observed with 8 wt % BSA. It appears that, depending on concentrations of biopolymers in the mixed systems, a balance between pectin and/or protein gel contribution exists.

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Figure 9. Similarity map obtained for CLSM micrographs taken after 1 h at 20 °C after the heat treatment (80 °C, 30 min) of 2 (a, a′), 4 (b, b′), and 8 (c, c′) wt % RITC-BSA gels without (a-c) and with 3 mM CaCl2 (a′-c′), in 0.1 M NaCl, pH 6.8. The numbers in parentheses correspond to G′ values after 1 h at 20 °C after the heat treatment.

Figure 10. Variations of G′ during the thermal process (dashed line ) temperature profile) for 2 wt % BSA (b) and BSA/LM pectin blends containing 0.21 (]), 0.43 (4) and 0.85 (0) wt % LM pectin in the presence of 3 mM CaCl2 and 0.1 M NaCl, pH 6.8.

The microstructure of the systems containing 4 and 8 wt % BSA is displayed in Figure 11 (columns 2 and 3). Whatever the pectin concentration, increasing protein concentration resulted in an increased number of dense fluorescent zones, reflecting an increase of protein aggregation. This suggests that proteins contribute most to the final gel modulus at high BSA/LM pectin ratio. However, the increase of local pectin concentration due to the phase separation process can also play a role in the increase of the gel strength. This could explain why BSA/LM pectin gels with calcium

are stronger than LM pectin gels and weaker than the corresponding BSA gels. In comparison with BSA/LM pectin systems without calcium, microscopic observations suggests that calcium ions established specific interactions with both protein and pectin that may be explained, depending on the biopolymer concentrations, by the balance between the biopolymer gel contribution observed in rheology. To evaluate how the presence of calcium ions and increasing protein concentration modify the microstructure of the BSA/LM pectin gel, texture image analysis has been performed on pictures obtained for 2, 4, and 8 wt % BSA in the presence of 0.43 wt % LM pectin systems, either with or without calcium ions. Figures 5 and 11 presented previously showed one of the 10 images taken for each system. The similarity map of the first two principal components obtained after texture image analysis of all images is presented in Figure 13. The first axis separated the systems as a function of the protein concentration with systems at low concentrations (2 wt %) located on the right part of the map and systems at high concentration (4 and 8 wt %) on the left part of the map. Parameters associated with systems containing low protein concentration (energy, maximum probability, inverse difference moment, correlation, and maximum probability) are indicative of complex and less homogeneous systems than highly concentrated gels. The images corresponding to these systems presented dense protein aggregates and small pores. The second axis did not provide any additional information. In the presence of calcium, images a′, b′, and c′ could be described as protein aggregates with a smaller size and a reduced connectivity compared to the pictures obtained in



Figure 11. CLSM micrographs for RITC-BSA and RITC-BSA/LM pectin blends in the presence of 3 mM CaCl2, in 0.1 M NaCl, pH 6.8, taken after 1 h at 20 °C after the heat treatment (80 °C, 30 min). Protein appears in bright. Scale bar is 25 µm.

Figure 12. G′ modulus of LM pectin ([) and BSA/LM pectin mixtures (measured after 1 h at 20 °C after the thermal process) as a function of LM pectin concentration for 2 (b), 4 (9), and 8 (2) wt % BSA in the presence of 3 mM CaCl2 and 0.1 M NaCl, pH 6.8.

the absence of calcium. As the images corresponding to systems with calcium are always on the right side of the

images corresponding to systems without calcium, the addition of calcium would have the same effect on texture image as a reduction of protein concentration. In comparison with rheological measurements (final G′ in parentheses), the first axis showed an increase of G′ for the different systems as protein concentration increased. Clearly, texture image analysis made it possible to classify more precisely all the different images, whereas visual observation was not sufficient to distinguish images from one to another. This results differs from those obtained by Beaulieu et al.29 in a recent study on a whey protein/LM pectin (8 wt %/1 wt %) mixture at pH 7 in water, which showed that addition of calcium (10 mM) increased gel strength of the mixture compared to the system without calcium. CLSM observations evidenced an increase in protein aggregation. A phase separation process also occurred, whether calcium was added to the system or not. The differences with our study could be explained first by the nature of the globular proteins, whey protein being a mixture of mainly β-lactoglobulin (∼50%), R-lactalbumin (∼20%), and BSA (∼10%), that behaved differently from individual proteins. The difference of ionic strength should also be considered. Indeed, electrostatic interactions such as salting-out effect are more

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Figure 13. Similarity map obtained for CLSM micrographs taken after 1 h at 20 °C after the heat treatment (80 °C, 30 min) of 2 (a, a′), 4 (b, b′), and 8 (c, c′) wt % RITC-BSA/0.43 wt % LM pectin gel without (a-c) and with 3 mM CaCl2 (a′-c′), in 0.1 M NaCl, pH 6.8. The numbers in parentheses correspond to G′ values after 1 h at 20 °C after the heat treatment.

probable at higher ionic strength and could have explained the loss of connectivity of the protein network during the heating process that we observed for pectin concentration higher than 0.43 wt % in the presence of calcium ions. However, the mode of interaction of the divalent ion calcium is probably different from that of monovalent ions and this specific effect should also be considered. Another study on β-Lg/sodium polypectate (SPP) (10 wt %/1 wt %) gel in the presence or absence of 15 mM CaCl2 in water at pH 6.5 showed that addition of calcium resulted in the formation of a translucent gel.27 This was attributed to gelled SPP entrapped in a uniform matrix of large protein aggregates. Higher G′ modulus for the mixed gel than those obtained for the same system and for protein only without calcium were obtained. In this case, it is important to consider that the structure of SPP was not clearly defined and may differ from the type of LM pectin used here. Parameters implied in the thermodynamics of phase-separated systems (polymer and calcium concentrations, medium conditions) have to be considered to explain the different behaviors observed. Conclusions Heat treatment of BSA/LM pectin mixtures induced protein gelation and a phase-separation process, which was kinetically trapped. This produced a weakening effect of the mixed gel compared to BSA gel only, due to a modification in protein aggregation. Addition of calcium ions had an effect on both biopolymers. Calcium ions induced pectin gelation and also affected BSA gel properties. The microstructure of the mixed system in the presence of calcium can be assumed

as interpenetrated structures of BSA and LM pectin gels generated both by a phase-separation process between the two components and by their specific interaction with ions (calcium and sodium) present in the systems. For protein concentrations of 4 and 8 wt % and for low pectin concentration (0.21 wt %), protein gelation seemed to govern the gelation of the system. For higher LM pectin concentrations, the polysaccharide controlled the gelation of the system at all protein concentrations. Depending on the concentration of biopolymers in the mixed system, a balance between pectin and/or protein gel contributions to final gel strength exists. As suggested by Beaulieu et al.,29 competition between protein and LM pectin for calcium ions can occur. Texture image analysis showed that for 0.43 wt % LM pectin added to BSA, addition of calcium to the mixed system has the same effect on the microstructure and final gel strength as the addition of LM pectin to BSA at a lower BSA concentration. To deepen the role of calcium ions in BSA/LM pectin systems and to explain more precisely the differences obtained from the literature, further studies need to be done, considering the importance of ionic strength. Indeed, Neiser et al.30 reported that ionic strength has a major effect on BSA/ alginate mixed gelled systems. Hence, mixed gelled systems can yield a variety of structures and textures depending on medium conditions (concentration, presence of calcium ions), which could lead to interesting properties in real food systems. Acknowledgment. We thank Philip Mugglestone for his kind proofreading of the manuscript.


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BM040061F

Low-Methoxyl Pectin

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