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low methoxyl pectins mixed gels with added calcium
International Dairy Journal 11 (2001) 961–967
Rheology, texture and microstructure of whey proteins/low methoxyl pectins mixed gels with added calcium Martin Beaulieua, Sylvie L. Turgeona,*, Jean-Louis Doublierb Centre de Recherche en Science et Technologie du Lait (STELA), Faculte! des Sciences de l’Agriculture et de l’Alimentation, ! Universite! Laval Pavillon Paul-Comtois, Sainte-Foy, Quebec, Canada G1K 7P4 b ! Institut National de la Recherche Scientifique (INRA), Centre de Recherche de Nantes, Laboratoire de Physicochimie des Macromolecules, B.P. 71627, 44316 Nantes Cedex 03, France a
Received 13 February 2001; accepted 4 August 2001
Abstract Gelation of a whey protein isolate mixed with various industrial low methoxyl pectins (LMPs) has been investigated. Ratios were adjusted to keep whey protein (WP) concentrations constant at 8%. Pectin and calcium concentrations were fixed at 0.1%, 0.5%, 1.0%, 1.5% and 0, 5, 10 mm; respectively. The pH was adjusted to 6.0. Heat treatment at 801C was used to induce WP gelation. Heating WP solution mixed with LMP allowed gel formation with lower protein concentration than normally observed; syneresis was also reduced. The type and proportion of pectin, as well as the calcium concentration affected gel hardness. Increasing the amount of pectin and the calcium concentration made mixed gels firmer. White gels were observed indicating aggregation of WPs. The gelation of the mixed systems appeared to have occurred through a phase separation and a competition between biopolymers for the binding of water and calcium. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Whey proteins; LM pectins; Calcium; Gel; Rheology
1. Introduction Food products are complex multi-component systems. Proteins and polysaccharides are largely responsible for the structural, mechanical, and physicochemical properties of foodstuffs (Tolstoguzov, 1991). As texture and stability are major criteria of food quality, investigations are primarily concerned with proteins and polysaccharides and their interactions in order to provide optimum structural quality to design new and attractive food (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). Mixed gels are formed from blends containing more than one gelling agent and may be classified into three types: interpenetrating, coupled, and phase-separated networks (Morris, 1986). Interpenetrating networks are formed when the two components gel separately and form independent networks. Both networks are continuous throughout the sample but any interaction *Corresponding author. Tel.: +1-418-656-2131 x4970; fax: +1-418656-3353. E-mail address: [email protected] (S.L. Turgeon).
between them is only topological (Morris, 1986). Coupled or complex coacervate networks are formed in the presence of favourable intermolecular interactions between the different types of polymers. In contrast, phase-separated gels are formed by incompatible polymers, where interactions between the different polymers are repulsive and/or when the two polymer types show varying affinity towards the solvent (Piculell & Lindman, 1992; Tolstoguzov, 1991, 1995). Whey proteins (WPs) are well known for their high nutritional value and versatile functional properties in food products (de Wit, 1998). Gelation is an important functional property of WPs (especially b-lactoglobulin) commonly used by the food industry. The gelation of WPs, as well as the factors affecting gelation, have been reviewed several times (Mulvihill & Kinsella, 1987; Stading & Hermansson, 1990, 1991; Gault & Fauquant, 1992; Hines & Foegeding, 1993; Aguilera, 1995; Boye, Alli, Ramaswamy, & Raghavan, 1997). Heat-induced gelation of WPs is affected by many factors, such as heating temperature, heating rate, pH, concentration, ionic strength, and presence of specific ions (Aguilera, 1995). Calcium greatly affects the functionality of WPs,
0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 2 7 - 3
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including their thermally induced gelation (Sherwin & Foegeding, 1997). WP gels made with dispersions containing 10–20 mm calcium have particulate microstructures, which give them an opaque appearance and low water-holding properties. Pectin is a linear polysaccharide built from polygalacturonic acid subunits that is partially esterified with methoxyl groups. The degree of methoxylation is very important as it influences pectin behaviour. It is defined as the average number of methoxyl groups per percent of the galacturonic acid units; this is commonly referred to as the degree of methoxylation (DM) and used to classify pectins into two groups. If the DM is greater than 50%, the pectin is referred to as high methoxyl pectin (HMP). Pectins with less than 50% DM are called low methoxyl pectin (LMP). LMP gels are formed as a result of interactions with calcium over a wide range of soluble solids and pH values. When calcium is added, cross-links begin to form between the pectin molecules causing a slight thickening of the solution. In practice, LMPs are gelled by using soluble calcium salts, which can be naturally present in the fruit or milk; or added as a dilute solution. LMP uses are varied, ranging from reduced sugar jams to soft confectionery jellies and spreadable processed cheeses. Very few studies exist on the interaction of pectins with WPs in mixed gel systems. The factors affecting the compatibility of bovine serum albumin (BSA) with HMP were reviewed by Semenova et al. (1991). Syrbe, Bauer, and Klostermeyer (1998) surveyed a large range of polysaccharides mixed with WPs including HMP. The rheology and microstructure of b-lactoglobulin and sodium polypectate mixed gels were investigated by Ndi, Swanson, Barbosa-Canovas, and Luedecke (1996). Considering that the WP and LMP gelation processes are affected by some common factors, behaviour of these biopolymers in mixed systems may be complementary. Reported here are the results of experimental investigations designed to study the effects of added calcium on ternary mixtures of whey protein isolate (WPI), LMP with different DM, and water.
2. Materials and methods
2.2. Preparation of solutions A stock WP solution ð16% w=vÞ was prepared by adding WP powder to deionized water and stirring gently for 2 h at room temperature. Solutions were then stored overnight at 41C to allow complete hydration. The pH of the protein solutions was adjusted to 6.0 using 1 n HCl: Pectin stock solutions (3% w=v) were made under strong stirring and heating at 601C until complete solubilization. In order to obtain desired calcium levels, other sets of WPI and pectin stock solutions were prepared by solubilizing in 5 and 10 mm (calcium ion) solutions of CaCl2 ; respectively. Blends, containing the right proportion of WP, different pectins, and calcium were mixed at 501C: The pH value was readjusted to 6.0 with 1 n NaOH: 2.3. Gel preparation Beakers (100 mL) were filled with 70 mL of final mixture and closed with aluminum foil that had been punctured for pinhole ventilation. Beakers were placed in an 801C water bath and heated for 30 min: Samples were then removed and cooled in a refrigerator where they were stored overnight at 41C: Gels were placed at 251C at least 2 h before texture analysis. 2.4. Visual aspect of the gel All heated samples were visually characterized by the same observer. Observations were made with the following characteristics in mind: solution or gel aspect (inversion test), clear or white opaque colouration, and notable aggregation. 2.5. Texture properties Gel texture parameters were determined by a Texture Analyzer TA XT-2 (Texture Technologies Corporation, NY, USA). The gels were penetrated with a 12 mm diameter cylinder probe (Ju & Kilara, 1998). A force– time curve was obtained at a crosshead speed of 60 mm min1 for a 10 mm displacement. The gel failure point was not reached. The resulting force–time curves were analyzed using the texture profile analysis (TPA) method.
2.1. Materials 2.6. Dynamic oscillatory measurements Dry WP isolates (92% w/w protein) were purchased from Davisco (Le Sueur, MN, USA). Citrus pectin samples (typical DM values: 28, 35, 40, 47, and 65, referred to as DE28, DE35, DE40, DE47, and DE65) were graciously provided by Hercules Co. DE65 was included in order to compare mixed gel behaviour made with LMP vs. HMP. CaCl2 was purchased from Sigma Chemical Company (St. Louis, MO, USA).
Small-deformation oscillatory measurements were performed, at a frequency of 1 Hz and a strain of 0.5%, on a controlled-strain rheometer (ARES 100 FRT, Rheometric Scientific, Piscataway, NJ, USA) equipped with a circulating water bath temperature controller. Couette device with a cup (35 mm diameter) and bob system (33 mm diameter, 35 mm length) was
M. Beaulieu et al. / International Dairy Journal 11 (2001) 961–967
used. Warmed solutions ð501CÞ were poured directly into the heated measuring system ð801CÞ of the rheometer. This temperature was maintained for 30 min: Samples were then cooled to 201C (1:51C min1 ) and kept at least 30 min at this temperature. Samples were covered with a thin film of paraffin oil to avoid evaporation during heat treatment. 2.7. Confocal laser scanning microscope (CLSM) observations The CLSM system consists of a Carl Zeiss LSM 310 Confocal Laser Scanning Microscope attached to a Zeiss Upright Axioplan Microscope. Images acquisition and analysis were managed using Zeiss software (Version 3.95). The light source was an argon laser with emission maxima of 488 and 514 nm: Excitation was performed at 514 nm: Staining of the samples was carried out by dissolving 0.001% (w/w) of Fast Green FCF (Sigma, St. Louis, MO, USA) in the solutions prior to heat treatment. Stained solution was poured onto a hollow slide which was then hermetically sealed. Heat treatment (801C for 30 min) was applied directly on these sealed hollow slides. Slides were then cooled in a refrigerator where they were stored overnight at 41C: Samples were placed at 251C at least 2 h before observation. 2.8. Data analysis Three different variables were used to characterize the behaviour of the WP/LMP system, namely, LMP concentration; LMP DM; and calcium concentration according to a factorial experimental design. Samples were produced and analysed at random. Every experimental condition was produced three times and at least three replicates of each measurement were made.
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molecules (Mulvihill & Kinsella, 1988; Kuhn & Foegeding, 1991). However, higher calcium concentration generated strong protein aggregation and weaker gels (Xiong, 1992; Sherwin & Foegeding, 1997; Ju & Kilara, 1998). Calcium-free pectin samples behaved like viscous solutions. Dynamic oscillatory measurements confirmed the liquid-like behaviour; the loss modulus ðG00 Þ was superior to the storage modulus ðG0 Þ: Pectin gels were obtained when calcium was added (Fig. 1). Increasing the calcium concentration from 0 to 10 mm resulted in firmer gels for pectins DE28 and DE35. Calcium is responsible for the formation of cross-links; higher calcium concentrations will generate stronger interactions and firmer gels (Whistler & BeMiller, 1997). Pectins DE40 and DE47 did not produce gels that could be analysed by TA XT-2, indicating that the formation of a pectin network via calcium cross-links was low with these higher DM pectins. However, Garnier, Axelos, and Thibault (1993) showed that gel could be obtained from LMP with higher DM by increasing the calcium concentration. It is important to remember that commercial pectins exhibited some structural variations depending on extraction process and subsequent treatments. Thus, pectins can produce different types of gels with various maximum firmness, depending on the environmental conditions (Whistler & BeMiller, 1997). The firmness of the pectin gels was not only influenced by the DM and the calcium concentration as shown in Fig. 1; at equal calcium concentration (10 mm), DE35 produced firmer gels than DE28. 3.2. Visual aspect of the gels WP, pectin, and mixed solutions were translucents prior to heat-treatment. Mixing of WP and pectin solutions resulted in stable single-phase systems. This co-solubilization of both biopolymers can be explained
3. Results and discussion 3.1. Gelation of WP and pectin solutions Gelation of WP solutions was not observed and a liquid-like behaviour was obtained in the dynamic oscillatory measurements. These results are not surprising since the solutions used had only an 8% (w/w) protein concentration. Boye et al. (1997) noticed that WP gels which formed below 15% (w/v) were very weak. On the other hand, the addition of calcium (up to 10 mm) to the 8% w/w WP solutions induced the formation of a gel (hardness 4:9 N), which was aggregated. It is well known that the calcium level up to 10 mm increases the strength of whey protein gels (i.e., stiffens the matrix) due to electrostatic interactions with the negatively charged and unfolded protein
Fig. 1. Effect of calcium on firmness of pectin gels ð3% w=wÞ at pH 6.0.
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by the stabilization of the system through electrostatic repulsions (Cai & Arntfield, 1997). On the other hand, it has been suggested that this behaviour could be ascribed to the formation of weak soluble complexes that are stabilized by hydrogen bonds (Tolstoguzov, 1991). Heating WP solutions generally induced clear gel networks via hydrophobic interactions and disulphide bonds (Hines & Foegeding, 1993). This type of gel is generally referred to as a fine-stranded gel, which is formed when the degree of repulsion between proteins is large (Langton & Hermansson, 1992). Heating mixed solutions generated gelation for all samples except for those with the lowest pectin concentrations (0.1%) and in which no calcium was added. A white colouration was observed for all gels (Fig. 2). A white opaque gel is an indication of protein aggregation; this type of gel is most often referred to as a particulate gel (Langton & Hermansson, 1992). Furthermore, raising the temperature increases protein–protein interactions and leads to more aggregation. Adding calcium (5 and 10 mm) led to an increase in protein aggregation. WPs have an isoelectric point of 5.2 and a total negative charge at pH 6.0. Adding cations, such as calcium, shields the negative charges on the proteins and the amount of energy needed for aggregation as well as the distance between the protein molecules (Gault & Fauquant, 1992). It is likely that pectins which presented a stronger affinity for calcium (DE28 and DE35) reduced protein aggregation by binding a portion of the calcium. On the other hand, pectin that could not bind calcium (DE47) resulted in stronger aggregated gels.
3.3. Gel texture Heating WP solution mixed with LMP resulted in gel formation. Ndi et al. (1996) observed the same behaviour for a mixture of b-lactoglobulin and sodium polypectate. As seen in Fig. 3, gel firmness was not only affected by the amount of LMP used, but by its type too. Mixed gels made with DE28 increased in firmness with the addition of pectin (from nil at 0.1% to 0:165 N at 1.5%). Similar results were obtained for DE35 and DE47 (maximum firmness of 0.187 and 0:278 N). However, DE40 generated stronger gels at all levels. The kinetics of gel formation of mixtures containing 8% WP and 1% LMP are illustrated in Fig. 4 from dynamic measurements. Gel formation was monitored in three steps. First, the temperature was maintained at 801C for 30 min; second, the temperature was decreased linearly from 801C to 201C at 1:51C min1 ; and finally, this temperature was maintained for 30 min: WP
Fig. 2. Visual aspect of heated mixed samples made with WP (8%) and LMP with different DM for different levels of added calcium (0, 5, and 10 mm). Liquid-like samples shown as white; white coloured solidlike samples shown as grey, and strongly aggregated solid-like samples shown as black.
WP 8% + DE28 1% WP 8% + DE35 1% WP 8% + DE40 1% WP 8% + DE47 1% WP 8% + DE65 1%
Temperature (˚C)
G' ( Pa )
Fig. 3. Firmness of mixed gels (pH 6.0) made with whey proteins ð8% w=wÞ and increasing concentrations of pectin DE28, DE35, DE40, and DE47 (0.1%, 0.5%, 1.0%, and 1:5% w=w).
Time (min) 0
Fig. 4. G profile for mixed gels (pH 6.0) made with 8% ðw=wÞ WP and pectins DE28, DE35, DE40, DE47, and DE65 ð1% w=wÞ:
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samples, with no added pectin, did not gel; G0 profile (not shown) was linear. Mixed gels with the five pectin samples resulted in similar profiles with a rapid increase for the first 5 min at 801C followed by a levelling. Upon cooling down to 201C an increase of G0 from B100 to B1000 Pa was measured. Overall, no differences between samples were observed. These results are consistent with the compression tests for a concentration of 1% added LMP. Mixed gels made with LM pectins are in all likelihood comparable in their structures and their solid-like behaviours after the heatinduced gelation. Considering the fact that these gels were made without adding calcium, it may seem reasonable to assume that WP is the only gelling agent in those mixed solutions. Higher pectin concentrations in the system will result in an increase in the amount of solvent that could be entrapped in the pectin phase, causing concentration of the protein phase and an increased gel firmness. However, we should also consider that water partition changed during the gelation process; Dickinson and McClements (1995) have mentioned that the solvent could be redistributed between the phases as a result of the polymeric conformational changes that accompany gelation, which suggest that the results obtained in this study could in fact be in attributed to the hydration level of the proteins during gelation as well as the solvent partition in each phase after the entire gelation process. On the other hand, one could be tempted to directly compare the results of texture analysis with those of dynamic measurements. Dynamic measurements were made using a small deformation (strain 0.5%) and texture analysis was made with a 25% deformation; small differences in the gel structure, which are not seen by small deformation measurements, can be detected by large deformation measurements (Stading & Hermansson, 1991). Large deformation measurements are related to the size of inhomogeneities in a material structure (Tang, McCarthy, & Munro, 1995).
Fig. 5. Firmness of mixed gels (pH 6.0) made with whey proteins ð8% w=wÞ; increasing concentrations of pectins DE28, DE35, DE40, and DE47 (0.1%, 0.5%, 1.0%, and 1:5% w=w), and 10 mm of added calcium.
ing the fact that large pores are the weakest part of a gel network (Stading, Langton, & Hermansson, 1993). The kinetics of gel formation of mixtures containing 8% WP and 1% pectin in the presence of 10 mm calcium are illustrated in Fig. 6 from dynamic measurements and compared to WP 8% alone. The four pectin samples yielded similar profiles as without Ca2þ (Fig. 4). These were quite similar to that of WP 8% suggesting that mixed gels are mainly generated by the WP phase. However, large differences were experienced in relation to the DM of the pectin sample; the lower the DM, the higher the storage modulus. As compared to Fig. 4, these values were much higher than in the absence of Ca2þ for the lowest DM while they were lower for the highest one. This shows clearly that Ca2þ plays a major role in the properties of the mixed gels. However, the
WP 8% + Ca10 WP 8% + DE35 1% + Ca10 WP 8% + DE40 1% + Ca10 WP 8% + DE47 1% + Ca10 WP 8% + DE65 1% + Ca10
Temperature (˚C)
Calcium increased the firmness of mixed gels made with DE28, DE35, and DE40 (Fig. 5); this increase of firmness was at least by a factor 10. Maximum firmness was reached for mixed gels made of DE35 regardless of the pectin content. Mixed gels presented a lower firmness than WP gels (hardness 4:9 N) obtained with the same calcium concentration. However, mixed gels were less brittle and retained more water; a decrease in the syneresis was also observed. Lower pectin concentrations as well as those made with pectins with a lower affinity for calcium, resulted in weaker gels. Higher calcium concentration was available for the WP phase generating strong aggregation and weak gels, consider-
G' (Pa)
3.4. Effect of added calcium
Time (min) Ti
Fig. 6. G0 profile for mixed gels (pH 6.0) made with 8% ðw=wÞ WP, pectins DE35, DE40, DE47, and DE65 ð1% w=wÞ; and 10 mm of added calcium.
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Fig. 7. CLSM observations of whey proteins gel structures stained by Fast Green FCF ð0:001% w=wÞ: (a) WP ð8% w=wÞ with 10 mm of added calcium resulted in low aggregation; (b) WP ð8% w=wÞ; and pectin DE47 ð1% w=wÞ; without added calcium shows aggregation; (c) WP ð8% w=wÞ; pectin DE47 ð1% w=wÞ; with 10 mm of added calcium shows strong aggregation. Protein phase is light grey (scale bar ¼ 10 mm).
storage modulus of the mixed systems was much lower (between 600 and 3000 Pa at 201C; depending upon the DM) than for WP alone ð6300 PaÞ at the same WP concentration. Addition of pectin resulted in a decrease of the rigidity of the protein gel in the presence of Ca2þ : Overall, these results with 1% LMP confirmed texture analysis for mixed gels as illustrated in Fig. 5. Indeed, adding calcium increased WP/WP interactions. High calcium concentration likely generated interactions between proteins by bridging negatively charged carboxyl groups, which increased the tendency towards coagulation rather than gelation (Zirbel & Kinsella, 1988). In the case of mixed systems, LMP likely compete with proteins with respect to Ca2þ : Therefore, part of Ca2þ is not available to the protein gel which may explain the decrease in the storage modulus of mixed gels.
3.5. CLSM observations Fig. 7 shows typical micrographs of WPs ð8% w=wÞ with 10 mm of added calcium (Fig. 7a); WPs ð8% w=wÞ with 1% pectin (DE47) (Fig. 7b); and WPs ð8% w=wÞ with 1% pectin (DE47) and 10 mm of added calcium (Fig. 7c). This illustrates clearly that quite different systems are obtained. In case of WP alone (Fig. 7a), proteins appear in white, almost regularly distributed. Addition of pectin (no Ca2þ added) apparently resulted in a stronger aggregation of proteins (Fig. 7b). A similar observation could be made in the presence of Ca2þ (Fig. 7c), the proteins being apparently aggregated. All pectins presented the same aggregation pattern. All mixed systems presented comparable type of aggregation for the same pectin and calcium concentration. These CLSM observations strongly suggest that WP and LMP are located in two different phases as a result of thermodynamic incompatibility, this phenomenon being
known to occur with a large number of protein/ polysaccharide systems (Grinberg & Tolstoguzov, 1997). Rheological measurements can therefore be interpreted from a phase separation process. In the absence of Ca2þ ; only the protein-enriched phase is gelled in a tridimensional porous network while the pectin-enriched phase is a liquid dispersion within this network. With respect to the original concentration, both biopolymers have been concentrated in separated phases as a result of the phase separation process. This explains why gels are obtained with WP 8% in the presence of pectin while WP 8% alone did not gel. In the presence of Ca2þ , the situation is much more complex owing to the competition of both biopolymers for Ca2þ : In the case of pectin of the lowest DM, the Ca2þ available to the protein is decreased so that the storage modulus (and hence the rigidity) of the gel is lower than with WP alone. This would make the properties of the composite system to strongly depend on the affinity of pectin to calcium. Parameters implied in the thermodynamics of phase separated mixed systems such as polymer molecular weight and concentration, heating and cooling kinetics, as well as ionic strength of the medium should be considered (Dickinson & McClements, 1995).
4. Conclusion Heating 8% WP solution mixed with LMP induced gel formation at lower protein concentration than normally observed for WP alone. Gelation of mixed solutions of WP and LMP occurs through a phase separation at pH 6.0. Competition for hydration and calcium partition between the two phases appeared to be an intrinsic part of the gelation process. Mixed gels firmness was affected by the concentration and the DM of the pectins used. Adding calcium increased gel firmness and generated strong protein aggregation.
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However, mixed gels were less brittle and presented a higher capacity to hold water. In order to obtain maximum firmness and minimum syneresis, the optimum WP/pectin/calcium ratio should exist. This ratio should include the right amount as well as the type of pectin, which will bind enough calcium to allow protein gelation without extensive aggregation; calcium/protein stoichiometry was found to be important in the WP gelation process (Sherwin & Foegeding, 1997).
Acknowledgements The financial support from FCAR, NSERC industrial chair and industrial partners: Agropur, CQVB, Novalait Inc. and Parmalat are gratefully acknowledged.
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Rheology, texture and microstructure of whey proteins/low methoxyl pectins mixed gels with added calcium Martin Beaulieua, Sylvie L. Turgeona,*, Jean-Louis Doublierb Centre de Recherche en Science et Technologie du Lait (STELA), Faculte! des Sciences de l’Agriculture et de l’Alimentation, ! Universite! Laval Pavillon Paul-Comtois, Sainte-Foy, Quebec, Canada G1K 7P4 b ! Institut National de la Recherche Scientifique (INRA), Centre de Recherche de Nantes, Laboratoire de Physicochimie des Macromolecules, B.P. 71627, 44316 Nantes Cedex 03, France a
Received 13 February 2001; accepted 4 August 2001
Abstract Gelation of a whey protein isolate mixed with various industrial low methoxyl pectins (LMPs) has been investigated. Ratios were adjusted to keep whey protein (WP) concentrations constant at 8%. Pectin and calcium concentrations were fixed at 0.1%, 0.5%, 1.0%, 1.5% and 0, 5, 10 mm; respectively. The pH was adjusted to 6.0. Heat treatment at 801C was used to induce WP gelation. Heating WP solution mixed with LMP allowed gel formation with lower protein concentration than normally observed; syneresis was also reduced. The type and proportion of pectin, as well as the calcium concentration affected gel hardness. Increasing the amount of pectin and the calcium concentration made mixed gels firmer. White gels were observed indicating aggregation of WPs. The gelation of the mixed systems appeared to have occurred through a phase separation and a competition between biopolymers for the binding of water and calcium. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Whey proteins; LM pectins; Calcium; Gel; Rheology
1. Introduction Food products are complex multi-component systems. Proteins and polysaccharides are largely responsible for the structural, mechanical, and physicochemical properties of foodstuffs (Tolstoguzov, 1991). As texture and stability are major criteria of food quality, investigations are primarily concerned with proteins and polysaccharides and their interactions in order to provide optimum structural quality to design new and attractive food (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). Mixed gels are formed from blends containing more than one gelling agent and may be classified into three types: interpenetrating, coupled, and phase-separated networks (Morris, 1986). Interpenetrating networks are formed when the two components gel separately and form independent networks. Both networks are continuous throughout the sample but any interaction *Corresponding author. Tel.: +1-418-656-2131 x4970; fax: +1-418656-3353. E-mail address: [email protected] (S.L. Turgeon).
between them is only topological (Morris, 1986). Coupled or complex coacervate networks are formed in the presence of favourable intermolecular interactions between the different types of polymers. In contrast, phase-separated gels are formed by incompatible polymers, where interactions between the different polymers are repulsive and/or when the two polymer types show varying affinity towards the solvent (Piculell & Lindman, 1992; Tolstoguzov, 1991, 1995). Whey proteins (WPs) are well known for their high nutritional value and versatile functional properties in food products (de Wit, 1998). Gelation is an important functional property of WPs (especially b-lactoglobulin) commonly used by the food industry. The gelation of WPs, as well as the factors affecting gelation, have been reviewed several times (Mulvihill & Kinsella, 1987; Stading & Hermansson, 1990, 1991; Gault & Fauquant, 1992; Hines & Foegeding, 1993; Aguilera, 1995; Boye, Alli, Ramaswamy, & Raghavan, 1997). Heat-induced gelation of WPs is affected by many factors, such as heating temperature, heating rate, pH, concentration, ionic strength, and presence of specific ions (Aguilera, 1995). Calcium greatly affects the functionality of WPs,
0958-6946/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 1 ) 0 0 1 2 7 - 3
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including their thermally induced gelation (Sherwin & Foegeding, 1997). WP gels made with dispersions containing 10–20 mm calcium have particulate microstructures, which give them an opaque appearance and low water-holding properties. Pectin is a linear polysaccharide built from polygalacturonic acid subunits that is partially esterified with methoxyl groups. The degree of methoxylation is very important as it influences pectin behaviour. It is defined as the average number of methoxyl groups per percent of the galacturonic acid units; this is commonly referred to as the degree of methoxylation (DM) and used to classify pectins into two groups. If the DM is greater than 50%, the pectin is referred to as high methoxyl pectin (HMP). Pectins with less than 50% DM are called low methoxyl pectin (LMP). LMP gels are formed as a result of interactions with calcium over a wide range of soluble solids and pH values. When calcium is added, cross-links begin to form between the pectin molecules causing a slight thickening of the solution. In practice, LMPs are gelled by using soluble calcium salts, which can be naturally present in the fruit or milk; or added as a dilute solution. LMP uses are varied, ranging from reduced sugar jams to soft confectionery jellies and spreadable processed cheeses. Very few studies exist on the interaction of pectins with WPs in mixed gel systems. The factors affecting the compatibility of bovine serum albumin (BSA) with HMP were reviewed by Semenova et al. (1991). Syrbe, Bauer, and Klostermeyer (1998) surveyed a large range of polysaccharides mixed with WPs including HMP. The rheology and microstructure of b-lactoglobulin and sodium polypectate mixed gels were investigated by Ndi, Swanson, Barbosa-Canovas, and Luedecke (1996). Considering that the WP and LMP gelation processes are affected by some common factors, behaviour of these biopolymers in mixed systems may be complementary. Reported here are the results of experimental investigations designed to study the effects of added calcium on ternary mixtures of whey protein isolate (WPI), LMP with different DM, and water.
2. Materials and methods
2.2. Preparation of solutions A stock WP solution ð16% w=vÞ was prepared by adding WP powder to deionized water and stirring gently for 2 h at room temperature. Solutions were then stored overnight at 41C to allow complete hydration. The pH of the protein solutions was adjusted to 6.0 using 1 n HCl: Pectin stock solutions (3% w=v) were made under strong stirring and heating at 601C until complete solubilization. In order to obtain desired calcium levels, other sets of WPI and pectin stock solutions were prepared by solubilizing in 5 and 10 mm (calcium ion) solutions of CaCl2 ; respectively. Blends, containing the right proportion of WP, different pectins, and calcium were mixed at 501C: The pH value was readjusted to 6.0 with 1 n NaOH: 2.3. Gel preparation Beakers (100 mL) were filled with 70 mL of final mixture and closed with aluminum foil that had been punctured for pinhole ventilation. Beakers were placed in an 801C water bath and heated for 30 min: Samples were then removed and cooled in a refrigerator where they were stored overnight at 41C: Gels were placed at 251C at least 2 h before texture analysis. 2.4. Visual aspect of the gel All heated samples were visually characterized by the same observer. Observations were made with the following characteristics in mind: solution or gel aspect (inversion test), clear or white opaque colouration, and notable aggregation. 2.5. Texture properties Gel texture parameters were determined by a Texture Analyzer TA XT-2 (Texture Technologies Corporation, NY, USA). The gels were penetrated with a 12 mm diameter cylinder probe (Ju & Kilara, 1998). A force– time curve was obtained at a crosshead speed of 60 mm min1 for a 10 mm displacement. The gel failure point was not reached. The resulting force–time curves were analyzed using the texture profile analysis (TPA) method.
2.1. Materials 2.6. Dynamic oscillatory measurements Dry WP isolates (92% w/w protein) were purchased from Davisco (Le Sueur, MN, USA). Citrus pectin samples (typical DM values: 28, 35, 40, 47, and 65, referred to as DE28, DE35, DE40, DE47, and DE65) were graciously provided by Hercules Co. DE65 was included in order to compare mixed gel behaviour made with LMP vs. HMP. CaCl2 was purchased from Sigma Chemical Company (St. Louis, MO, USA).
Small-deformation oscillatory measurements were performed, at a frequency of 1 Hz and a strain of 0.5%, on a controlled-strain rheometer (ARES 100 FRT, Rheometric Scientific, Piscataway, NJ, USA) equipped with a circulating water bath temperature controller. Couette device with a cup (35 mm diameter) and bob system (33 mm diameter, 35 mm length) was
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used. Warmed solutions ð501CÞ were poured directly into the heated measuring system ð801CÞ of the rheometer. This temperature was maintained for 30 min: Samples were then cooled to 201C (1:51C min1 ) and kept at least 30 min at this temperature. Samples were covered with a thin film of paraffin oil to avoid evaporation during heat treatment. 2.7. Confocal laser scanning microscope (CLSM) observations The CLSM system consists of a Carl Zeiss LSM 310 Confocal Laser Scanning Microscope attached to a Zeiss Upright Axioplan Microscope. Images acquisition and analysis were managed using Zeiss software (Version 3.95). The light source was an argon laser with emission maxima of 488 and 514 nm: Excitation was performed at 514 nm: Staining of the samples was carried out by dissolving 0.001% (w/w) of Fast Green FCF (Sigma, St. Louis, MO, USA) in the solutions prior to heat treatment. Stained solution was poured onto a hollow slide which was then hermetically sealed. Heat treatment (801C for 30 min) was applied directly on these sealed hollow slides. Slides were then cooled in a refrigerator where they were stored overnight at 41C: Samples were placed at 251C at least 2 h before observation. 2.8. Data analysis Three different variables were used to characterize the behaviour of the WP/LMP system, namely, LMP concentration; LMP DM; and calcium concentration according to a factorial experimental design. Samples were produced and analysed at random. Every experimental condition was produced three times and at least three replicates of each measurement were made.
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molecules (Mulvihill & Kinsella, 1988; Kuhn & Foegeding, 1991). However, higher calcium concentration generated strong protein aggregation and weaker gels (Xiong, 1992; Sherwin & Foegeding, 1997; Ju & Kilara, 1998). Calcium-free pectin samples behaved like viscous solutions. Dynamic oscillatory measurements confirmed the liquid-like behaviour; the loss modulus ðG00 Þ was superior to the storage modulus ðG0 Þ: Pectin gels were obtained when calcium was added (Fig. 1). Increasing the calcium concentration from 0 to 10 mm resulted in firmer gels for pectins DE28 and DE35. Calcium is responsible for the formation of cross-links; higher calcium concentrations will generate stronger interactions and firmer gels (Whistler & BeMiller, 1997). Pectins DE40 and DE47 did not produce gels that could be analysed by TA XT-2, indicating that the formation of a pectin network via calcium cross-links was low with these higher DM pectins. However, Garnier, Axelos, and Thibault (1993) showed that gel could be obtained from LMP with higher DM by increasing the calcium concentration. It is important to remember that commercial pectins exhibited some structural variations depending on extraction process and subsequent treatments. Thus, pectins can produce different types of gels with various maximum firmness, depending on the environmental conditions (Whistler & BeMiller, 1997). The firmness of the pectin gels was not only influenced by the DM and the calcium concentration as shown in Fig. 1; at equal calcium concentration (10 mm), DE35 produced firmer gels than DE28. 3.2. Visual aspect of the gels WP, pectin, and mixed solutions were translucents prior to heat-treatment. Mixing of WP and pectin solutions resulted in stable single-phase systems. This co-solubilization of both biopolymers can be explained
3. Results and discussion 3.1. Gelation of WP and pectin solutions Gelation of WP solutions was not observed and a liquid-like behaviour was obtained in the dynamic oscillatory measurements. These results are not surprising since the solutions used had only an 8% (w/w) protein concentration. Boye et al. (1997) noticed that WP gels which formed below 15% (w/v) were very weak. On the other hand, the addition of calcium (up to 10 mm) to the 8% w/w WP solutions induced the formation of a gel (hardness 4:9 N), which was aggregated. It is well known that the calcium level up to 10 mm increases the strength of whey protein gels (i.e., stiffens the matrix) due to electrostatic interactions with the negatively charged and unfolded protein
Fig. 1. Effect of calcium on firmness of pectin gels ð3% w=wÞ at pH 6.0.
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by the stabilization of the system through electrostatic repulsions (Cai & Arntfield, 1997). On the other hand, it has been suggested that this behaviour could be ascribed to the formation of weak soluble complexes that are stabilized by hydrogen bonds (Tolstoguzov, 1991). Heating WP solutions generally induced clear gel networks via hydrophobic interactions and disulphide bonds (Hines & Foegeding, 1993). This type of gel is generally referred to as a fine-stranded gel, which is formed when the degree of repulsion between proteins is large (Langton & Hermansson, 1992). Heating mixed solutions generated gelation for all samples except for those with the lowest pectin concentrations (0.1%) and in which no calcium was added. A white colouration was observed for all gels (Fig. 2). A white opaque gel is an indication of protein aggregation; this type of gel is most often referred to as a particulate gel (Langton & Hermansson, 1992). Furthermore, raising the temperature increases protein–protein interactions and leads to more aggregation. Adding calcium (5 and 10 mm) led to an increase in protein aggregation. WPs have an isoelectric point of 5.2 and a total negative charge at pH 6.0. Adding cations, such as calcium, shields the negative charges on the proteins and the amount of energy needed for aggregation as well as the distance between the protein molecules (Gault & Fauquant, 1992). It is likely that pectins which presented a stronger affinity for calcium (DE28 and DE35) reduced protein aggregation by binding a portion of the calcium. On the other hand, pectin that could not bind calcium (DE47) resulted in stronger aggregated gels.
3.3. Gel texture Heating WP solution mixed with LMP resulted in gel formation. Ndi et al. (1996) observed the same behaviour for a mixture of b-lactoglobulin and sodium polypectate. As seen in Fig. 3, gel firmness was not only affected by the amount of LMP used, but by its type too. Mixed gels made with DE28 increased in firmness with the addition of pectin (from nil at 0.1% to 0:165 N at 1.5%). Similar results were obtained for DE35 and DE47 (maximum firmness of 0.187 and 0:278 N). However, DE40 generated stronger gels at all levels. The kinetics of gel formation of mixtures containing 8% WP and 1% LMP are illustrated in Fig. 4 from dynamic measurements. Gel formation was monitored in three steps. First, the temperature was maintained at 801C for 30 min; second, the temperature was decreased linearly from 801C to 201C at 1:51C min1 ; and finally, this temperature was maintained for 30 min: WP
Fig. 2. Visual aspect of heated mixed samples made with WP (8%) and LMP with different DM for different levels of added calcium (0, 5, and 10 mm). Liquid-like samples shown as white; white coloured solidlike samples shown as grey, and strongly aggregated solid-like samples shown as black.
WP 8% + DE28 1% WP 8% + DE35 1% WP 8% + DE40 1% WP 8% + DE47 1% WP 8% + DE65 1%
Temperature (˚C)
G' ( Pa )
Fig. 3. Firmness of mixed gels (pH 6.0) made with whey proteins ð8% w=wÞ and increasing concentrations of pectin DE28, DE35, DE40, and DE47 (0.1%, 0.5%, 1.0%, and 1:5% w=w).
Time (min) 0
Fig. 4. G profile for mixed gels (pH 6.0) made with 8% ðw=wÞ WP and pectins DE28, DE35, DE40, DE47, and DE65 ð1% w=wÞ:
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samples, with no added pectin, did not gel; G0 profile (not shown) was linear. Mixed gels with the five pectin samples resulted in similar profiles with a rapid increase for the first 5 min at 801C followed by a levelling. Upon cooling down to 201C an increase of G0 from B100 to B1000 Pa was measured. Overall, no differences between samples were observed. These results are consistent with the compression tests for a concentration of 1% added LMP. Mixed gels made with LM pectins are in all likelihood comparable in their structures and their solid-like behaviours after the heatinduced gelation. Considering the fact that these gels were made without adding calcium, it may seem reasonable to assume that WP is the only gelling agent in those mixed solutions. Higher pectin concentrations in the system will result in an increase in the amount of solvent that could be entrapped in the pectin phase, causing concentration of the protein phase and an increased gel firmness. However, we should also consider that water partition changed during the gelation process; Dickinson and McClements (1995) have mentioned that the solvent could be redistributed between the phases as a result of the polymeric conformational changes that accompany gelation, which suggest that the results obtained in this study could in fact be in attributed to the hydration level of the proteins during gelation as well as the solvent partition in each phase after the entire gelation process. On the other hand, one could be tempted to directly compare the results of texture analysis with those of dynamic measurements. Dynamic measurements were made using a small deformation (strain 0.5%) and texture analysis was made with a 25% deformation; small differences in the gel structure, which are not seen by small deformation measurements, can be detected by large deformation measurements (Stading & Hermansson, 1991). Large deformation measurements are related to the size of inhomogeneities in a material structure (Tang, McCarthy, & Munro, 1995).
Fig. 5. Firmness of mixed gels (pH 6.0) made with whey proteins ð8% w=wÞ; increasing concentrations of pectins DE28, DE35, DE40, and DE47 (0.1%, 0.5%, 1.0%, and 1:5% w=w), and 10 mm of added calcium.
ing the fact that large pores are the weakest part of a gel network (Stading, Langton, & Hermansson, 1993). The kinetics of gel formation of mixtures containing 8% WP and 1% pectin in the presence of 10 mm calcium are illustrated in Fig. 6 from dynamic measurements and compared to WP 8% alone. The four pectin samples yielded similar profiles as without Ca2þ (Fig. 4). These were quite similar to that of WP 8% suggesting that mixed gels are mainly generated by the WP phase. However, large differences were experienced in relation to the DM of the pectin sample; the lower the DM, the higher the storage modulus. As compared to Fig. 4, these values were much higher than in the absence of Ca2þ for the lowest DM while they were lower for the highest one. This shows clearly that Ca2þ plays a major role in the properties of the mixed gels. However, the
WP 8% + Ca10 WP 8% + DE35 1% + Ca10 WP 8% + DE40 1% + Ca10 WP 8% + DE47 1% + Ca10 WP 8% + DE65 1% + Ca10
Temperature (˚C)
Calcium increased the firmness of mixed gels made with DE28, DE35, and DE40 (Fig. 5); this increase of firmness was at least by a factor 10. Maximum firmness was reached for mixed gels made of DE35 regardless of the pectin content. Mixed gels presented a lower firmness than WP gels (hardness 4:9 N) obtained with the same calcium concentration. However, mixed gels were less brittle and retained more water; a decrease in the syneresis was also observed. Lower pectin concentrations as well as those made with pectins with a lower affinity for calcium, resulted in weaker gels. Higher calcium concentration was available for the WP phase generating strong aggregation and weak gels, consider-
G' (Pa)
3.4. Effect of added calcium
Time (min) Ti
Fig. 6. G0 profile for mixed gels (pH 6.0) made with 8% ðw=wÞ WP, pectins DE35, DE40, DE47, and DE65 ð1% w=wÞ; and 10 mm of added calcium.
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Fig. 7. CLSM observations of whey proteins gel structures stained by Fast Green FCF ð0:001% w=wÞ: (a) WP ð8% w=wÞ with 10 mm of added calcium resulted in low aggregation; (b) WP ð8% w=wÞ; and pectin DE47 ð1% w=wÞ; without added calcium shows aggregation; (c) WP ð8% w=wÞ; pectin DE47 ð1% w=wÞ; with 10 mm of added calcium shows strong aggregation. Protein phase is light grey (scale bar ¼ 10 mm).
storage modulus of the mixed systems was much lower (between 600 and 3000 Pa at 201C; depending upon the DM) than for WP alone ð6300 PaÞ at the same WP concentration. Addition of pectin resulted in a decrease of the rigidity of the protein gel in the presence of Ca2þ : Overall, these results with 1% LMP confirmed texture analysis for mixed gels as illustrated in Fig. 5. Indeed, adding calcium increased WP/WP interactions. High calcium concentration likely generated interactions between proteins by bridging negatively charged carboxyl groups, which increased the tendency towards coagulation rather than gelation (Zirbel & Kinsella, 1988). In the case of mixed systems, LMP likely compete with proteins with respect to Ca2þ : Therefore, part of Ca2þ is not available to the protein gel which may explain the decrease in the storage modulus of mixed gels.
3.5. CLSM observations Fig. 7 shows typical micrographs of WPs ð8% w=wÞ with 10 mm of added calcium (Fig. 7a); WPs ð8% w=wÞ with 1% pectin (DE47) (Fig. 7b); and WPs ð8% w=wÞ with 1% pectin (DE47) and 10 mm of added calcium (Fig. 7c). This illustrates clearly that quite different systems are obtained. In case of WP alone (Fig. 7a), proteins appear in white, almost regularly distributed. Addition of pectin (no Ca2þ added) apparently resulted in a stronger aggregation of proteins (Fig. 7b). A similar observation could be made in the presence of Ca2þ (Fig. 7c), the proteins being apparently aggregated. All pectins presented the same aggregation pattern. All mixed systems presented comparable type of aggregation for the same pectin and calcium concentration. These CLSM observations strongly suggest that WP and LMP are located in two different phases as a result of thermodynamic incompatibility, this phenomenon being
known to occur with a large number of protein/ polysaccharide systems (Grinberg & Tolstoguzov, 1997). Rheological measurements can therefore be interpreted from a phase separation process. In the absence of Ca2þ ; only the protein-enriched phase is gelled in a tridimensional porous network while the pectin-enriched phase is a liquid dispersion within this network. With respect to the original concentration, both biopolymers have been concentrated in separated phases as a result of the phase separation process. This explains why gels are obtained with WP 8% in the presence of pectin while WP 8% alone did not gel. In the presence of Ca2þ , the situation is much more complex owing to the competition of both biopolymers for Ca2þ : In the case of pectin of the lowest DM, the Ca2þ available to the protein is decreased so that the storage modulus (and hence the rigidity) of the gel is lower than with WP alone. This would make the properties of the composite system to strongly depend on the affinity of pectin to calcium. Parameters implied in the thermodynamics of phase separated mixed systems such as polymer molecular weight and concentration, heating and cooling kinetics, as well as ionic strength of the medium should be considered (Dickinson & McClements, 1995).
4. Conclusion Heating 8% WP solution mixed with LMP induced gel formation at lower protein concentration than normally observed for WP alone. Gelation of mixed solutions of WP and LMP occurs through a phase separation at pH 6.0. Competition for hydration and calcium partition between the two phases appeared to be an intrinsic part of the gelation process. Mixed gels firmness was affected by the concentration and the DM of the pectins used. Adding calcium increased gel firmness and generated strong protein aggregation.
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However, mixed gels were less brittle and presented a higher capacity to hold water. In order to obtain maximum firmness and minimum syneresis, the optimum WP/pectin/calcium ratio should exist. This ratio should include the right amount as well as the type of pectin, which will bind enough calcium to allow protein gelation without extensive aggregation; calcium/protein stoichiometry was found to be important in the WP gelation process (Sherwin & Foegeding, 1997).
Acknowledgements The financial support from FCAR, NSERC industrial chair and industrial partners: Agropur, CQVB, Novalait Inc. and Parmalat are gratefully acknowledged.
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