Biomacromolecules 2003, 4, 1142-1149
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Gelatin vs Polysaccharide in Mixture with Sugar Stefan Kasapis* and Insaf M. Al-Marhoobi Department of Food Science & Nutrition, College of Agricultural & Marine Sciences, Sultan Qaboos University, PO Box 34, Al-Khod 123, Sultanate of Oman
Marcin Deszczynski and John R. Mitchell Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, U.K.
Rukmal Abeysekera Institute for Applied Biology, University of York, Yorkshire YO1 5DD, U.K. Received November 12, 2002
The structural behavior of a well-characterized gelatin sample has been revisited to investigate the morphology of its network in the presence of sugar. This was then contrasted with the corresponding properties of the gelling polysaccharides agarose, κ-carrageenan, and deacylated gellan. Small deformation dynamic oscillation, differential scanning calorimetry in plain and modulated mode, visual observations, and transmission electron microscopy were used to identify the structural characteristics of the biopolymers from the rubbery plateau through the transition region to the glassy state. In contrast to the collapse of the polysaccharide gels at intermediate levels of co-solute, gelatin forms reinforced networks. The drop in polysaccharide network strength is accompanied by a decline in the enthalpy of the coil-to-helix transition, whereas the transition enthalpy is more pronounced in gelatin gels in accordance with their strengthening. Tangible evidence of the molecular transformations was obtained using microscopy, with polysaccharides disaggregating and dissolving in the saturated sugar environment. Gelatin, on the other hand, is visualized in an aggregated form thus producing a phase-separated topology with sugar. Introduction Over the last 20 years, we have witnessed the development of a materials science approach to food structure in high solids formulations.1-3 This has involved the construction of state diagrams and the use of these diagrams to understand the kinetics of physicochemical changes occurring on storage of materials. Central to this development is the idea of molecular mobility governing the kinetics of phase/state transitions and chemical reactions. Molecular mobility is often, though not invariably, associated with the macroscopic viscosity of the material,4 and the approach is relevant to the low water conditions of confectionery and ice cream products. The science governing the behavior of hydrocolloids in these sugar, low water systems is much less understood than in the high water environment where they are widely employed.5,6 In the former, the most frequently used hydrocolloids are gelatin, starch, or pectin. The structural attributes imparted by these molecules are different, though starch manufacturers are putting considerable effort into developing ingredients that can match the functional properties of gelatin. There is also substantial research effort in developing gelatin alternatives from other hydrocolloids such as pectin, carrageenan, agarose, or gellan gum. * To whom correspondence should be addressed: Fax: + 968 513 418. E-mail:
[email protected].
Confectionery manufacturers, ingredient suppliers, and academics consider how to extend the sophisticated synthetic polymer approach with the intention of obtaining a better understanding of hydrocolloid behavior in high-solid systems.7-9 Thus, ideas of polymer physics which form the backbone of the materials science were used to interpret the mechanical, thermal, and spectroscopic characteristics of biological glasses and melts. Three critical scientific issues were addressed, as summarized below: The transition from the rubbery to the glassy state was clearly identified allowing definition of the glass transition temperature (Tg) as the conjunction of two molecular mechanisms. In terms of the temperature dependence of relaxation processes,10 these were based on the configurational rearrangements of segments of the polymeric backbone occurring in the glass transition region and the energetic barrier to rotation of submolecular groups in the glassy state. Second, the glass transition temperature measured by calorimetry remains unaltered by the presence of low levels of polysaccharide suggesting that the mobility of the sugar is unaffected by the presence of the macromolecule. However, the mechanical profile of the rubber-to-glass transition is strongly influenced by the polysaccharide particularly if it is network forming. Thus, it is possible to represent the magnitude of this polysaccharide contribution to rheology by a “network Tg”, the greater the extent to which this differs
10.1021/bm0201237 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/26/2003
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Sugar/Biopolymer Mixtures Table 1. Data on the Physicochemical Characterization of Our Gelatin Sample sample
PS1
Blooma isoelectric point (pI) % moisture (wwb) calcium (ppm) sulfate (%) chloride (%) phosphate (ppm) Mnb M. wt. > 106 kD M. wt. > 540 kD tetra + penta gamma beta alpha subunits 1 subunits 2 subunits 3 subunits 4
305 8.7 10 80 70%). This is seen as an increasingly vitrified system with decreasing temperature or time of measurement. Figure 1 reproduces the mechanical manifestation of vitrification, which includes a progressive five-to-six-decade enhancement of shear modulus from about 104 Pa in the rubbery plateau to 1010 Pa in the glassy state. This, of course, has significant implications for the prediction and control of the structural properties of several foodstuffs and pharmaceuticals. Increasing amounts of co-solute in the gelatin samples also induce changes in small deformation properties from conventional hydrogels to profiles that cover the four regions of the master curve of viscoelasticity. Figure 3a illustrates the rubbery spectrum of a 7% gelatin gel in the presence of 53% sugar. A flat G′ trace is discernible, accompanied by a dynamic viscosity (η*) gradient that approaches the value of -1 and a loss modulus (G′′) trace that shows a dip toward
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Figure 3. Frequency variation of storage modulus, loss modulus and complex dynamic viscosity for 7% gelatin in the presence of (a) 53 and (b) 63% sugar at 0°C.
the low frequency range. This corresponds to the blending of long topological changes (chain slippage), mainly contributing to the relaxation in the terminal zone, with short configurational vibrations of the chain segments, primarily seen in the transition region.32 Incorporation of an extra 10% sugar in the mixture (70% total solids) creates a mechanical spectrum at 0 °C which is shifted closer to the onset of the glass transition region (Figure 3b). Thus, the G′′ trace shows a pronounced frequency dependence and the G′ values starts taking off at frequencies higher than 1 rad s-1. At 80% solids, the gelatin network acquires considerable thermal stability and the G′ trace has taken off at temperatures below 10 °C in Figure 2. The acceleration of vitrification properties at subzero temperatures yields shear modulus values which are coincident with those of polysaccharides in the upper range of sugar concentration in Figure 1. Thus, the gap observed in the development of structure between the two types of systems at intermediate levels of co-solute is now bridged. An example of the master curve of viscoelasticity as a function of temperature for the upper range of solids is given in Figure 4. At the highest experimental temperatures, G′′ dominates G′ thus unveiling part of the flow region. Cooling sees a rapid development in G′ with the two traces crossing over at a point which demarcates the onset of the rubbery region (∼32 °C). The storage modulus now flattens out but the damping factor (tan δ ) G′′/G′) never falls below 0.5. This is very different from the thermal profile of the protein in an aqueous environment forming cohesive networks with tan δ values as low as 0.01.33 On further cooling, the loss modulus develops rapidly and overtakes the storage modulus once more (tan δ ) 1 at about -0.5 °C). This region is known as the glass transition and covers modulus values stretching to 108.5 Pa. Here polymer backbone adjustments are diminishing, but short-range movement of pendant groups or molecular vibrations are present. At the lowest temperature, there is yet another development and a hard solid response is obtained (G′ > G′′ with values approaching 1010 Pa). This is the fourth region of the master curve, the so-called glassy state where
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Figure 4. Cooling run of G′, G′′ and their ratio (tan δ) for 7% gelatin in the presence of 73% sugar. The solid lines between 30 and 50°C represent the heating profiles of G′ and G′′ which no longer overlap with their cooling counterparts because of thermal hysteresis (scan rate: 1 °C min-1; strain: 1 to 0.00072%).
Figure 5. Heat flow variation as a function of temperature for 7% gelatin plus 73% sugar obtained at a scan rate of 1 °C min-1. The asymptotes to the experimental trace demarcate the limits and the middle of the calorimetric glass transition region.
mainly stretching and bending of chemical bonds, and “β transitions” are observed.32,34 Thermal Properties and the Glass Transition Temperature. Thermal analysis routines measured the temperature and heat flow of the transitions of our materials in an effort to contrast underlying patterns with the mechanical evidence described in the foregoing sections. Direct measurements of heat capacity were performed with MDSC at 1 °C min-1, and a typical case is shown in Figure 5 for the gelatin sample at 80% solids. During cooling, the heat capacity decreases as the material’s excess volume because of thermal motions and the molecular mobility diminish. Conventionally, the onset and end of the heat capacity change are considered to demarcate the glass transition region thus affording pinpoint-
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ing of the calorimetrically derived glass transition temperature at the middle of the transition. This is recorded in Figure 5 at about -43 °C (Tgc). It is well documented that the thermal spectrum of proteins or polysaccharides in the presence of a high sugar environment is dominated by the vitrification of the latter.11,35 Thus, values of Tgc for single sugar preparations or protein/ polysaccharide-sugar mixtures can be predicted by the total level of solids in the system. Furthermore, the calorimetric glass transition temperature is comparable to its rheological counterpart for sugar samples.12 It appears, however, that small additions of gelatin or gelling polysaccharide to a sugar preparation alter dramatically the mechanical manifestation of the rubber-to-glass transition. The acceleration of vitrification properties in the presence of biopolymer is evidenced in Figure 1, at which there is a considerable gap in the development of shear modulus values between sugar preparations and their mixture with biopolymer at level of solids above 60%. According to the postulates of Ferry’s free volume theory and Ngai’s coupling model, a fundamental definition of the rheological glass transition temperature (Tgr) can be advanced at the conjunction of the transition region and the glassy state.10,16,32 Examining the rheological and thermal spectra of vitrification for the gelatin/sugar sample at 80% solids, it is clear that the Tgr in Figure 4 is recorded at higher temperatures than the Tgc in Figure 5 (about -20 and -43°C, respectively). Therefore, the glass transition temperature measured by calorimetry remains unaltered by the presence of low levels of biopolymer suggesting that the “micromobility” of the sugar is unaffected by the presence of the macromolecule. Calorimetric Evidence of the Changing Nature of High Sugar/Biopolymer Networks. Figures 1 and 2 summarize the effect of co-solute on the mechanical properties of gelatin and polysaccharide thus unveiling structural disparities in the two types of networks. DSC experiments were also recorded at 1 °C min-1 for the same samples as in the rheological studies. Cooling runs are particularly revealing for polysaccharides, and in all cases, there are well-defined exothermic events, but with considerable variation in size, shape, and temperature range. As shown in Figure 6, aqueous preparations of 3% agarose yield a relatively sharp transition indicative of a co-operative process of coil to helix formation. The end of the transition is quite broad and forms a tail, which in accordance with optical rotation profiles, is attributed to molecular polydispersity and a range of temperature-induced relaxation processes.36 The positive development in the temperature dependence of shear modulus at low levels of co-solute in Figure 1 is matched in Figure 6 by a rise in enthalpy from 23.1 to 29.1 J g-1 at 0 and 27% sugar, respectively. In contrast, increasing the sugar concentration to 57% creates a broad exotherm of 20.1 J g-1 suggesting a gradual ordering process and a reduction in the cooperativity of helix formation. Thus, the negative development in the magnitude of the thermal event is congruent with the drop in network strength of the polysaccharide at intermediate levels of co-solute (Figure 1). Similar trends have also been reported for 0.5 to 1%
Sugar/Biopolymer Mixtures
Figure 6. Cooling exotherms of 3% agarose samples at levels of sugar shown by the individual traces (0 and 27% sugar, left y axis; 57% sugar, right y axis; scan rate: 1 deg min-1).
Figure 7. Heating endotherms at 1 deg min-1 of 7% gelatin samples without and with 53% sugar (left and right y axis, respectively).
κ-carrageenan and deacylated gellan gum at sugar contents extending to 85% in the formulation.13,14 Gelatin, on the other hand, exhibits a positive trend in the development of energy holding associations in a co-solute environment. Figure 7 depicts endothermic events of the “equilibrium” heating process afforded by the early melting temperature of the protein assemblies. In all cases, similar results were obtained from well-defined exotherms on cooling scans, but with a reduced size of the thermal event of about 25%. This result is paralleled in the increase in melting temperature of gelatin networks observed rheologically and argues for a certain thermal hysteresis in the threedimensional structure. For example, the empirical tform and tmelt are 32 and 46 °C, respectively, in Figure 4. DSC heating scans of 7% gelatin remain sharp in the presence of 53% sugar in Figure 7, an outcome which corroborates the reinforcement of networks at intermediate levels of co-solute (Figures 1 and 2).
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Figure 8. Enthalpy changes as a function of total level of solids for biopolymer/sugar mixtures containing 3% agarose, 0.5% κ-carrageenan (10 mM KCl), and 7% gelatin.
Figure 9. Turbidity loss in 2% agarose gels at 0, 20, 30, 40, 60, and 70% glucose syrup (left to right). The test tube on the right is a 70% glucose syrup solution.
Visual Observations in Support of Calorimetry and Rheology. Integration of the area under the peak of the DSC scans allows estimation of the enthalpy change (∆H) of the gelation process in biopolymers. This is reproduced as a function of sugar content for agarose, κ-carrageenan, and gelatin in Figure 8. Strikingly, the thermal profiles of the polymers fall into three distinct patterns of behaviour. There is an early drop in the values of the transition enthalpy in agarose gels, which reach a maximum at 30% solids and at 77% solids have lost about 70% of their original stabilizing intermolecular associations. Aggregation of the uncharged agarose helices is quite extensive and results in turbid aqueous gels which exhibit considerable levels of syneresis and brittleness. As demonstrated in Figure 9, however, gradual addition of co-solute reduces dramatically the turbidity of the systems, which are transformed into elastic gels at 72% solids. Therefore, it appears that aggregation and its stabilizing effect on the individual helices is severely disrupted at relatively low additions of sugar, in accordance with the conclusions from the DSC work. [In Figure 9, there
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are issues of changing refractive index with increasing level of solids in the sample but we believe that the result is consistent with our hypothesis.] The extent of aggregation and thermal hysteresis decreases, as the charge density of the polygalactan increases, through the series: ι-carrageenan < κ-carrageenan < agarose.37,38 Furthermore, low levels of potassium ions (10 mM) were added to 0.5% κ-carrageenan which was ion-exchanged in the sodium form to “temper” aggregation of the polysaccharide.39 The morphology of such a network is compatible with additional molecular interactions, and the dominant feature is a rise in the transition enthalpy at levels of sugar higher than those of agarose (Figure 8). Eventually, the threshold of thermodynamic stability of structure formation is exceeded, and considerable parts of the network “dissolve” in the saturated sugar environment with concomitant collapse in the values of ∆H. The preferential exclusion of co-solute from the domain of gelatin prohibits the formation of a solvation sheath around the protein which is forced to adopt an increasingly ordered state. That should result in rising values of the transition enthalpy as, indeed, are recorded for gelatin in the presence of sugar (Figure 8). Tangible evidence of the disparity in the topology of the polysaccharide and gelatin phases is obtained using transmission electron microscopy (Figure 10). The micrograph on the top shows an aqueous gellan gel in the presence of added calcium ions. A polymer network of fibrillar thickness between 20 and 30 nm is obtained which should comprise up to 10 or 20 double helical strands per filament. The morphology of long-range structures of the gellan molecule in an aqueous environment was also probed by V. J. Morris and his colleagues using atomic force microscopy. The fibrous structure of the polysaccharide was confirmed in the absence of gel-forming counterions (TMA-salt form), with filaments appearing to be of constant height and width.40 This result argues against lateral aggregation of the helical assemblies in these conditions. The presence of moderate amounts of gel-forming counterions (e.g., K+) or addition of GDL to reduce the pH below that of the pKa of the glucuronic acid residues produces networks with fibrillar dimensions comparable to our work at the top of Figure 10.41 Thus, microscopy evidence indicates side-by-side association of gellan helices. Nevertheless, gellan gels remain transparent, a result which is contrasted with the extensive aggregation and concomitant opacity of agarose gels at equivalent levels of solids.37,38 In contrast, addition of high levels of sugar produces microscopy images congruent with the postulate of reduced aggregation in the polysaccharide network. Although the polymer forms a continuous phase that supports the mixture in the form of a cohesive gel, dense fibrillar structures are not visible (Figure 10 middle). Gelatin appears to respond differently to gelling polysaccharides in a high solids environment, with sugar promoting chain association rather than inhibiting it. Thus, there are demixed gelatin and sugar rich domains of considerable size in the bottom micrograph of Figure 10, as opposed to the homogeneous polysaccharide assemblies at high levels of
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Figure 10. Transmission electron micrographs of 0.7% deacylated gellan with 10 mN Ca2+ in aqueous environment (top) and in the presence of 80% glucose syrup (middle) and of a 7% gelatin preparation with 77% sugar showing sugar rich (light) and gelatin rich (dark) regions.
co-solute. The heterogeneity in the protein/sugar mixture is also evident in the small deformation properties in Figure 4, which show a tan δ trace covering a broad rubber-to-glass transition due to an extended spectrum of relaxation processes. It is speculated that this behavior may have implications and lie behind the difficulties encountered in the
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commercial replacement of gelatin with small additions of gelling polysaccharide in high sugar foodstuffs. Acknowledgment. Support from the Department of Environment and Rural Affairs and several industrial companies through the Link project “Viscoelasticity, crystallinity and phase behavior of high sugar polysaccharide systems relevant to the food industry” is gratefully acknowledged. We are grateful to Dr. Alan Parker currently of Firmenich SA for GPC measurements of the gelatin sample. References and Notes (1) Slade, L.; Levine, H. Crit. ReV. Food Sci. Nutr. 1991, 30, 115. (2) Blanshard, J. M. V.; Lillford, P. J. The glassy state in foods; Nottingham University Press: Nottingham, U.K., 1993. (3) Roos, Y. H. Phase transitions in food; Academic Press: San Diego, CA, 1995. (4) Champion, D.; Hervet, H.; Blond, G.; Le Meste, M.; Simatos, D. J. Phys. Chem. B 1997, 101, 10674. (5) Huang, Y.; Szleifer, I.; Peppas, N. A. Macromolecules 2002, 35, 1373. (6) Kavanagh, G. M.; Clark, A. H.; Ross-Murphy, S. B. Int. J. Biol. Macromol. 2000, 28, 41. (7) Kasapis, S.; Al-Alawi, A.; Guizani, N.; Khan, A. J.; Mitchell, J. R. Carbohydr. Res. 2000, 329, 399. (8) Kasapis, S.; Mitchell, J. R. Int. J. Biol. Macromol. 2001, 29, 315. (9) Kasapis, S.; Al-Marhoobi, I. M. A. In Gums and stabilisers for the food industry 11; Williams, P. A., Phillips, G. O., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2002, 39. (10) Plazek, D. J.; Ngai, K. L. Macromolecules 1991, 24, 1222. (11) Goff, H. D.; Caldwell, K. B.; Stanley, D. W. J. Dairy Sci. 1993, 76, 1268. (12) Kasapis, S.; Mitchell, J. R.; Abeysekera, R.; MacNaughton, W. Trends Food Sci. Technol. 2003, in press. (13) Whittaker, L. E.; Al-Ruqaie, I. M.; Kasapis, S.; Richardson, R. K. Carbohydr. Polym. 1997, 33, 39. (14) Evageliou, V.; Kasapis, S.; Hember, M. W. N. Polymer 1998, 39, 3909. (15) Tsoga, A.; Kasapis, S.; Richardson, R. K. Biopolymers 1999, 49, 267. (16) Ngai, K. L.; Plazek, D. J. Rubber Chem. Technol. 1995, 68, 376. (17) Plazek, D. J.; Chay, I. C.; Ngai, K. L.; Roland, C. M. Macromolecules 1995, 28, 6432.
Biomacromolecules, Vol. 4, No. 5, 2003 1149 (18) Kasapis, S.; Al-Marhoobi, I. M.; Mitchell, J. R. Carbohydr. Res. 2003, in press. (19) Atkin, N.; Abeysekera, R. M.; Kronestedt-Robards, E. C.; Robards, A. W. Biopolymers 2000, 54, 195. (20) Nishinari, K.; Watase, M.; Williams, P. A.; Phillips, G. O. J. Agric. Food Chem. 1990, 38, 1188. (21) Watase, M.; Nishinari, K.; Williams, P. A.; Phillips, G. O. J. Agric. Food Chem. 1990, 38, 1181. (22) Sworn, G.; Kasapis, S. Food Hydrocolloids 1998, 12, 283. (23) Pezron, I.; Herning, T.; Djabourov, M.; Leblond, J. In Physical networks, polymers and gels; Burchard, W., Ross-Murphy S. B., Eds.; Elsevier: London, 1990; p 231. (24) Djabourov, M.; Lechaire, J.-P.; Gaill, F. Biorheology 1993, 30, 191. (25) Busnel, J. P.; Clegg, S. M.; Morris, E. R. In Gums and stabilisers for the food industry 4; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, U.K., 1988; p 105. (26) te Nijenhuis, K. AdV. Polym. Sci. 1997, 130, 1. (27) Gilsenan, P. M.; Ross-Murphy, S. B. Int. J. Biol. Macromol. 2001, 29, 53. (28) Tait, M. J.; Suggett, A.; Franks, F.; Ablett, S.; Quickenden, P. A. J. Solution Chem. 1972, 1, 131. (29) Chandrasekaran, R.; Radha, A. Trends Food Sci. Technol. 1995, 6, 143. (30) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4667. (31) Oakenfull, D.; Scott, A. Food Hydrocolloids 1986, 1, 163. (32) Ferry, J. D. Viscoelastic properties of polymers; John Wiley: New York, 1980; p 224. (33) Papageorgiou, M.; Kasapis, S.; Richardson, R. K. Food Hydrocolloids 1994, 8, 97. (34) Kasapis, S.; Desbrie`res, J.; Al-Marhoobi, I. M.; Rinaudo, M. Carbohydr. Res. 2002, 337, 595. (35) Roos, Y. Carbohydr. Res. 1993, 238, 39. (36) McKinnon.; A. A.; Rees, D. A.; Williamson, F. B. Chem. Commun. 1969, 701. (37) Dea, I. C. M. Pure Appl. Chem. 1989, 61, 1315. (38) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R.; Gidley, M. J. Biopolymers 1995, 36, 643. (39) Hermansson, A.-M. Carbohydr. Polym. 1989, 10, 163. (40) Morris, V. J.; Kirby, A. R.; Gunning, A. P. Prog. Colloid Polym. Sci. 1999, 114, 102. (41) Gunning, A. P.; Kirby, A. R.; Ridout, M. J.; Brownsey, G. J.; Morris, V. J. Macromolecules 1996, 29, 6791.
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