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Rheological behaviour of an amide pectin
Journal of Food Engineering 55 (2002) 123–129 www.elsevier.com/locate/jfoodeng
Rheological behaviour of an amide pectin Manuel Alonso-Moug an a, Francisco Meijide a, Aida Jover a, ~ez b,*, Jose V Eugenio Rodrıguez-N un azquez-Tato a a b
Departamento de Quımica Fısica, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain Departamento de Fısica Aplicada, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain Received 2 March 2001; accepted 6 June 2001
Abstract Forced-oscillation measurements of aqueous dispersions of an amide pectin were carried out at pH 3.83 in the presence of 50% sucrose. The results show a predominant viscous behaviour corresponding to the so-called terminal region of the general mechanical spectrum of biopolymer solutions. The polymer chains have been described by the bead-spring model with dominant hydrodynamic interactions among beads. At pH 3.31, samples are more elastic than viscous and the mechanical spectra reveal an entanglement network (weak gel), although not dense enough to store the stress during a long period of time after having been deformed. At low pectin concentration, the plot of loss moduli versus frequency shows a minimum that suggest monodisperse junction zones and the existence of two regions with different relaxation times. The short relaxation process corresponds to movements of galacturonic units inside the junction zones (local configuration adjustment), and the longer one to movements of the junction zone as a whole (rearrangements of couplings through entanglement and cross-linking). The comparison of the results obtained here at different sucrose concentrations and temperatures with those for methoxy pectins, suggests that the hydrogen bonds formed by amide groups have an important role in the formation of the gel. The role of the hydrophobic interactions in the stability of the junction zones has also been discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Amide pectin; Rheological properties; Viscoelastic behaviour; Gelation
1. Introduction Pectins are structural polysaccharides of vegetable origin. The polymer backbone is based on (1 ! 4)linked a-D -galacturonate residues interrupted by insertion of (1 ! 2)-linked rhamnosyl residues where neutral sugar side chains are attached. During the industrial extraction of pectins the degree of esterification is modified resulting in low or high methoxy pectins (LMP, HMP) depending on the degree of esterification. LMP are gelled by controlled addition of calcium ions. The gelation of both LMP and HMP can also be induced by lowering the pH and adding co-solutes as sucrose, glucose or fructose (Morris, 1986). Therefore, pectins are used as gelling and thickening agents in the food industry.
*
Corresponding author. Tel.: +34-82-223-325x24080; fax: +34-82224-904. ~ez). E-mail address: [email protected] (E. Rodrıguez-N un
Different intermolecular interactions, acting as driving forces for association, are involved in the gelation of pectins. For instance, the gelation of LMP in the presence of calcium ions, involves electrostatic interactions between the cations and the negative charged cavities formed by polymer chains, where the cations are inserted (egg box model). This process is reminiscent of the gelation of alginates (Clark & Ross-Murphy, 1987), a non-surprising fact since galacturonic blocks in pectates are almost mirror images of guluronic blocks in alginates. According to Kohn (1975), the minimum sequence length required for a stable association is seven calcium ions bound to consecutive unesterified residues on each of the participating strands. The egg box model has been supported by different experimental techniques by various authors (Gidley, Morris, Murray, Powell, & Rees, 1979; Powell, Morris, Gidley, & Rees, 1982). The HMP gelation takes place in conditions where electrostatic repulsions and water activity are reduced, i.e., in the presence of co-solutes at low pH. At this stage, the driving forces are hydrogen bonding and
0260-8774/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 2 ) 0 0 0 2 6 - 2
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hydrophobic interactions. It appears that the crosslinking at the junction zones takes place through the formation of aggregates of chains of varying sizes (Clark & Ross-Murphy, 1987), with no definite stoichiometry involved. Although other co-solutes as glycerol or ammonium sulfate can also be used (Rees, 1969), the possibility of a specific role for sucrose or other polyols, cannot be ruled out, i.e., the function of the co-solute seems more complex than simple competition for the available water (Ptitchkina, Danilova, Doxastakis, Kasapis, & Morris, 1994). Classical techniques have extensively been used to characterise the rheological behaviour of HMP dispersions and gels. These methods (Ikkala, 1986) imply large-deformation measurements and allow the determination of full stress–strain profiles and failure properties. Other static viscoelastic methods, such as creep (Dahme, 1985; Kawabata, 1977; Plashchina, Fomina, Braudo, & Tolstoguzov, 1979) and stress relaxation (Comby, Doublier, & Lefebvre, 1986) have also been used. More recently, modern controlled-stress rheometers have measured the performance of dynamic tests under small deformation (Beveridge & Timbers, 1989; Dahme, 1992; Ikkala, 1986; Lopes da Silva & Goncßalves, 1994; Lopes da Silva, Goncßalves, & Rao, 1993, 1994; Rao & Cooley, 1993, 1994; Rao, Van Buren, & Cooley, 1993). These non-destructive methods give a better insight into the rheological behaviour of the systems under investigation. On the other hand, since structural changes, ionic strength, pH and temperature affect the intermolecular interactions in different ways, they will also have consequences in the rheological behaviour of the samples. Therefore, studies of varying temperature (Beveridge and Timbers, 1989; Dahme, 1992; Lopes da Silva et al., 1994; Oakenfull & Scott, 1984; Rao & Cooley, 1993, 1994; Rao et al., 1993), co-solute (Lopes da Silva & Goncßalves, 1994; Rao & Cooley, 1994), degree of esterification (Lopes da Silva et al., 1993, 1994), and side branches in pectins (Hwang & Kokini, 1992), curing time of gels (Lopes da Silva & Goncßalves, 1994; Rao & Cooley, 1993; Rao et al., 1993) and the gelation process itself (Beveridge & Timbers, 1989; Dahme, 1992; Lopes da Silva & Goncßalves, 1994; Rao et al., 1993) have been carried out. Papers by Lopes da Silva and Goncßalves (1994), Lopes da Silva et al. (1993, 1994), and Oakenfull and Scott (1984) are particularly important for the purposes of this work. Oakenfull and Scott have concluded that ‘‘the junction zones consist of two adjacent segments of polysaccharide chain varying in length from 18 to about 250 galacturonic acid units, increasing with the degree of methoxylation’’. This well-defined stoichiometry contrasts with less precise situation commented on above. Such a discrepancy is explained in terms of the different materials used and/or different gelation conditions (Clark & Ross-Murphy (1987)). Although, the forma-
tion of hydrogen bonds contributes as much as twice the hydrophobic interactions in stabilising the junction zones, it is not sufficient enough to stabilise them. The dynamic rheological measurements carried out by Lopes da Silva et al. (1993) with LMP and HMP dispersions, show that both pectins have a quite different behaviour. This has been related to a lower intermolecular association and a higher hydrodynamic volume (due to the higher charge density) of LMP. In studying the influence of temperature on the storage and loss moduli (Lopes da Silva et al., 1994), they also observed that the frequency-temperature or time-temperature superposition principle fails. So, structural changes in the aggregates with different relaxation mechanisms and different dependence on temperature, have to be involved. These authors (Lopes da Silva & Goncßalves, 1994) have also studied the influence of the temperature on the nature of the HMP/sucrose networks. The observed dependence for the storage modulus was interpreted by assuming non-permanent cross-links and by the opposite effects of temperature on the hydrophobic interactions and hydrogen bonding that stabilise them. The substitution of some ester by amide groups offers a great chance to investigate some points mentioned by Lopes da Silva et al. (1993, 1994), since the presence of amide groups will facilitate the formation of hydrogen bonds. To carry out the rheological study, we have chosen an amide pectin with both kinds of groups. The results reported in this work by the authors include the effects of pectin and sucrose concentrations, pH and temperature.
2. Experimental The study was made on a commercial amide pectin Genu LM-104 AS (Copenhagen Pectin Factory). According to the supplier the degree of methoxyl and amide groups in the pectin molecule were 30% and 20%. The samples were prepared as follows: Pectin and sucrose were added to solutions at pH 3.83 and 3.31 of lactic/lactate buffer (Fluka) and sodium azide (Merck, for preserving microbial growth, 80 ppm) and, finally, the appropriate weight of water (Milli-Q) was added. For one hour and half the dispersion was magnetically stirred and slowly heated until the temperature reached 87 °C (held at this temperature less than 5 min) in a sealed flask to obtain a homogeneous solution. The samples were kept at room temperature (22–25 °C) for 24 h, where they have gelled. Dynamic rheological measurements were carried out in a Haake RS100 rheometer, with a cone and plate geometry (diameter 35 mm, angle 4°). The samples were transferred to the plate and, once in the measurement position, their contour surface was covered with a thin layer of paraffin oil to avoid dehydration. The temper-
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ature was kept constant by circulating water (through the bottom plate) from a Haake K15 thermocryostat. All dynamic measurements were done in the linear viscoelastic region, to assure that the calculated parameters correspond to an intact network structure. The operation frequencies were varied at least by a factor of 103 . Duplicated experiments showed an excellent reproducibility. Studies of some samples were repeated after two months from their preparation. The mechanical spectra (results not shown) were identical to those recorded after 24 h. Therefore gel stability and curing were completed after one day. The following moduli were determined, in forcedoscillation experiments, stress (s) and strain (c) are related via the complex shear modulus, G : G ¼ sðtÞ=cðtÞ
ð1Þ
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Fig. 2. Influence of x on the storage and loss moduli for samples of 0.75% (w/w) pectin at pH 3.83.
Samples with a constant sucrose content of 50% w/w and pectin concentration in the range 0.5–2.5% w/w were studied at this pH value and at 25 °C. Fig. 1 shows
that at operation frequency of 0.628 s1 (x ¼ 3:946 rad s1 ) the complex shear modulus, G , is constant in the range of stress 0.1–2 Pa, which is included in the linear viscoelastic region. Therefore, all experimental results were obtained under applied stresses from 0.5 to 2 Pa. As an example, Figs. 2 and 3 show the mechanical spectrum ðG0 and G00 versus xÞ for pectin concentrations of 0.75% and 2.5%. For both concentrations, G00 > G0 along the complete time scale, indicating that the samples are more viscous than elastic. It must be noticed that the range of frequencies covered in both concentrations is quite different. For the dilute system (Fig. 2), the storage modulus has a quadratic dependence with x (slope 2:00 0:02), while the loss modulus depends linearly with x (slope 0:98 0:01). Consequently, the complex viscosity g is frequency independent. This is the so-called terminal region, in which the polymer concentration belongs to the semi-dilute regime, typical for biopolymer
Fig. 1. Dependence of the complex shear modulus with the applied stress at pH 3.83.
Fig. 3. Influence of x on the storage and loss moduli for samples of 2.5% (w/w) pectin at pH 3.83.
which may be partitioned into real and imaginary components: G ¼ G0 þ iG00
ð2Þ
where G0 and G00 are the storage and loss moduli, respectively. The ratio G00 =G0 is the tangent of the phase angle. Alternatively, the complex viscosity, g , can be defined as: g ¼ G =f
ð3Þ
where f is the operation frequency (x ¼ 2pf ). g measures the total resistance to a dynamic shear. 3. Results and discussion 3.1. Results at pH 3.83
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dispersions revealing an entangled system. This behaviour was found for galactomannan solutions and other disordered random-coil polysaccharides (Robinson, Ross-Murphy, & Morris, 1982; Richardson & RossMurphy, 1987), and for locust bean gum and LMP, 3%, without added sugars (Lopes da Silva et al., 1994). For a pectin concentration of 2.5% and the highest frequencies, Fig. 3 shows that both moduli are similar in magnitude (G00 being yet greater than G0 ). Fitting the experimental data to a power law in x give exponent values of 0:61 0:01 and 0:67 0:01 for G0 and G00 , respectively. These results can be understood under the beadspring models for linear flexible random coils. Rouse’s theory does not consider hydrodynamic interactions among beads while in the theory of Zimm the hydrodynamic interaction is dominant. In the limit of low frequencies, both theories give the same result, predicting the well-known quadratic and linear dependencies for G0 and G00 , respectively. At higher frequencies, they predict slopes for both moduli of 1/2 (Rouse theory) and 2/3 (Zimm theory). Therefore, in the present experimental conditions, samples obey the Zimm model, which means that, in this region, the molecular relaxation implies a gradual stress relaxation in which the brownian motion moves the chain segments farther and farther along the backbone polymer to reach their final equilibrium configuration. Sometimes in biological polymers exist a transition from viscous fluid to a relatively elastic system, that is characterised by a crossover of the storage and loss moduli and can be achieved by increasing either the angular frequencies employed or the pectin concentration. Lopes da Silva et al. (1993) have observed such a transition in LMP, without sugars added, but at higher pectin concentrations; for HMP, in similar conditions, they estimated a crossover frequency >63 rad s1 for pectin contents between 2.8% and 3.9%. In our experimental conditions the transition from a viscous fluid to an elastic system was not found. The behaviour of pectin samples with intermediate concentrations is similar to those shown in previous figures, with power law exponents in the mentioned range. Furthermore, the magnitude of these moduli increase with pectin concentration. All this suggest that increasing the pectin concentration promotes the transition from a viscous system to a more entangled solution showing intermolecular interactions occurring on long time scales (Frangou, Morris, Rees, Richardson, & Ross-Murphy, 1982; Rocherfort & Middleman, 1987).
Fig. 4. Dependence of the complex shear modulus with the applied stress at pH 3.31.
those at pH 3.83. Now at operation frequency of 4.279 s1 (x ¼ 26:886 rad s1 ) the linear viscoelastic region extends at least up till 15 Pa (Fig. 4). Consequently, all the following results were obtained under an applied stress in the range of 2–10 Pa. Figs. 5 and 6 show the variation of G0 and G00 moduli with x for different pectin concentrations. For all samples, the storage modulus is one order of magnitude greater than the loss modulus, and G0 is less dependence on x that G00 . This behaviour corresponds to the socalled entanglement network with a high-frequency rubbery plateau for G0 , typical of more entangled (with respect to the previous pH) biopolymer solutions. The frequency dependence of both moduli implies that the complex viscosity g , diminishes with x, having found that the exponent of a potential law is independent on the pectin concentration, with a value of (0.8– 0.9). Similar qualitative results were found for gels of HMP/sucrose (Lopes da Silva & Goncßalves, 1994) and HMP/glucose and fructose (Rao & Cooley, 1994).
3.2. Results at pH 3.31 The range of pectin concentration and the sucrose content studied at pH 3.31 and at 25 °C were the same as
Fig. 5. Dependence on x of the storage modulus for samples at pH 3.31 and different pectin concentrations.
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Fig. 6. Dependence on x of the loss modulus for samples at pH 3.31 and different pectin concentrations.
These facts suggest that the samples have a solid-like behaviour, but since the rubbery plateau is not completely reached, the physical entanglement is not dense enough to store the stress during a long period of time after having been deformed. According with the classification criterion proposed by Clark and Ross-Murphy (1987) these samples are weak gels, while those prepared at pH 3.83 are entanglement network systems. Potentiometric titration of the pectin allowed us to estimate a value of 3.6 for the pKa . The carboxylic groups of the molecule are less ionised at the lower pH studied, and, therefore, the repulsive electrostatic interactions are reduced. It causes an increase in the number and density of the entanglements, implying a lower relative effect of some broken junctions on the G0 value, and a lower time necessary to create a new entanglement zone immediately after breaking the old one. This suggests an explanation of the observed differences in the linear viscoelastic region at both pH values. For the most diluted samples, G00 shows a minimum at x 6¼ 0:02 s1 (Fig. 7), which is normally of polymers that exhibit effects of entanglement coupling (te Nijenhuis, 1997). Following Leung and Goddard (1991) this minimum separates two regions with different relaxation processes. We suggest the short relaxation process corresponding to movements of galacturonic units inside the junction zones (local configuration adjustment), and the longer one to movements of the junction zone as a whole (rearrangements of couplings through entanglement and cross-linking). On the other hand, maybe the existence of the minimum evidences that the junction zones are rather monodisperse, in spite of the molecular mass polydispersity of the polymer. This conclusion is in agreement with the narrow molecular mass distribution observed by Powell et al. (1982) for the homopolymer blocks length in non-amidated pectins. However the earlier comments are spec-
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Fig. 7. Storage and loss moduli at pH 3.31 as a function of frequency for a sample of 0.75% (w/w) pectin.
ulative and it should be needed more information in addition to rheological data to understand the physical basis of such behaviour. Fig. 8 resumes the results obtained for a polymer concentration of 2% at different sucrose contents. For sucrose contents at 40%, G00 > G0 and g has a slight dependence with x. The opposite behaviour is observed at concentrations higher than 50%, where G0 > G00 and the mechanical spectrum is the typical one of a polysaccharide weak gel. It may be concluded that in the 40– 50% region there is a transition from an entanglement network to a weak gel, as a result of an increase in the polymer entanglement when the sucrose content raises. This strengthening of the pectin entanglement may be caused by three main effects: (a) the stabilising effect of the disaccharide on the hydrophobic interactions between ester methyl groups, (b) the dehydrating effect of the sugar and (c) the formation of new hydrogen bonds through the decrease in the water activity caused by the
Fig. 8. Influence of the sucrose content on the storage and loss moduli of samples with 2% (w/w) pectin at pH 3.31.
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presence of more co-solute molecules. As it was noticed in the Section 1, the formation of hydrogen-bonded bridges through sucrose molecules between pectin chains is not probable. Back, Oakenfull, and Smith (1979) have shown that the first effect explains the higher thermal stability of globular proteins in the presence of certain sugars or polyols. Oakenfull and Scott (1984) have explained the stability of HMP/sugars gels by this effect, which would be dependent on the molecular geometry of the sugar. These authors have also argued that the traditional view that sugar acts non-specifically by partly dehydrating (point b) the pectin molecules (Rees, 1969) does not seem probable. However, the network is not formed if the water activity is not reduced. Aqueous sucrose is apparently a poorer solvent than water for pectin. In terms of osmosis, the network cannot be formed in the absence of sucrose, because it could not withstand the osmotic pressure of the pure solvent. In other words, the presence of sucrose makes the water less able to solvate the polysaccharide chains and any tendency for them to separate from solution (to form junction zones, for example) is enhanced (Rees, 1969). Under this situation, hydrogen bonding through amide and acid groups between chains (point c) may be facilitated. Further insights on the role of these effects have been deduced by studying the influence of the temperature. For a pectin concentration of 0.75% and sucrose 50%, the influence of temperature was studied from 15 to 45 °C by analysing different samples at each temperature. The dependence of G0 , G00 and g with x is the typical of biopolymer gels near the rubbery plateau. Fig. 9 shows a plot of the storage modulus versus temperature at x ¼ 0:20 rad s1 . Identical profiles are also observed at other frequencies. The rise in G0 is slow within the temperature interval 45–35 °C, it remains constant between 35 and 25 °C. At lower temperatures a remarkable increment is observed (the ratio of G0 (25 °C)/G0 (15 °C) is 6).
Fig. 9. Storage modulus at x ¼ 0:20 rad s1 as a function of temperature.
Similar qualitative results, although with much lower effects, were also observed by Lopes da Silva and Goncßalves (1994) in studying high-methoxyl (64%) pectin gels in the presence of 60% sucrose. Oakenfull and Scott (1984) have also reported similar trends for the rupture strength of other HMP gels (methoxyl degree, 69.7%; sucrose, 55%). It is known that the hydrogen bonds are weakened (Joesten & Schaad, 1974) and the hydrophobic interactions are strengthened (Ben-Naim, 1980; Oakenfull & Fenwick, 1977) when the temperature increases. It has been argued (Lopes da Silva & Goncßalves, 1994; Oakenfull & Scott, 1984) that these two opposite effects explain the observed profile. At low temperatures hydrogen bonds are favoured, reinforcing the junction zones and the storage modulus increases. At higher temperatures, the loss of hydrogen bond association is compensated with the reinforced hydrophobic interactions. Here the electrostatic interactions are not considered because their contribution should be small due to the low degree of ionisation of the carboxylic groups of the polysaccharide. The greater effect in the G0 values on cooling the samples found for the amidate pectin with respect to the esterified ones (Lopes da Silva & Goncßalves, 1994; Oakenfull & Scott, 1984) must be related with the higher ability of the amide group to form the hydrogen bonds which stabilise the junction zones. Therefore, the amide groups might play an important role on gelation, namely promoting H-bonding.
Acknowledgements The authors thank the DGICYT (Spain) for financial support (Project PB90-0758).
References Back, J. F., Oakenfull, D., & Smith, M. B. (1979). Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry, 18, 5191–5196. Ben-Naim, A. (1980). Hydrophobic interactions. New York, USA: Plenum Press, p. 185. Beveridge, T., & Timbers, G. E. (1989). Small amplitude oscillatory testing (SAOT). Application to pectin gelation. Journal of Texture Studies, 20, 317–324. Clark, A. H., & Ross-Murphy, S. B. (1987). Structural and mechanical properties of biopolymer gels. Advances in Polymer Science, 83, 57–192. Comby, S., Doublier, J. L., & Lefebvre, J. (1986). Strees-relaxation study of high-methoxyl pectin gels. In G. O. Phillips, D. J. Wedlock, & P. A. Williams (Eds.), Gums and stabilisers for the food industry 3 (pp. 203–212). London, UK: Elsevier Applied Science. Dahme, A. (1985). Characterization of linear and nonlinear deformation behavior of a weak standard gel made from high-methoxyl citrus pectin. Journal of Texture Studies, 16, 227–239. Dahme, A. (1992). Gelpoint measurements on high-methoxyl pectin gels by different techniques. Journal of Texture Studies, 23, 1–11.
M. Alonso-Mougan et al. / Journal of Food Engineering 55 (2002) 123–129 Frangou, S. A., Morris, E. R., Rees, D. A., Richardson, R. K., & Ross-Murphy, S. B. (1982). Molecular origin of xanthan solution rheology: effect of urea on chain conformation and interactions. Journal of Polymer Science. Polymer Letters Edition, 20, 531– 538. Gidley, M. J., Morris, E. R., Murray, E. J., Powell, D. A., & Rees, D. A. (1979). Spectroscopy and stoichiometric characterization of the calcium-mediated association of pectate in gels and in the solid state. Journal of Chemical Society. Chemical Communications, 22, 990–992. Hwang, J., & Kokini, J. L. (1992). Contribution of the sides branches to rheological properties of pectins. Carbohydrate Polymers, 20, 41–50. Ikkala, P. (1986). Characterization of Pectins. A different way of looking at pectins. In G. O. Phillips, D. J. Wedlock, & P. A. Williams (Eds.), Gums and stabilisers for the food industry 3 (pp. 253–267). London, UK: Elsevier Applied Science. Joesten, M. D., & Schaad, L. J. (1974). Hydrogen bonding (p. 182). New York, USA: Marcel Dekker. Kawabata, A. (1977). Studies on chemical and physical properties of pectic substances from fruits. Memoirs of the Tokyo University of Agricultural, 19, 115–200. Kohn, R. (1975). Ion binding on polyuronates. Alginate and pectin. Pure and Applied Chemistry, 42, 371–397. Leung, P. S., & Goddard, E. D. (1991). Gels from dilute polymer/ surfactant solutions. Langmuir, 7, 608–609. Lopes da Silva, J. A., & Goncßalves, M. P. (1994). Rheological study into the ageing process of high methoxy pectin/sucrose aqueous gel. Carbohydrate Polymers, 24, 235–245. Lopes da Silva, J. A., Goncßalves, M. P., & Rao, M. A. (1993). Viscoelastic behaviour of mixtures of locust bean gum and pectin dispersions. Journal of Food Engineering, 18, 211–228. Lopes da Silva, J. A., Goncßalves, M. P., & Rao, M. A. (1994). Influence of temperature on the dynamic and steady-shear rheology of pectin dispersions. Carbohydrate Polymers, 23, 77–87. Morris, V. J. (1986). Gelation of polysaccharide. In J. R. Mitchell, & D. A. Ledward (Eds.), Functional properties of food macromolecules. London, UK: Elsevier Applied Science. Oakenfull, D. G., & Fenwick, D. E. (1977). Thermodynamics and mechanism of hydrophobic interaction. Australian Journal of Chemistry, 30, 741–752.
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Oakenfull, D. G., & Scott, A. (1984). Hydrophobic interaction in the gelation of high methoxyl pectins. Journal of Food Science, 49, 1093–1098. Plashchina, I. G., Fomina, O. A., Braudo, E. E., & Tolstoguzov, V. B. (1979). Creep study of high-esterified pectin gels. I. The creep of saccharose-containing gels. Colloid and Polymer Science, 257, 1180–1187. Powell, D. A., Morris, E. R., Gidley, M. J., & Rees, D. A. (1982). Conformations and interactions of pectins. II. Influence of residue sequence on chain association in calcium pectate gels. Journal of Molecular Biology, 155, 517–531. Ptitchkina, N. M., Danilova, I. A., Doxastakis, G., Kasapis, S., & Morris, E. R. (1994). Pumpkin pectin: gel formation at unusually low concentration. Carbohydrate Polymers, 23, 265–273. Rao, M. A., & Cooley, H. J. (1993). Dynamic rheological measurement of structure development in high-methoxyl pectin/fructose gels. Journal of Food Science, 58, 876–879. Rao, M. A., & Cooley, H. J. (1994). Influence of glucose and fructose on high-methoxyl pectin gel strength and structure development. Journal of Food Quality, 17, 21–31. Rao, M. A., Van Buren, J. P., & Cooley, H. J. (1993). Rheological changes during gelation of high-methoxyl pectin/fructose dispersions: effect of temperature and aging. Journal of Food Science, 58, 173–176,185. Rees, D. A. (1969). Structure, conformation and mechanism in the formation of polysaccharide gels and networks. Advances in Carbohydrate Chemistry and Biochemistry, 24, 267–332. Richardson, R. K., & Ross-Murphy, S. B. (1987). Non-linear viscoelasticity of polysaccharide solutions. 2. Xanthan polysaccharide solutions. International Journal of Biological Macromolecules, 9, 257–264. Robinson, G., Ross-Murphy, S. B., & Morris, E. R. (1982). Viscositymolecular weight relationships, intrinsic chain flexibility, and dynamic solution properties of guar galactomannan. Carbohydrate Research, 107, 17–32. Rocherfort, W. E., & Middleman, S. (1987). Rheology of xanthan gum: salt, temperature, and strain effects in oscillatory and steady shear experiments. Journal of Rheology, 31, 337–369. te Nijenhuis, K. (1997). Thermoreversible networks. Viscoelastic properties and structure of gels. Advances in polymer science (vol. 30) (p. 267). Berlin, Germany: Springer-Verlag.
Rheological behaviour of an amide pectin Manuel Alonso-Moug an a, Francisco Meijide a, Aida Jover a, ~ez b,*, Jose V Eugenio Rodrıguez-N un azquez-Tato a a b
Departamento de Quımica Fısica, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain Departamento de Fısica Aplicada, Facultad de Ciencias, Universidad de Santiago de Compostela, Campus Universitario, 27002 Lugo, Spain Received 2 March 2001; accepted 6 June 2001
Abstract Forced-oscillation measurements of aqueous dispersions of an amide pectin were carried out at pH 3.83 in the presence of 50% sucrose. The results show a predominant viscous behaviour corresponding to the so-called terminal region of the general mechanical spectrum of biopolymer solutions. The polymer chains have been described by the bead-spring model with dominant hydrodynamic interactions among beads. At pH 3.31, samples are more elastic than viscous and the mechanical spectra reveal an entanglement network (weak gel), although not dense enough to store the stress during a long period of time after having been deformed. At low pectin concentration, the plot of loss moduli versus frequency shows a minimum that suggest monodisperse junction zones and the existence of two regions with different relaxation times. The short relaxation process corresponds to movements of galacturonic units inside the junction zones (local configuration adjustment), and the longer one to movements of the junction zone as a whole (rearrangements of couplings through entanglement and cross-linking). The comparison of the results obtained here at different sucrose concentrations and temperatures with those for methoxy pectins, suggests that the hydrogen bonds formed by amide groups have an important role in the formation of the gel. The role of the hydrophobic interactions in the stability of the junction zones has also been discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Amide pectin; Rheological properties; Viscoelastic behaviour; Gelation
1. Introduction Pectins are structural polysaccharides of vegetable origin. The polymer backbone is based on (1 ! 4)linked a-D -galacturonate residues interrupted by insertion of (1 ! 2)-linked rhamnosyl residues where neutral sugar side chains are attached. During the industrial extraction of pectins the degree of esterification is modified resulting in low or high methoxy pectins (LMP, HMP) depending on the degree of esterification. LMP are gelled by controlled addition of calcium ions. The gelation of both LMP and HMP can also be induced by lowering the pH and adding co-solutes as sucrose, glucose or fructose (Morris, 1986). Therefore, pectins are used as gelling and thickening agents in the food industry.
*
Corresponding author. Tel.: +34-82-223-325x24080; fax: +34-82224-904. ~ez). E-mail address: [email protected] (E. Rodrıguez-N un
Different intermolecular interactions, acting as driving forces for association, are involved in the gelation of pectins. For instance, the gelation of LMP in the presence of calcium ions, involves electrostatic interactions between the cations and the negative charged cavities formed by polymer chains, where the cations are inserted (egg box model). This process is reminiscent of the gelation of alginates (Clark & Ross-Murphy, 1987), a non-surprising fact since galacturonic blocks in pectates are almost mirror images of guluronic blocks in alginates. According to Kohn (1975), the minimum sequence length required for a stable association is seven calcium ions bound to consecutive unesterified residues on each of the participating strands. The egg box model has been supported by different experimental techniques by various authors (Gidley, Morris, Murray, Powell, & Rees, 1979; Powell, Morris, Gidley, & Rees, 1982). The HMP gelation takes place in conditions where electrostatic repulsions and water activity are reduced, i.e., in the presence of co-solutes at low pH. At this stage, the driving forces are hydrogen bonding and
0260-8774/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 2 ) 0 0 0 2 6 - 2
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hydrophobic interactions. It appears that the crosslinking at the junction zones takes place through the formation of aggregates of chains of varying sizes (Clark & Ross-Murphy, 1987), with no definite stoichiometry involved. Although other co-solutes as glycerol or ammonium sulfate can also be used (Rees, 1969), the possibility of a specific role for sucrose or other polyols, cannot be ruled out, i.e., the function of the co-solute seems more complex than simple competition for the available water (Ptitchkina, Danilova, Doxastakis, Kasapis, & Morris, 1994). Classical techniques have extensively been used to characterise the rheological behaviour of HMP dispersions and gels. These methods (Ikkala, 1986) imply large-deformation measurements and allow the determination of full stress–strain profiles and failure properties. Other static viscoelastic methods, such as creep (Dahme, 1985; Kawabata, 1977; Plashchina, Fomina, Braudo, & Tolstoguzov, 1979) and stress relaxation (Comby, Doublier, & Lefebvre, 1986) have also been used. More recently, modern controlled-stress rheometers have measured the performance of dynamic tests under small deformation (Beveridge & Timbers, 1989; Dahme, 1992; Ikkala, 1986; Lopes da Silva & Goncßalves, 1994; Lopes da Silva, Goncßalves, & Rao, 1993, 1994; Rao & Cooley, 1993, 1994; Rao, Van Buren, & Cooley, 1993). These non-destructive methods give a better insight into the rheological behaviour of the systems under investigation. On the other hand, since structural changes, ionic strength, pH and temperature affect the intermolecular interactions in different ways, they will also have consequences in the rheological behaviour of the samples. Therefore, studies of varying temperature (Beveridge and Timbers, 1989; Dahme, 1992; Lopes da Silva et al., 1994; Oakenfull & Scott, 1984; Rao & Cooley, 1993, 1994; Rao et al., 1993), co-solute (Lopes da Silva & Goncßalves, 1994; Rao & Cooley, 1994), degree of esterification (Lopes da Silva et al., 1993, 1994), and side branches in pectins (Hwang & Kokini, 1992), curing time of gels (Lopes da Silva & Goncßalves, 1994; Rao & Cooley, 1993; Rao et al., 1993) and the gelation process itself (Beveridge & Timbers, 1989; Dahme, 1992; Lopes da Silva & Goncßalves, 1994; Rao et al., 1993) have been carried out. Papers by Lopes da Silva and Goncßalves (1994), Lopes da Silva et al. (1993, 1994), and Oakenfull and Scott (1984) are particularly important for the purposes of this work. Oakenfull and Scott have concluded that ‘‘the junction zones consist of two adjacent segments of polysaccharide chain varying in length from 18 to about 250 galacturonic acid units, increasing with the degree of methoxylation’’. This well-defined stoichiometry contrasts with less precise situation commented on above. Such a discrepancy is explained in terms of the different materials used and/or different gelation conditions (Clark & Ross-Murphy (1987)). Although, the forma-
tion of hydrogen bonds contributes as much as twice the hydrophobic interactions in stabilising the junction zones, it is not sufficient enough to stabilise them. The dynamic rheological measurements carried out by Lopes da Silva et al. (1993) with LMP and HMP dispersions, show that both pectins have a quite different behaviour. This has been related to a lower intermolecular association and a higher hydrodynamic volume (due to the higher charge density) of LMP. In studying the influence of temperature on the storage and loss moduli (Lopes da Silva et al., 1994), they also observed that the frequency-temperature or time-temperature superposition principle fails. So, structural changes in the aggregates with different relaxation mechanisms and different dependence on temperature, have to be involved. These authors (Lopes da Silva & Goncßalves, 1994) have also studied the influence of the temperature on the nature of the HMP/sucrose networks. The observed dependence for the storage modulus was interpreted by assuming non-permanent cross-links and by the opposite effects of temperature on the hydrophobic interactions and hydrogen bonding that stabilise them. The substitution of some ester by amide groups offers a great chance to investigate some points mentioned by Lopes da Silva et al. (1993, 1994), since the presence of amide groups will facilitate the formation of hydrogen bonds. To carry out the rheological study, we have chosen an amide pectin with both kinds of groups. The results reported in this work by the authors include the effects of pectin and sucrose concentrations, pH and temperature.
2. Experimental The study was made on a commercial amide pectin Genu LM-104 AS (Copenhagen Pectin Factory). According to the supplier the degree of methoxyl and amide groups in the pectin molecule were 30% and 20%. The samples were prepared as follows: Pectin and sucrose were added to solutions at pH 3.83 and 3.31 of lactic/lactate buffer (Fluka) and sodium azide (Merck, for preserving microbial growth, 80 ppm) and, finally, the appropriate weight of water (Milli-Q) was added. For one hour and half the dispersion was magnetically stirred and slowly heated until the temperature reached 87 °C (held at this temperature less than 5 min) in a sealed flask to obtain a homogeneous solution. The samples were kept at room temperature (22–25 °C) for 24 h, where they have gelled. Dynamic rheological measurements were carried out in a Haake RS100 rheometer, with a cone and plate geometry (diameter 35 mm, angle 4°). The samples were transferred to the plate and, once in the measurement position, their contour surface was covered with a thin layer of paraffin oil to avoid dehydration. The temper-
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ature was kept constant by circulating water (through the bottom plate) from a Haake K15 thermocryostat. All dynamic measurements were done in the linear viscoelastic region, to assure that the calculated parameters correspond to an intact network structure. The operation frequencies were varied at least by a factor of 103 . Duplicated experiments showed an excellent reproducibility. Studies of some samples were repeated after two months from their preparation. The mechanical spectra (results not shown) were identical to those recorded after 24 h. Therefore gel stability and curing were completed after one day. The following moduli were determined, in forcedoscillation experiments, stress (s) and strain (c) are related via the complex shear modulus, G : G ¼ sðtÞ=cðtÞ
ð1Þ
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Fig. 2. Influence of x on the storage and loss moduli for samples of 0.75% (w/w) pectin at pH 3.83.
Samples with a constant sucrose content of 50% w/w and pectin concentration in the range 0.5–2.5% w/w were studied at this pH value and at 25 °C. Fig. 1 shows
that at operation frequency of 0.628 s1 (x ¼ 3:946 rad s1 ) the complex shear modulus, G , is constant in the range of stress 0.1–2 Pa, which is included in the linear viscoelastic region. Therefore, all experimental results were obtained under applied stresses from 0.5 to 2 Pa. As an example, Figs. 2 and 3 show the mechanical spectrum ðG0 and G00 versus xÞ for pectin concentrations of 0.75% and 2.5%. For both concentrations, G00 > G0 along the complete time scale, indicating that the samples are more viscous than elastic. It must be noticed that the range of frequencies covered in both concentrations is quite different. For the dilute system (Fig. 2), the storage modulus has a quadratic dependence with x (slope 2:00 0:02), while the loss modulus depends linearly with x (slope 0:98 0:01). Consequently, the complex viscosity g is frequency independent. This is the so-called terminal region, in which the polymer concentration belongs to the semi-dilute regime, typical for biopolymer
Fig. 1. Dependence of the complex shear modulus with the applied stress at pH 3.83.
Fig. 3. Influence of x on the storage and loss moduli for samples of 2.5% (w/w) pectin at pH 3.83.
which may be partitioned into real and imaginary components: G ¼ G0 þ iG00
ð2Þ
where G0 and G00 are the storage and loss moduli, respectively. The ratio G00 =G0 is the tangent of the phase angle. Alternatively, the complex viscosity, g , can be defined as: g ¼ G =f
ð3Þ
where f is the operation frequency (x ¼ 2pf ). g measures the total resistance to a dynamic shear. 3. Results and discussion 3.1. Results at pH 3.83
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dispersions revealing an entangled system. This behaviour was found for galactomannan solutions and other disordered random-coil polysaccharides (Robinson, Ross-Murphy, & Morris, 1982; Richardson & RossMurphy, 1987), and for locust bean gum and LMP, 3%, without added sugars (Lopes da Silva et al., 1994). For a pectin concentration of 2.5% and the highest frequencies, Fig. 3 shows that both moduli are similar in magnitude (G00 being yet greater than G0 ). Fitting the experimental data to a power law in x give exponent values of 0:61 0:01 and 0:67 0:01 for G0 and G00 , respectively. These results can be understood under the beadspring models for linear flexible random coils. Rouse’s theory does not consider hydrodynamic interactions among beads while in the theory of Zimm the hydrodynamic interaction is dominant. In the limit of low frequencies, both theories give the same result, predicting the well-known quadratic and linear dependencies for G0 and G00 , respectively. At higher frequencies, they predict slopes for both moduli of 1/2 (Rouse theory) and 2/3 (Zimm theory). Therefore, in the present experimental conditions, samples obey the Zimm model, which means that, in this region, the molecular relaxation implies a gradual stress relaxation in which the brownian motion moves the chain segments farther and farther along the backbone polymer to reach their final equilibrium configuration. Sometimes in biological polymers exist a transition from viscous fluid to a relatively elastic system, that is characterised by a crossover of the storage and loss moduli and can be achieved by increasing either the angular frequencies employed or the pectin concentration. Lopes da Silva et al. (1993) have observed such a transition in LMP, without sugars added, but at higher pectin concentrations; for HMP, in similar conditions, they estimated a crossover frequency >63 rad s1 for pectin contents between 2.8% and 3.9%. In our experimental conditions the transition from a viscous fluid to an elastic system was not found. The behaviour of pectin samples with intermediate concentrations is similar to those shown in previous figures, with power law exponents in the mentioned range. Furthermore, the magnitude of these moduli increase with pectin concentration. All this suggest that increasing the pectin concentration promotes the transition from a viscous system to a more entangled solution showing intermolecular interactions occurring on long time scales (Frangou, Morris, Rees, Richardson, & Ross-Murphy, 1982; Rocherfort & Middleman, 1987).
Fig. 4. Dependence of the complex shear modulus with the applied stress at pH 3.31.
those at pH 3.83. Now at operation frequency of 4.279 s1 (x ¼ 26:886 rad s1 ) the linear viscoelastic region extends at least up till 15 Pa (Fig. 4). Consequently, all the following results were obtained under an applied stress in the range of 2–10 Pa. Figs. 5 and 6 show the variation of G0 and G00 moduli with x for different pectin concentrations. For all samples, the storage modulus is one order of magnitude greater than the loss modulus, and G0 is less dependence on x that G00 . This behaviour corresponds to the socalled entanglement network with a high-frequency rubbery plateau for G0 , typical of more entangled (with respect to the previous pH) biopolymer solutions. The frequency dependence of both moduli implies that the complex viscosity g , diminishes with x, having found that the exponent of a potential law is independent on the pectin concentration, with a value of (0.8– 0.9). Similar qualitative results were found for gels of HMP/sucrose (Lopes da Silva & Goncßalves, 1994) and HMP/glucose and fructose (Rao & Cooley, 1994).
3.2. Results at pH 3.31 The range of pectin concentration and the sucrose content studied at pH 3.31 and at 25 °C were the same as
Fig. 5. Dependence on x of the storage modulus for samples at pH 3.31 and different pectin concentrations.
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Fig. 6. Dependence on x of the loss modulus for samples at pH 3.31 and different pectin concentrations.
These facts suggest that the samples have a solid-like behaviour, but since the rubbery plateau is not completely reached, the physical entanglement is not dense enough to store the stress during a long period of time after having been deformed. According with the classification criterion proposed by Clark and Ross-Murphy (1987) these samples are weak gels, while those prepared at pH 3.83 are entanglement network systems. Potentiometric titration of the pectin allowed us to estimate a value of 3.6 for the pKa . The carboxylic groups of the molecule are less ionised at the lower pH studied, and, therefore, the repulsive electrostatic interactions are reduced. It causes an increase in the number and density of the entanglements, implying a lower relative effect of some broken junctions on the G0 value, and a lower time necessary to create a new entanglement zone immediately after breaking the old one. This suggests an explanation of the observed differences in the linear viscoelastic region at both pH values. For the most diluted samples, G00 shows a minimum at x 6¼ 0:02 s1 (Fig. 7), which is normally of polymers that exhibit effects of entanglement coupling (te Nijenhuis, 1997). Following Leung and Goddard (1991) this minimum separates two regions with different relaxation processes. We suggest the short relaxation process corresponding to movements of galacturonic units inside the junction zones (local configuration adjustment), and the longer one to movements of the junction zone as a whole (rearrangements of couplings through entanglement and cross-linking). On the other hand, maybe the existence of the minimum evidences that the junction zones are rather monodisperse, in spite of the molecular mass polydispersity of the polymer. This conclusion is in agreement with the narrow molecular mass distribution observed by Powell et al. (1982) for the homopolymer blocks length in non-amidated pectins. However the earlier comments are spec-
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Fig. 7. Storage and loss moduli at pH 3.31 as a function of frequency for a sample of 0.75% (w/w) pectin.
ulative and it should be needed more information in addition to rheological data to understand the physical basis of such behaviour. Fig. 8 resumes the results obtained for a polymer concentration of 2% at different sucrose contents. For sucrose contents at 40%, G00 > G0 and g has a slight dependence with x. The opposite behaviour is observed at concentrations higher than 50%, where G0 > G00 and the mechanical spectrum is the typical one of a polysaccharide weak gel. It may be concluded that in the 40– 50% region there is a transition from an entanglement network to a weak gel, as a result of an increase in the polymer entanglement when the sucrose content raises. This strengthening of the pectin entanglement may be caused by three main effects: (a) the stabilising effect of the disaccharide on the hydrophobic interactions between ester methyl groups, (b) the dehydrating effect of the sugar and (c) the formation of new hydrogen bonds through the decrease in the water activity caused by the
Fig. 8. Influence of the sucrose content on the storage and loss moduli of samples with 2% (w/w) pectin at pH 3.31.
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presence of more co-solute molecules. As it was noticed in the Section 1, the formation of hydrogen-bonded bridges through sucrose molecules between pectin chains is not probable. Back, Oakenfull, and Smith (1979) have shown that the first effect explains the higher thermal stability of globular proteins in the presence of certain sugars or polyols. Oakenfull and Scott (1984) have explained the stability of HMP/sugars gels by this effect, which would be dependent on the molecular geometry of the sugar. These authors have also argued that the traditional view that sugar acts non-specifically by partly dehydrating (point b) the pectin molecules (Rees, 1969) does not seem probable. However, the network is not formed if the water activity is not reduced. Aqueous sucrose is apparently a poorer solvent than water for pectin. In terms of osmosis, the network cannot be formed in the absence of sucrose, because it could not withstand the osmotic pressure of the pure solvent. In other words, the presence of sucrose makes the water less able to solvate the polysaccharide chains and any tendency for them to separate from solution (to form junction zones, for example) is enhanced (Rees, 1969). Under this situation, hydrogen bonding through amide and acid groups between chains (point c) may be facilitated. Further insights on the role of these effects have been deduced by studying the influence of the temperature. For a pectin concentration of 0.75% and sucrose 50%, the influence of temperature was studied from 15 to 45 °C by analysing different samples at each temperature. The dependence of G0 , G00 and g with x is the typical of biopolymer gels near the rubbery plateau. Fig. 9 shows a plot of the storage modulus versus temperature at x ¼ 0:20 rad s1 . Identical profiles are also observed at other frequencies. The rise in G0 is slow within the temperature interval 45–35 °C, it remains constant between 35 and 25 °C. At lower temperatures a remarkable increment is observed (the ratio of G0 (25 °C)/G0 (15 °C) is 6).
Fig. 9. Storage modulus at x ¼ 0:20 rad s1 as a function of temperature.
Similar qualitative results, although with much lower effects, were also observed by Lopes da Silva and Goncßalves (1994) in studying high-methoxyl (64%) pectin gels in the presence of 60% sucrose. Oakenfull and Scott (1984) have also reported similar trends for the rupture strength of other HMP gels (methoxyl degree, 69.7%; sucrose, 55%). It is known that the hydrogen bonds are weakened (Joesten & Schaad, 1974) and the hydrophobic interactions are strengthened (Ben-Naim, 1980; Oakenfull & Fenwick, 1977) when the temperature increases. It has been argued (Lopes da Silva & Goncßalves, 1994; Oakenfull & Scott, 1984) that these two opposite effects explain the observed profile. At low temperatures hydrogen bonds are favoured, reinforcing the junction zones and the storage modulus increases. At higher temperatures, the loss of hydrogen bond association is compensated with the reinforced hydrophobic interactions. Here the electrostatic interactions are not considered because their contribution should be small due to the low degree of ionisation of the carboxylic groups of the polysaccharide. The greater effect in the G0 values on cooling the samples found for the amidate pectin with respect to the esterified ones (Lopes da Silva & Goncßalves, 1994; Oakenfull & Scott, 1984) must be related with the higher ability of the amide group to form the hydrogen bonds which stabilise the junction zones. Therefore, the amide groups might play an important role on gelation, namely promoting H-bonding.
Acknowledgements The authors thank the DGICYT (Spain) for financial support (Project PB90-0758).
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