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Shear creep of gelatin gels from mammalian and piscine collagens
International Journal of Biological Macromolecules 29 (2001) 53 – 61 www.elsevier.com/locate/ijbiomac
Shear creep of gelatin gels from mammalian and piscine collagens Paula M. Gilsenan, Simon B. Ross-Murphy Biopolymers Group, Di6ision of Life Sciences, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo, London SE1 9NN, UK Received 27 November 2000; accepted 18 April 2001
Abstract This paper describes new measurements on the creep rheological behaviour of gelatin gels from both traditional mammalian and piscine sources. Measurements on a series of concentrations of gels were obtained using a high-precision controlled stress rheometer. Results for the concentration dependence of compliance are close to those expected from dynamic oscillatory measurements of gel modulus, assuming ideal elasticity. The concentration dependence of viscosity approximates power law behaviour, with p 8C 2 – 3, lower than the exponent expected for semi-dilute solutions. The apparent contradiction implied by this is discussed and a novel gel viscosity versus concentration state diagram presented. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Creep; Gelatin; Rheology
1. Introduction During the last century, a great deal of experimental work has been performed investigating the structural and mechanical properties of gelatin gels [1 – 3]. Both the structural and mechanical behaviour of thermoreversible gels from the polypeptide gelatin have been widely studied, and the review by te Nijenhuis (1997) [3] summarises much of these endeavours. All the work in his review and almost all in the literature as a whole have, however, been performed on traditional mammalian gelatins with very little focus on samples from alternative sources. Most of this work is also concerned with macroscopic gels, although the approach of de Carvalho and Djabourov [4] tackles the problem of the rheology of gelatin gelation under shear. Only very recently has there been interest in gelatins from nonmammalian, mainly piscine, sources. In the present paper, we summarise some of these data and also examine previous creep measurements performed on conventional mammalian gels. Surprisingly, despite the ready availability of both the samples and of modern * Corresponding author. Tel.: + 44-207-8484081; fax: +44-2078484082. E-mail address: [email protected] (S.B. Ross-Murphy).
controlled stress rheometers, ideal for performing the creep experiment, since the work of Higgs [5] and one of the present authors, very little seems to have been published in the latter area. This is despite the renewed interest in long-time behaviour of gelatin gels and its possible implications for gel healing. In this section, we briefly summarise the main points of gelatin gelation, which are now widely accepted, and then present data on the concentration dependence of creep behaviour and creep recovery for both traditional mammalian samples and a range of fish gelatins.
1.1. Molecular structure of gelatin sols and gels Gelatin is a water-soluble protein that is widely used in the food, pharmaceutical and photographic industries. It is derived by hydrolytic degradation of collagen, the principal component of white fibrous connective tissue [6]. Commercially, both acid and alkaline hydrolysis routes are employed, and most commercial samples are extracted from either bovine or porcine sources. The basic molecular unit of collagen is a triple-helical rod (tropocollagen triple helix). This consists of three a-chains arranged in a left-handed axis, with the whole structure wound into a right-handed super helix. The extraction process involves partially
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hydrolysing the collagen towards the individual a chains, although commercial samples tend to contain a-, b- and g-chains. It is now accepted that the gelation of gelatin involves, at least partly, regeneration of the collagen molecule i.e. reformation of triple helical ‘junction zones’ (renaturation), separated by regions of flexible polypeptide chain. This occurs on cooling a warm solution to below room temperature; a transparent, thermoreversible gel is formed provided that the concentration, C, is greater than the critical gelation concentration, Co [7]. This is typically 0.4– 1.0 w/w% for commercial mammalian gelatins. Above 40°C, a solution of flexible, random coils is obtained. The source and type of collagen influence the properties of the resulting gelatins. The amino acid content and sequence vary from one source to another, but collagen is an unusual protein in that it always consists of large amounts of the peptides proline, hydroxyproline and glycine [8]. The melting and gelling temperature of gelatin have been found to correlate with the proportion of imino acids proline and hydroxyproline (both with a five-membered pyrrolidine ring) in the original collagen [9]. The proline content plays an important role in the stability of gelatin gels, since it promotes formation of the polyproline II helix. This in turn determines the form of the tropocollagen trimer.
Creep measurements [19] can be used to study the long-time behaviour of gelatin gels [5]. In a creep experiment, a fixed stress is applied to the sample ‘instantaneously’, held constant for a given time and then removed. The time-dependent increase in strain is measured as a function of this stress. The behaviour of compliance, J(t), where:
1.2. Gelatins from alternati6e sources
J(t)=
As mentioned above, most commercial gelatins are obtained from either cowhide or pigskin. Consequently, almost all of the work in the literature has been carried out on traditional mammalian samples. Recently, however, there has been considerable interest in using alternative substitutes. It has been known for some years that the un-hydrolysed collagen coil-helix temperature depends upon the proportion of proline and hydroxyproline [9]. This is considerably lower for fish (16– 18%) than for mammals (24%). Cold-water fish, for example cod, have a very low hydroxyproline content and because of this a very low gelling and melting temperature B 15°C. In practice, a typical mammalian gelatin of concentration 5 – 10% w/w will form around ambient temperatures, whilst a cod gelatin under equivalent conditions will only just gel at 2°C [10]. The industrial interest in non-mammalian gelatins in the food industry has followed the recent bovine spongiform encephalopathy (BSE) crisis in the UK. Non-mammalian gelatins, e.g. those derived from fish collagen, have obvious advantages in both minority and ethnic food products (‘vegetarian’, halal and kosher). They also have potential advantages in applications such as the production of pharmaceutical capsules and the photographic industries. In volume terms, the mar-
k(t) is the change (increase) in shear strain with time, t, and ~ is the applied stress, is particularly valuable since it can be used to understand the behaviour of physically cross-linked gels. However, since our work in this journal in 1990 [5], there seems to have been little in the way of subsequent investigation. The paper by Normand and Ravey [20] is, in rheological terms, a fairly complete characterisation using both oscillatory shear and creep data, particularly since they were able to use a numerical transform to superpose time- and frequency-domain data over approximately five decades from t10 − 2 to 103 s. It is, nevertheless, limited to two concentrations of a single (alkaline extracted) gelatin sample. A limited amount of data have also been collected on creep of chemically cross-linked gelatin gels [11,21]. In the present work, we have adopted a strategy more in keeping with our earlier paper [5] by examining a number of different samples, at several different concentrations, but employing a standard rheological protocol. The following practical aspects must be considered when performing such creep experiments on gelatin gels: 1. Time-scale of gel formation. Since gelatin gelation is a kinetic process and continues indefinitely long after the initial ‘setting’ of the gel [3,22,23], sufficient
ket for gelatin is, in fact, split almost equally between food (55%) and non-food, i.e. photographic, pharmaceutical and assorted technical applications, such as adhesives [11]. However, the higher-grade products used in photographic and pharmaceutical applications are of considerably more value to the gelatin producer. A number of papers have appeared recently describing the extraction and purification of gelatins, particularly from fish (and usually from fish skins). Some key references include work on hake and trout [12], plaice, shark and cod [13– 15]. The article by Norland [16], an early pioneer, summarises most of the work prior to 1990. Our own publications [10,17,18] describe the oscillatory shear characterisation of a number of different mammalian and piscine gelatin samples; the present work extends these by employing time domain shear creep measurements.
1.3. Creep measurements on gelatin gels
k(t) , ~
(1)
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time must elapse before beginning the experiment so that any further change during the experiment is negligible. Failure to consider this aspect can mean that the kinetics of gel modulus growth are convoluted (hopelessly) with the time domain data collection. 2. Thermal history. The thermal history has a great influence over the properties of a gelatin gel [3]. Differential scanning calorimetry measurements, for example, have shown considerable differences between gels cooled directly to a low temperature and those cooled in two stages [3,24]. There are also differences for gels cooled to the same temperature at different rates. Thus, the temperature and cooling procedure must be carefully controlled. 3. Applied stress le6el. The stress must be chosen so that the strain is measurable but still remains within the linear viscoelastic region. This requires an estimate of the linear strain. Gel moduli vary with concentration, and at high concentrations, an approximate C 2 dependence (see Section 4) is seen [7]. An estimate of the required stress can therefore be made from the modulus, using the second approximation G( [ 0) = 1/J(t [ ) (also Section 4).
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tures are below room temperature) cod gelatin samples (IC and 2747), and the tilapia (tropical fish) gelatin. The latter is intermediate in behaviour between the cod and bovine samples. Samples were supplied either as the usual coarse gelatin granules or as finer granulated powders. Relevant physico-chemical properties of these are given in Table 1. Solutions were prepared by soaking the pre-weighed gelatin granules in water for 2–3 h at ambient temperature, followed by mechanical stirring for 10–15 min at 70°C to give a final range of concentrations (C) between 1 and 10 w/w%. Shear creep experiments were performed using a Rheometrics Dynamic Stress Rheometer (DSR500, Rheometric Scientific Ltd., UK) with 40 mm diameter and 1.5 mm gap parallel plate geometry. The bottom plate temperature was controlled (heated and/cooled) using a Peltier system with a heatsink, consisting of a Haake thermostatted water bath set at 15°C, to ensure a reproducible time and cooling regime. The Peltier-controlled bottom plate was set to 35°C before adding the hot gelatin solution. The top plate was lowered to give the correct gap and the excess solution removed. A thin layer of silicone oil (Dow Corning 200/10cs) was placed around the exposed surface of the sample to prevent loss of moisture by evaporation. The sample was then cooled at a rate of 0.5°C/min to 2°C. It was maintained at this temperature for exactly 19 h before beginning the creep phase of the experiment. Stresses (~) between 5 and 1000 Pa, depending on the concentration, were applied ‘instantaneously’ to the sample. At higher concentrations, ~ was increased, assuming it to be proportional to C 2. Consequently, the instantaneous strain was never \15% so that, at least by normal criteria, the linear viscoelastic region was not exceeded. The developing strain was measured for 5000 s, the stress was then removed and the recoverable strain measured for a further 5000 s. Data collection made use of the instrument’s ‘zone’ feature, in which the total experiment was divided into eight semi-logarithmic zones (four creep, four recovery). The same number of points is collected in each zone (say 100). In this manner, more data can be gathered in the early stages of the experiment. The instrument converts the instantaneous voltage from the position (strain) trans-
2. Materials and methods The work was carried out as part of a collaborative project funded by the EC, and our partners included commercial suppliers of gelatin as well as biochemists expert in the extraction of gelatin samples from fish species (see Acknowledgements). Two samples of mammalian gelatin (OC1 and AP1) were supplied by SKW Biosystems, France. In this project, the fish samples studied were tilapia, tuna, megrim and cod, ranging in order from tropical to cold water. Most of these are not available commercially, although two of the fish gelatins — cod (2747) and tilapia (7056) — were supplied by Croda Colloids, Luton, UK. In the present work, and following the conclusions of an earlier paper [18], we studied only a representative sub-sample of these. One was a mammalian (bovine) sample OC1, which behaves quite similarly to the porcine sample, and two were ‘non-gelling’ (i.e. their gelation temperaTable 1 Sample specifications (data supplied by collaborators) Sample
Code
Mw
Mw/Mn
Percentage imino groups
pI
Bovine Tilapia Cod Cod
OC1 7056 2747 IC
145 700 89 540 60 000 120 500
1.68 1.57 1.33 1.93
25.3 23.3 17.9 17.0
5.00 8.58 7.92 8.90
pI= isoelectric point of gelatin sample. Mw = weight average molecular weight, determined by HPLC. Mw/Mn =polydispersity.
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Fig. 1. Creep compliance J(t) plotted against time, for a 10 w/w% gelatin gel from cod collagen (sample 2747) after 19 h at 2°C. The instantaneous compliance, Jo, the maximum compliance, Jm, and the irrecoverable compliance, Ji, respectively, are indicated.
ducer into the strain value k(t) using the appropriate geometry constant and then evaluates J(t) using Eq. (1), and the applied stress, ~. Buffered values are stored in the instruments own memory, and then downloaded, as required, to an attached PC. In this way, the timedependent shear strain k(t) and compliance J(t), at a given instant, t, in creep and creep recovery, were automatically calculated and stored.
3. Results Fig. 1 shows a typical plot of J(t) and k(t) (righthand axis) vs. t for 10 w/w% cod gelatin sample (2747) at 2°C. Both the creep and recovery phase on removal of the stress can be seen. After the stress is applied, most of the deformation occurs ‘instantaneously’ — actually allowing for instrument and sample inertia, in 0.01s — followed by a slow creep phase. Jo, the instantaneous compliance, is taken as the first stable reading. The maximum compliance, Jm, is measured just before the stress is removed at 5000 s. When the stress is removed, there is an instantaneous elastic response followed by a further, slower, recovery phase. Here, Ji is the irrecoverable compliance and is the value taken at the end of the experiment. The creep curve for the same sample on an expanded scale is shown in Fig. 2. This plot shows some similarity to that of a viscous liquid with linear J(t) at large t. This curve can be used to estimate the viscosity in two ways: (i) from the inverse of the gradient in the creep phase and (ii) from the expression p= t/Ji, where t is the load time (5000 s) [25], and Ji is the irrecoverable compliance defined above.
Fig. 3a and b show double log plots of J(t) vs. t for different concentrations of tilapia (7056) and cod (2747) gelatins, respectively. These plots are relatively flat and close to horizontal with the measurements appearing parallel except for the lowest concentration (1% w/w) of the tilapia gelatin. (In reality, the J(t) versus t data are neither quite parallel nor horizontal, and the slope can be used to estimate the concentration dependence of the creep phase viscosity as we shall see below.) For the 1% w/w tilapia system, there is evidence of damped shear strain oscillations induced by the sudden increase in stress, coupled with the instrument and sample inertia. This is the form of data that Normand and Ravey [20] used to calculate the dynamic moduli, although we have not attempted this because, in our case, data are convoluted with the stress rise time (not instantaneous, but actually around 0.02 s). The oscillations for the higher concentration, lower compliance systems, are damped out more efficiently and are not shown here. For a perfectly elastic (as opposed to viscoelastic) or Hookean system G, the (static) shear modulus must be equal to 1/J, that is when both are time-/frequency-independent, or are measured in the zero frequency limit. The value of either this apparent time independent compliance or the initial compliance, J0, can be compared with estimates of the gel modulus. This procedure is shown (Fig. 4) for four different samples — OC1, tilapia (7056) and the two cod gelatin samples (2747, IC). The compliance has been compared with previously measured small controlled strain oscillatory measurements (as 1/G%) [18]. The closed symbols show the concentration dependence of Jo, whilst the open symbols show how 1/G% varies with concentration. Both sets of data for each sample have been fitted to a
P.M. Gilsenan, S.B. Ross-Murphy / International Journal of Biological Macromolecules 29 (2001) 53–61
second-order polynomial, which shows quite a good fit. Overall, though, there is good agreement between the concentration dependence of compliance and that of the gel modulus from dynamic measurements. The final plot (Fig. 5) shows the concentration dependence of creep phase viscosity. This has been estimated using the two methods described earlier (i) from the creep phase (open symbols) and (ii) from the recovery phase (closed symbols). As we [5] and others [25] have pointed out such a comparison is quite testing experimentally and may, indeed, be used as to assess the instrument reliability. The agreement here, which, except for one of the cod gelatin samples (IC) at the lowest concentration is excellent, can be regarded as creditable. The two, essentially independent, measurements for each sample concentration are in close agreement with each other, with the recovery phase data being slightly higher in each case, a result to be expected experimentally [5]. The slopes of the log p versus log C plots vary from 2 to 3. The difference in absolute values between the instantaneous compliance and creep viscosity for the gelatins from the normal mammalian source — in this case bovine (OC1), the tuna sample (7056) and the two cod gelatin samples (2747 and IC) — reflect the overall difference in gelling behaviour, which is in turn related to their composition. As we showed in earlier papers, the propensity to gel, at a given concentration and MW, increases with the imino acid composition. An extensive discussion of this is given in Ref. [18], but to summarise, although there are significant differences, the gelation characteristics can be related to these factors and the overall behaviour understood if we use the extension of the Eldridge– Ferry gel melting theory, due to Takahashi [26].
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4. Discussion The interest in creep measurements on gelatin gels stems from the fact that over typical oscillatory frequencies (say \ 10 − 2/s), there is no indication of ‘terminal flow’, at least for concentrations above 4C0, and reasonably below the gel melting temperature. Such gels are thus much more solid than typical entangled melts of linear chains. This then begs the question of the exact behaviour at very long times. In our earlier paper [5], we examined the creep behaviour of a series of concentrations from one gelatin sample. We have now expanded our measurements to include a broader range of gelatin samples from different sources. The main practical difference between mammalian and fish gelatins is that fish gelatins have a higher critical gelling concentration, C0, and a lower melting temperature, Tm. (The two are, of course, related since the measured C0 will depend upon the difference, T− Tm and, assuming Tm to be greater than the glass-transition temperature, Tg, C0 will [ \ 100% at T=Tm). It is important, therefore, to choose conditions where the systems can be compared. For example, at say 15°C, some of the present materials are gels, and others are sols. Because of this, a temperature of 2°C was chosen. We were therefore able to include the ‘non-gelling’ cod sample (2747) in our study. As expected, the values of compliance for the cold-water fish are much higher (i.e. the modulus, G, is lower) than for mammalian gelatins. Nevertheless, the approximation J0 1/G% works well (Fig. 4), and fits of log (J0, 1/G%) versus log gelatin concentration to a second-order polynomial are reasonable. As mentioned above, and according to much of the early (and, sadly, still some more recent) literature, the behaviour of gelatin gels can be described by:
Fig. 2. J(t) vs. t for a 10 w/w% cod (2747) gelatin gel on an expanded scale.
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Fig. 3. (a) Log J(t) vs. log t for different concentrations of tilapia gelatin (7056). (b) Log J(t) vs. log t for different concentrations of cod gelatin (2747).
G%: kC 2.
(2)
This equation is only true if C \ C0. Other workers have replaced the C 2 term by C n, where n is an exponent, say 2–3. Unfortunately, this is also incorrect, because as C [ C0, gelation theory predicts that n [ . We would not, therefore, expect single-power (scaling) law behaviour in this regime. Although the theoretical behaviour is more complex, and cannot be written explicitly [7], we would expect that approximately parabolic behaviour would be observed for C 1–5C0, with an apparent low concentration asymptote. This is precisely what is seen in Fig. 4. The viscosity measurements from both the creep phase and the recovery phase are in close agreement for
all samples; the viscosity values for the cold-water fish are much lower than the mammalian samples, as expected. The slopes from each phase are quite similar and vary from 2 to 3. In our earlier paper [5], the creep phase viscosity, p, was found to be 8 C 1.1. The sample used was a high-molecular-weight alkaline processed ossein gelatin (Mw 1.55× 105 and Mw/Mn 2.17), comparable with the bovine OC1 sample used here (Table 1) and the concentration range (2.5–15% w/w was also similar). The values of creep phase viscosity were also of the same order ( 108 Pa s). Experimental conditions were slightly different, however; the curing temperature was 2°C here, as opposed to 15°C in the earlier work, although the same time span of 19
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h was used, before beginning the creep experiment. The creep phase viscosity, p, for all of the OC1 samples was found to be 8C 3.6, although this high value is influenced by the low p for the lowest concentration sample. (If we neglect this point, as we have in Fig. 5, the slope is 2.24). We have no firm explanation for the difference between this and earlier results, other than that the current instrument is both more sophisticated and more sensitive (in strain detection) than that used in the earlier paper. Indeed, there are very few comparable data in the literature; Braudo and co-workers obtained p8 C 3.3 for a range of polysaccharide gels [25] with values in the range of 108 –1011 Pa s, and they comment on the apparent congruence of this exponent with that expected for non-gelling semi-dilute solutions and melts. We argued previously that this could not be a universal result [5], nor was the agreement necessarily more than coincidental, since, at higher concentrations, we would have expected the exponent to decrease. Indeed, there is some evidence in our data of the apparent exponent decreasing at higher concentrations. However, as far as we aware, there is no theory available to predict this exponent for the creep phase viscosity of gels. What is interesting is to consider the various mechanisms that give rise to the creep-phase viscosity, and its high value. For a permanently cross-linked network close to critical gelling conditions, there will a substantial sol fraction — material that is itself of high molecular weight but is not attached to the gel mega-molecule. As the degree of cross-linking increases, both MW and the absolute amount of the sol fraction will decrease (to zero), and the gel fraction will increase.
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Nevertheless, flow of the sol fraction can occur. Even within the gel fraction, not all material is completely attached, and relaxation of dangling ends will occur. Moreover, the amount of material forming dangling ends will also decrease as the degree of cross-linking increases. For physical gels, such as gelatin, there are further complications. Within the branching theory (percolation based) model, which has been applied to gelatin amongst other physical gel systems, the degree of cross-linking is an increasing function of the concentration, so we would predict that the creep-phase viscosity would increase with concentration, although the concentration dependence is less clear. Although there are some structural similarities with, for example, entangled, long-chain branched melts, there are also real differences, and we would not expect (nor do we see) exponential behaviour [27]. The other possibility is that the creep-phase viscosity reflects non-permanence of the physical cross-links, and subsequent re-healing [the modulus of these gels is known to increase even when plotted against log (time)]. As noted above, there is some evidence in our data that the apparent exponent decreases with increasing concentration. However, that cannot hold for all concentrations, since, at some point, we will cross into a glass transition regime. We would expect a still greater viscosity here. Overall, we can only speculate on the effect of the various terms above, but Fig. 6 illustrates our expectation for this behaviour during the transition from sol to gel to glass. Pre-gel, the creep phase viscosity is, of course, simply the zero shear rate viscosity of the whole system.
Fig. 4. Log Jo (closed symbols) and log 1/G% (open symbols) plotted against log concentration; tilapia (triangles), bovine gelatin OC1 (stars), and cod samples IC (circles) and 2747 (squares).
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Fig. 5. Log p vs. log concentration; (open symbols) from creep phase, (closed symbols) from recovery phase; tilapia (triangles), bovine gelatin OC1 (stars), cod samples IC (circles) and 2747 (squares).
5. Conclusions
Fig. 6. Postulated creep viscosity versus concentration behaviour for a gelling system. The extent of the creep-phase regime, and where the glass transition occurs, will depend upon the system and the temperature.
Nevertheless, this is an important issue since it addresses the (quasi-philosophical) question of whether a gelatin gel is really just a highly entangled liquid. This is discussed in more detail in the review of te Nijenhuis [3] and in our recent paper [18]. It would appear that rheological experiments alone cannot answer this question unequivocally, since the time scales are too long; time –temperature superposition cannot be used, and sample stability limits also have to be considered. As we suggested earlier, perhaps the time is now ripe to consider alternative approaches such as gel ‘healing’ experiments [18,28].
Creep measurements have been carried out on gelatins from fish and typical mammalian (bovine) sources. The concentration dependence of the compliance closely mirrors that of the reciprocal gel moduli, suggesting that these gels are not far from being ideal elastic solids. Nevertheless, the concentration dependence of the creep phase viscosity is similar to that of entangled solutions. Further theoretical work is clearly needed to rationalise the apparent contradictions. Despite this, the similarities and differences between piscine and mammalian samples, previously observed by us in oscillatory shear experiments, are maintained.
Acknowledgements The authors acknowledge the European Commission for funding this work under FAIR CT97-3055, and their partners including Dr M. Gudmundsson (IceTec, Iceland), Professor M. Djabourov (ESPCI, Paris, France), Dr P. Montero (Instituto del Frio, Madrid, Spain) and Dr G. Takerkart (SKW Biosystems, Paris, France) for providing both samples and insights, without which this paper could not have been written. We are also grateful to Dr D.S. Field of Croda Colloids, Luton, England for provision of fish gelatin samples. The controlled stress rheometer was provided by the BBSRC under grant F06419.
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[15] Gudmundsson M, Hafsteinsson H. J Food Sci 1997;62:37. [16] Norland RE. In: Voight MN, Botta JK, editors. Advances in Fisheries Technology and Biotechnology for Increased Profitability. Lancaster, PA: Technomic Publishing, 1990:325. [17] Gilsenan PM, Ross-Murphy SB. Food Hydrocolloids 2000;14:191. [18] Gilsenan PM, Ross-Murphy SB. J Rheol 2000;44:871. [19] Ferry JD. Viscoelastic Properties of Polymers, third ed. New York: Wiley, 1980. [20] Normand V, Ravey JC. Rheol Acta 1997;36:610. [21] Kuijpers AJ, Engbers GHM, Feijen J, De Smedt SC, Meyvis TKL, Demeester J, et al. Macromolecules 1999;32:3325. [22] Djabourov M, Leblond J, Papon P. J Phys (France) 1988;49:319. [23] Pezron I, Herning T, Djabourov M, Leblond J. In: Burchard W, Ross-Murphy SB, editors. Physical Networks — Polymers and Gels. London: Elsevier Applied Science, 1990:231. [24] Busnel JP, Clegg SM, Morris ER. In: Phillips GO, Wedlock DJ, Williams PA, editors. Gums and Stabilizers for the Food Industry IV. Oxford: IRL Press, 1988:105. [25] Braudo EE, Plashchina IG, Tolstoguzov VB. Carbohydr Polym 1984;4:23. [26] Takahashi A. Polym J 1972;3:207. [27] McLeish TCB, Milner ST. Adv Polym Sci 1999;143:195. [28] Mowery CL, Crosby AJ, Ahn D, Shull KR. Langmuir 1997;13:6101.
Shear creep of gelatin gels from mammalian and piscine collagens Paula M. Gilsenan, Simon B. Ross-Murphy Biopolymers Group, Di6ision of Life Sciences, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, Waterloo, London SE1 9NN, UK Received 27 November 2000; accepted 18 April 2001
Abstract This paper describes new measurements on the creep rheological behaviour of gelatin gels from both traditional mammalian and piscine sources. Measurements on a series of concentrations of gels were obtained using a high-precision controlled stress rheometer. Results for the concentration dependence of compliance are close to those expected from dynamic oscillatory measurements of gel modulus, assuming ideal elasticity. The concentration dependence of viscosity approximates power law behaviour, with p 8C 2 – 3, lower than the exponent expected for semi-dilute solutions. The apparent contradiction implied by this is discussed and a novel gel viscosity versus concentration state diagram presented. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Creep; Gelatin; Rheology
1. Introduction During the last century, a great deal of experimental work has been performed investigating the structural and mechanical properties of gelatin gels [1 – 3]. Both the structural and mechanical behaviour of thermoreversible gels from the polypeptide gelatin have been widely studied, and the review by te Nijenhuis (1997) [3] summarises much of these endeavours. All the work in his review and almost all in the literature as a whole have, however, been performed on traditional mammalian gelatins with very little focus on samples from alternative sources. Most of this work is also concerned with macroscopic gels, although the approach of de Carvalho and Djabourov [4] tackles the problem of the rheology of gelatin gelation under shear. Only very recently has there been interest in gelatins from nonmammalian, mainly piscine, sources. In the present paper, we summarise some of these data and also examine previous creep measurements performed on conventional mammalian gels. Surprisingly, despite the ready availability of both the samples and of modern * Corresponding author. Tel.: + 44-207-8484081; fax: +44-2078484082. E-mail address: [email protected] (S.B. Ross-Murphy).
controlled stress rheometers, ideal for performing the creep experiment, since the work of Higgs [5] and one of the present authors, very little seems to have been published in the latter area. This is despite the renewed interest in long-time behaviour of gelatin gels and its possible implications for gel healing. In this section, we briefly summarise the main points of gelatin gelation, which are now widely accepted, and then present data on the concentration dependence of creep behaviour and creep recovery for both traditional mammalian samples and a range of fish gelatins.
1.1. Molecular structure of gelatin sols and gels Gelatin is a water-soluble protein that is widely used in the food, pharmaceutical and photographic industries. It is derived by hydrolytic degradation of collagen, the principal component of white fibrous connective tissue [6]. Commercially, both acid and alkaline hydrolysis routes are employed, and most commercial samples are extracted from either bovine or porcine sources. The basic molecular unit of collagen is a triple-helical rod (tropocollagen triple helix). This consists of three a-chains arranged in a left-handed axis, with the whole structure wound into a right-handed super helix. The extraction process involves partially
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hydrolysing the collagen towards the individual a chains, although commercial samples tend to contain a-, b- and g-chains. It is now accepted that the gelation of gelatin involves, at least partly, regeneration of the collagen molecule i.e. reformation of triple helical ‘junction zones’ (renaturation), separated by regions of flexible polypeptide chain. This occurs on cooling a warm solution to below room temperature; a transparent, thermoreversible gel is formed provided that the concentration, C, is greater than the critical gelation concentration, Co [7]. This is typically 0.4– 1.0 w/w% for commercial mammalian gelatins. Above 40°C, a solution of flexible, random coils is obtained. The source and type of collagen influence the properties of the resulting gelatins. The amino acid content and sequence vary from one source to another, but collagen is an unusual protein in that it always consists of large amounts of the peptides proline, hydroxyproline and glycine [8]. The melting and gelling temperature of gelatin have been found to correlate with the proportion of imino acids proline and hydroxyproline (both with a five-membered pyrrolidine ring) in the original collagen [9]. The proline content plays an important role in the stability of gelatin gels, since it promotes formation of the polyproline II helix. This in turn determines the form of the tropocollagen trimer.
Creep measurements [19] can be used to study the long-time behaviour of gelatin gels [5]. In a creep experiment, a fixed stress is applied to the sample ‘instantaneously’, held constant for a given time and then removed. The time-dependent increase in strain is measured as a function of this stress. The behaviour of compliance, J(t), where:
1.2. Gelatins from alternati6e sources
J(t)=
As mentioned above, most commercial gelatins are obtained from either cowhide or pigskin. Consequently, almost all of the work in the literature has been carried out on traditional mammalian samples. Recently, however, there has been considerable interest in using alternative substitutes. It has been known for some years that the un-hydrolysed collagen coil-helix temperature depends upon the proportion of proline and hydroxyproline [9]. This is considerably lower for fish (16– 18%) than for mammals (24%). Cold-water fish, for example cod, have a very low hydroxyproline content and because of this a very low gelling and melting temperature B 15°C. In practice, a typical mammalian gelatin of concentration 5 – 10% w/w will form around ambient temperatures, whilst a cod gelatin under equivalent conditions will only just gel at 2°C [10]. The industrial interest in non-mammalian gelatins in the food industry has followed the recent bovine spongiform encephalopathy (BSE) crisis in the UK. Non-mammalian gelatins, e.g. those derived from fish collagen, have obvious advantages in both minority and ethnic food products (‘vegetarian’, halal and kosher). They also have potential advantages in applications such as the production of pharmaceutical capsules and the photographic industries. In volume terms, the mar-
k(t) is the change (increase) in shear strain with time, t, and ~ is the applied stress, is particularly valuable since it can be used to understand the behaviour of physically cross-linked gels. However, since our work in this journal in 1990 [5], there seems to have been little in the way of subsequent investigation. The paper by Normand and Ravey [20] is, in rheological terms, a fairly complete characterisation using both oscillatory shear and creep data, particularly since they were able to use a numerical transform to superpose time- and frequency-domain data over approximately five decades from t10 − 2 to 103 s. It is, nevertheless, limited to two concentrations of a single (alkaline extracted) gelatin sample. A limited amount of data have also been collected on creep of chemically cross-linked gelatin gels [11,21]. In the present work, we have adopted a strategy more in keeping with our earlier paper [5] by examining a number of different samples, at several different concentrations, but employing a standard rheological protocol. The following practical aspects must be considered when performing such creep experiments on gelatin gels: 1. Time-scale of gel formation. Since gelatin gelation is a kinetic process and continues indefinitely long after the initial ‘setting’ of the gel [3,22,23], sufficient
ket for gelatin is, in fact, split almost equally between food (55%) and non-food, i.e. photographic, pharmaceutical and assorted technical applications, such as adhesives [11]. However, the higher-grade products used in photographic and pharmaceutical applications are of considerably more value to the gelatin producer. A number of papers have appeared recently describing the extraction and purification of gelatins, particularly from fish (and usually from fish skins). Some key references include work on hake and trout [12], plaice, shark and cod [13– 15]. The article by Norland [16], an early pioneer, summarises most of the work prior to 1990. Our own publications [10,17,18] describe the oscillatory shear characterisation of a number of different mammalian and piscine gelatin samples; the present work extends these by employing time domain shear creep measurements.
1.3. Creep measurements on gelatin gels
k(t) , ~
(1)
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time must elapse before beginning the experiment so that any further change during the experiment is negligible. Failure to consider this aspect can mean that the kinetics of gel modulus growth are convoluted (hopelessly) with the time domain data collection. 2. Thermal history. The thermal history has a great influence over the properties of a gelatin gel [3]. Differential scanning calorimetry measurements, for example, have shown considerable differences between gels cooled directly to a low temperature and those cooled in two stages [3,24]. There are also differences for gels cooled to the same temperature at different rates. Thus, the temperature and cooling procedure must be carefully controlled. 3. Applied stress le6el. The stress must be chosen so that the strain is measurable but still remains within the linear viscoelastic region. This requires an estimate of the linear strain. Gel moduli vary with concentration, and at high concentrations, an approximate C 2 dependence (see Section 4) is seen [7]. An estimate of the required stress can therefore be made from the modulus, using the second approximation G( [ 0) = 1/J(t [ ) (also Section 4).
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tures are below room temperature) cod gelatin samples (IC and 2747), and the tilapia (tropical fish) gelatin. The latter is intermediate in behaviour between the cod and bovine samples. Samples were supplied either as the usual coarse gelatin granules or as finer granulated powders. Relevant physico-chemical properties of these are given in Table 1. Solutions were prepared by soaking the pre-weighed gelatin granules in water for 2–3 h at ambient temperature, followed by mechanical stirring for 10–15 min at 70°C to give a final range of concentrations (C) between 1 and 10 w/w%. Shear creep experiments were performed using a Rheometrics Dynamic Stress Rheometer (DSR500, Rheometric Scientific Ltd., UK) with 40 mm diameter and 1.5 mm gap parallel plate geometry. The bottom plate temperature was controlled (heated and/cooled) using a Peltier system with a heatsink, consisting of a Haake thermostatted water bath set at 15°C, to ensure a reproducible time and cooling regime. The Peltier-controlled bottom plate was set to 35°C before adding the hot gelatin solution. The top plate was lowered to give the correct gap and the excess solution removed. A thin layer of silicone oil (Dow Corning 200/10cs) was placed around the exposed surface of the sample to prevent loss of moisture by evaporation. The sample was then cooled at a rate of 0.5°C/min to 2°C. It was maintained at this temperature for exactly 19 h before beginning the creep phase of the experiment. Stresses (~) between 5 and 1000 Pa, depending on the concentration, were applied ‘instantaneously’ to the sample. At higher concentrations, ~ was increased, assuming it to be proportional to C 2. Consequently, the instantaneous strain was never \15% so that, at least by normal criteria, the linear viscoelastic region was not exceeded. The developing strain was measured for 5000 s, the stress was then removed and the recoverable strain measured for a further 5000 s. Data collection made use of the instrument’s ‘zone’ feature, in which the total experiment was divided into eight semi-logarithmic zones (four creep, four recovery). The same number of points is collected in each zone (say 100). In this manner, more data can be gathered in the early stages of the experiment. The instrument converts the instantaneous voltage from the position (strain) trans-
2. Materials and methods The work was carried out as part of a collaborative project funded by the EC, and our partners included commercial suppliers of gelatin as well as biochemists expert in the extraction of gelatin samples from fish species (see Acknowledgements). Two samples of mammalian gelatin (OC1 and AP1) were supplied by SKW Biosystems, France. In this project, the fish samples studied were tilapia, tuna, megrim and cod, ranging in order from tropical to cold water. Most of these are not available commercially, although two of the fish gelatins — cod (2747) and tilapia (7056) — were supplied by Croda Colloids, Luton, UK. In the present work, and following the conclusions of an earlier paper [18], we studied only a representative sub-sample of these. One was a mammalian (bovine) sample OC1, which behaves quite similarly to the porcine sample, and two were ‘non-gelling’ (i.e. their gelation temperaTable 1 Sample specifications (data supplied by collaborators) Sample
Code
Mw
Mw/Mn
Percentage imino groups
pI
Bovine Tilapia Cod Cod
OC1 7056 2747 IC
145 700 89 540 60 000 120 500
1.68 1.57 1.33 1.93
25.3 23.3 17.9 17.0
5.00 8.58 7.92 8.90
pI= isoelectric point of gelatin sample. Mw = weight average molecular weight, determined by HPLC. Mw/Mn =polydispersity.
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Fig. 1. Creep compliance J(t) plotted against time, for a 10 w/w% gelatin gel from cod collagen (sample 2747) after 19 h at 2°C. The instantaneous compliance, Jo, the maximum compliance, Jm, and the irrecoverable compliance, Ji, respectively, are indicated.
ducer into the strain value k(t) using the appropriate geometry constant and then evaluates J(t) using Eq. (1), and the applied stress, ~. Buffered values are stored in the instruments own memory, and then downloaded, as required, to an attached PC. In this way, the timedependent shear strain k(t) and compliance J(t), at a given instant, t, in creep and creep recovery, were automatically calculated and stored.
3. Results Fig. 1 shows a typical plot of J(t) and k(t) (righthand axis) vs. t for 10 w/w% cod gelatin sample (2747) at 2°C. Both the creep and recovery phase on removal of the stress can be seen. After the stress is applied, most of the deformation occurs ‘instantaneously’ — actually allowing for instrument and sample inertia, in 0.01s — followed by a slow creep phase. Jo, the instantaneous compliance, is taken as the first stable reading. The maximum compliance, Jm, is measured just before the stress is removed at 5000 s. When the stress is removed, there is an instantaneous elastic response followed by a further, slower, recovery phase. Here, Ji is the irrecoverable compliance and is the value taken at the end of the experiment. The creep curve for the same sample on an expanded scale is shown in Fig. 2. This plot shows some similarity to that of a viscous liquid with linear J(t) at large t. This curve can be used to estimate the viscosity in two ways: (i) from the inverse of the gradient in the creep phase and (ii) from the expression p= t/Ji, where t is the load time (5000 s) [25], and Ji is the irrecoverable compliance defined above.
Fig. 3a and b show double log plots of J(t) vs. t for different concentrations of tilapia (7056) and cod (2747) gelatins, respectively. These plots are relatively flat and close to horizontal with the measurements appearing parallel except for the lowest concentration (1% w/w) of the tilapia gelatin. (In reality, the J(t) versus t data are neither quite parallel nor horizontal, and the slope can be used to estimate the concentration dependence of the creep phase viscosity as we shall see below.) For the 1% w/w tilapia system, there is evidence of damped shear strain oscillations induced by the sudden increase in stress, coupled with the instrument and sample inertia. This is the form of data that Normand and Ravey [20] used to calculate the dynamic moduli, although we have not attempted this because, in our case, data are convoluted with the stress rise time (not instantaneous, but actually around 0.02 s). The oscillations for the higher concentration, lower compliance systems, are damped out more efficiently and are not shown here. For a perfectly elastic (as opposed to viscoelastic) or Hookean system G, the (static) shear modulus must be equal to 1/J, that is when both are time-/frequency-independent, or are measured in the zero frequency limit. The value of either this apparent time independent compliance or the initial compliance, J0, can be compared with estimates of the gel modulus. This procedure is shown (Fig. 4) for four different samples — OC1, tilapia (7056) and the two cod gelatin samples (2747, IC). The compliance has been compared with previously measured small controlled strain oscillatory measurements (as 1/G%) [18]. The closed symbols show the concentration dependence of Jo, whilst the open symbols show how 1/G% varies with concentration. Both sets of data for each sample have been fitted to a
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second-order polynomial, which shows quite a good fit. Overall, though, there is good agreement between the concentration dependence of compliance and that of the gel modulus from dynamic measurements. The final plot (Fig. 5) shows the concentration dependence of creep phase viscosity. This has been estimated using the two methods described earlier (i) from the creep phase (open symbols) and (ii) from the recovery phase (closed symbols). As we [5] and others [25] have pointed out such a comparison is quite testing experimentally and may, indeed, be used as to assess the instrument reliability. The agreement here, which, except for one of the cod gelatin samples (IC) at the lowest concentration is excellent, can be regarded as creditable. The two, essentially independent, measurements for each sample concentration are in close agreement with each other, with the recovery phase data being slightly higher in each case, a result to be expected experimentally [5]. The slopes of the log p versus log C plots vary from 2 to 3. The difference in absolute values between the instantaneous compliance and creep viscosity for the gelatins from the normal mammalian source — in this case bovine (OC1), the tuna sample (7056) and the two cod gelatin samples (2747 and IC) — reflect the overall difference in gelling behaviour, which is in turn related to their composition. As we showed in earlier papers, the propensity to gel, at a given concentration and MW, increases with the imino acid composition. An extensive discussion of this is given in Ref. [18], but to summarise, although there are significant differences, the gelation characteristics can be related to these factors and the overall behaviour understood if we use the extension of the Eldridge– Ferry gel melting theory, due to Takahashi [26].
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4. Discussion The interest in creep measurements on gelatin gels stems from the fact that over typical oscillatory frequencies (say \ 10 − 2/s), there is no indication of ‘terminal flow’, at least for concentrations above 4C0, and reasonably below the gel melting temperature. Such gels are thus much more solid than typical entangled melts of linear chains. This then begs the question of the exact behaviour at very long times. In our earlier paper [5], we examined the creep behaviour of a series of concentrations from one gelatin sample. We have now expanded our measurements to include a broader range of gelatin samples from different sources. The main practical difference between mammalian and fish gelatins is that fish gelatins have a higher critical gelling concentration, C0, and a lower melting temperature, Tm. (The two are, of course, related since the measured C0 will depend upon the difference, T− Tm and, assuming Tm to be greater than the glass-transition temperature, Tg, C0 will [ \ 100% at T=Tm). It is important, therefore, to choose conditions where the systems can be compared. For example, at say 15°C, some of the present materials are gels, and others are sols. Because of this, a temperature of 2°C was chosen. We were therefore able to include the ‘non-gelling’ cod sample (2747) in our study. As expected, the values of compliance for the cold-water fish are much higher (i.e. the modulus, G, is lower) than for mammalian gelatins. Nevertheless, the approximation J0 1/G% works well (Fig. 4), and fits of log (J0, 1/G%) versus log gelatin concentration to a second-order polynomial are reasonable. As mentioned above, and according to much of the early (and, sadly, still some more recent) literature, the behaviour of gelatin gels can be described by:
Fig. 2. J(t) vs. t for a 10 w/w% cod (2747) gelatin gel on an expanded scale.
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Fig. 3. (a) Log J(t) vs. log t for different concentrations of tilapia gelatin (7056). (b) Log J(t) vs. log t for different concentrations of cod gelatin (2747).
G%: kC 2.
(2)
This equation is only true if C \ C0. Other workers have replaced the C 2 term by C n, where n is an exponent, say 2–3. Unfortunately, this is also incorrect, because as C [ C0, gelation theory predicts that n [ . We would not, therefore, expect single-power (scaling) law behaviour in this regime. Although the theoretical behaviour is more complex, and cannot be written explicitly [7], we would expect that approximately parabolic behaviour would be observed for C 1–5C0, with an apparent low concentration asymptote. This is precisely what is seen in Fig. 4. The viscosity measurements from both the creep phase and the recovery phase are in close agreement for
all samples; the viscosity values for the cold-water fish are much lower than the mammalian samples, as expected. The slopes from each phase are quite similar and vary from 2 to 3. In our earlier paper [5], the creep phase viscosity, p, was found to be 8 C 1.1. The sample used was a high-molecular-weight alkaline processed ossein gelatin (Mw 1.55× 105 and Mw/Mn 2.17), comparable with the bovine OC1 sample used here (Table 1) and the concentration range (2.5–15% w/w was also similar). The values of creep phase viscosity were also of the same order ( 108 Pa s). Experimental conditions were slightly different, however; the curing temperature was 2°C here, as opposed to 15°C in the earlier work, although the same time span of 19
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h was used, before beginning the creep experiment. The creep phase viscosity, p, for all of the OC1 samples was found to be 8C 3.6, although this high value is influenced by the low p for the lowest concentration sample. (If we neglect this point, as we have in Fig. 5, the slope is 2.24). We have no firm explanation for the difference between this and earlier results, other than that the current instrument is both more sophisticated and more sensitive (in strain detection) than that used in the earlier paper. Indeed, there are very few comparable data in the literature; Braudo and co-workers obtained p8 C 3.3 for a range of polysaccharide gels [25] with values in the range of 108 –1011 Pa s, and they comment on the apparent congruence of this exponent with that expected for non-gelling semi-dilute solutions and melts. We argued previously that this could not be a universal result [5], nor was the agreement necessarily more than coincidental, since, at higher concentrations, we would have expected the exponent to decrease. Indeed, there is some evidence in our data of the apparent exponent decreasing at higher concentrations. However, as far as we aware, there is no theory available to predict this exponent for the creep phase viscosity of gels. What is interesting is to consider the various mechanisms that give rise to the creep-phase viscosity, and its high value. For a permanently cross-linked network close to critical gelling conditions, there will a substantial sol fraction — material that is itself of high molecular weight but is not attached to the gel mega-molecule. As the degree of cross-linking increases, both MW and the absolute amount of the sol fraction will decrease (to zero), and the gel fraction will increase.
59
Nevertheless, flow of the sol fraction can occur. Even within the gel fraction, not all material is completely attached, and relaxation of dangling ends will occur. Moreover, the amount of material forming dangling ends will also decrease as the degree of cross-linking increases. For physical gels, such as gelatin, there are further complications. Within the branching theory (percolation based) model, which has been applied to gelatin amongst other physical gel systems, the degree of cross-linking is an increasing function of the concentration, so we would predict that the creep-phase viscosity would increase with concentration, although the concentration dependence is less clear. Although there are some structural similarities with, for example, entangled, long-chain branched melts, there are also real differences, and we would not expect (nor do we see) exponential behaviour [27]. The other possibility is that the creep-phase viscosity reflects non-permanence of the physical cross-links, and subsequent re-healing [the modulus of these gels is known to increase even when plotted against log (time)]. As noted above, there is some evidence in our data that the apparent exponent decreases with increasing concentration. However, that cannot hold for all concentrations, since, at some point, we will cross into a glass transition regime. We would expect a still greater viscosity here. Overall, we can only speculate on the effect of the various terms above, but Fig. 6 illustrates our expectation for this behaviour during the transition from sol to gel to glass. Pre-gel, the creep phase viscosity is, of course, simply the zero shear rate viscosity of the whole system.
Fig. 4. Log Jo (closed symbols) and log 1/G% (open symbols) plotted against log concentration; tilapia (triangles), bovine gelatin OC1 (stars), and cod samples IC (circles) and 2747 (squares).
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Fig. 5. Log p vs. log concentration; (open symbols) from creep phase, (closed symbols) from recovery phase; tilapia (triangles), bovine gelatin OC1 (stars), cod samples IC (circles) and 2747 (squares).
5. Conclusions
Fig. 6. Postulated creep viscosity versus concentration behaviour for a gelling system. The extent of the creep-phase regime, and where the glass transition occurs, will depend upon the system and the temperature.
Nevertheless, this is an important issue since it addresses the (quasi-philosophical) question of whether a gelatin gel is really just a highly entangled liquid. This is discussed in more detail in the review of te Nijenhuis [3] and in our recent paper [18]. It would appear that rheological experiments alone cannot answer this question unequivocally, since the time scales are too long; time –temperature superposition cannot be used, and sample stability limits also have to be considered. As we suggested earlier, perhaps the time is now ripe to consider alternative approaches such as gel ‘healing’ experiments [18,28].
Creep measurements have been carried out on gelatins from fish and typical mammalian (bovine) sources. The concentration dependence of the compliance closely mirrors that of the reciprocal gel moduli, suggesting that these gels are not far from being ideal elastic solids. Nevertheless, the concentration dependence of the creep phase viscosity is similar to that of entangled solutions. Further theoretical work is clearly needed to rationalise the apparent contradictions. Despite this, the similarities and differences between piscine and mammalian samples, previously observed by us in oscillatory shear experiments, are maintained.
Acknowledgements The authors acknowledge the European Commission for funding this work under FAIR CT97-3055, and their partners including Dr M. Gudmundsson (IceTec, Iceland), Professor M. Djabourov (ESPCI, Paris, France), Dr P. Montero (Instituto del Frio, Madrid, Spain) and Dr G. Takerkart (SKW Biosystems, Paris, France) for providing both samples and insights, without which this paper could not have been written. We are also grateful to Dr D.S. Field of Croda Colloids, Luton, England for provision of fish gelatin samples. The controlled stress rheometer was provided by the BBSRC under grant F06419.
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