Critical helix content in gelatin gels

Critical helix content in gelatin gels J. Y. Chatellier*, D. Durand* and J. R. Emery~ *Laboratoire de Chimie et Physicochimie Macromol&ulaire, tLaborawire de Physique de rEtat Condense, Equipes de Recherche Associ~es au CNRS, Universit~ du Maine, Route de Laval, 72017 Le Mans Cedex, France

(Received 18 January 1985; revised 23 April 1985)

Aqueous gelatin solutions of different concentrations have been investiflated at various quench temperatures by viscosity measurements to determine the gel times and by optical rotation measurements to derive the evolution of the helix content by reference to native collaoen. As a result, it appears that the gelation of the different aqueous gelatin solutions tested takes place at a common helix concentration independent of the initial gelatin concentration and quench temperature. Further, for each concentration, the dependence of gel time as a function of quench temperature has revealed the existence of two domains: a higher temperature domain where gel times increase strongly with quench temperature and a lower temperature domain where gel times are short and only slightly dependent on quench temperature. Keywords: Gelatin; gels; viscosity; optical rotation

Introduction The reversible formation of weak gels (physical gelation) is a very important process in living systems and more specifically in biopolymers. The thermoreversible sol-gel transition of aqueous gelatin solutions is an in vitro example of such a phenomenon. Because of the technological importance of gelatin gels and the fundamental aspects involved in the gelation process, much attention has been paid in the literature to the renaturation process of dilute gelatin solutions (see for example Rds 1 and 2). Recent investigations by polarimetric experiments3'4, theological properties measurements 5,6'7, d.s.c, a and n.m.r. 9 have been devoted to the elucidation of the formation process of these physical gels; but the detailed mechanism is still not fully understood. Gelatin is a protein resulting from the denaturation of collagen. The basic structure of the collagen molecule is made of three strands, each having a molecular weight of 10~. Each strand is twisted into a left-handed helix of about 9 A pitch. All three strands are themseves wrapped into a super-right-handed helix with a pitch of 86 A. This structure is stabilized by intramolecular hydrogen bonds. At a temperature above its melting point, Tm=36°C 3, collagen loses its ordered structure and is soluble in water; the chains collapse and adopt a random coil conformation identical to gelatin in solution above 40°C. Inversely, on cooling an aqueous gelatin solution below 40°C, gelation will occur if the concentration is above a certain limit depending on the nature of the gelatin used. During this sol-gel transition, the viscosity of the medium increases strongly and an elastic behaviour appears. Electron microscopy of the gel discloses the presence of fibrillar crystals made up of strands of regenerated collagen 1°. Thus the gelation process involves the for0141--8130/85/050311--04503.00 © 1985 Butterworth & Co. (Publishers) Ltd

mation of junction zones containing mainly assemblies of physically associated triple helices. The transition from coil to helix is easily detected by an important change of optical rotation. In this paper, attention is focused on the helix concentration at gel of aqueous gelatin solutions for different concentrations and quench temperatures. The gel times have been determined from viscosity measurements and the associated helix concentrations from polarimetric measurements. Experimental

Material The gelatin used was provided by Prolabo and its characteristics kindly determined by Society Rousselot are as follows: moisture content, 11%; pH at the isoelectric point, 4.9; bloom test, 175; ashes, 0%. The molecular weight distribution was obtained by high pressure size exclusion chromatography 14 (Figure 1) and the number and weight average molecular weights are respectively Mn = 38.00 and M,,-- 150.000. Techniques Optical rotation. The optical rotation measurements were performed with a Jobin-Yvon ILl numerical micropolarimeter. The optical rotation angle at was determined at wavelength 2 = 383 nm and was directly recorded. The cell used had a volume of 0.2 ml and an optical path of 0.17 din. The temperature regulation was provided by an external bath circulating into the jacketed cell. The temperature was directly measured in the cell by a thermocouple. The time required for complete thermal equilibration was estimated at about 30 s.

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14scosity. The viscosity measurements were performed rapidly (measure time, 15 s) every 2 min by using a classical rotational viscometer at a low shear rate. The temperature was regulated at 0.1°C. Sample preparation The solutions were prepared by weighing gelatin in an Erlenmeyer flask and adding the amount of water needed. (In this study, the concentrations are determined as the weight fraction of gelatin.) The swollen gelatin was dissolved by heating the mixture at 50°C for about 30 min. As it is well known in gelatin gels 3'5, some parameters characterizing the gels, especially the gel time, depend on the thermal history of the sample. Initially, the experimental conditions to eradicate the thermal history of the tested samples were determined by size exclusion chromatography (s.e.c.) analysis. As Figure 2 points out, the s.e.c. chromatograms of samples having never gelled and samples having undergone one and two gelations (by quenching at 15°C during 30 min), are not identical after heating at 45°C for 30 min (Figure 2a). Inversely, the s.e.c. chromatograms of identical samples heated at 50°C for 30min are quite similar (Figure 2b). So the holding of samples at 50°C for 30 min is required to erase the thermal history of the material.

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Results Optical rotation measurements The variation of optical rotation of gelatin solutions has been observed as a function of time for four concentrations 1.3, 2, 6 and 11% w/w at different quench temperatures. The ageing time, t, is defined as the time which elapses after the quench of the sample from 50°C to the temperature of study, T. In this work, the specific optical rotation, ~, measured at 589 nm is defined by:

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Critical helix content in gelatin gels: J. Y. Chatellier, D. Durand and J. R. Emery and in solution at T > 4 0 ° C (completely denatured or gelatin) respectively. At 589 nm, the values used in this study are [g]ho,x = - 3 5 0 ° C (Ref. 12) and [~t]coil= - 140°C. F o r any concentration and ageing time, [ot] = - 140 ° for any quench temperature 40°C < T < 50°C. As examples, Figures 3 and 4 give the evolution of the helix content for 6 and 11~o w/w aqueous gelatin solutions, respectively quenched at different temperatures over a period of 4 h. The evolution of the helix contents exhibited by these plots shows that the kinetics of renaturation are strongly dependent on the quench temperature: in these cases, a higher concentration

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Hscosity measurements Changes in the viscosity of aqueous gelatin solutions were investigated as a function of the ageing time for the different concentrations and temperatures studied in polarimetric measurements. F o r a given sample, the absolute values of viscosity were found to depend on experimental conditions (shear rate, frequency of measurements, nature of the viscometer used) indicating that the measuring method partially destroys the gel structure. However, for each sample tested, the viscosity shows a maximum which occurs at the same time independent of the experimental conditions; these experimental conditions affect only the value of the measured viscosity. As examples, Figures 5 and 6 show the evolution of the viscosity as a function of time for 6 and 11% w/w aqueous gelatin solutions. This maximum has been attributed to gelation threshold and the associated time defined as gel time;

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Critical helix content in 9elatin 9els: J. Y. Chatellier, D. Durand and J. R. Emery IO

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indeed, for all the tested concentrations and temperatures, this time corresponds to the exact moment when a test, sample, placed under the same experimental conditions, stops flowing. In addition, this time corresponds also to the maximum of viscosity measured by using a moving sphere microrheometer at very low shear rate (typically 10 - a S - 1). Figure 7 shows the gel times as a function of temperature for the four tested concentrations.

Discussion For each concentration, the helix contents connected with the gel times, called critical helix contents hs, are independent of temperature as shown in Figures 3 and 4. At a lower temperature, the helix content increases rapidly and, as the gel time is short, the determination of the critical helix content is less accurate than at higher temperatures. But the evolution of the helix content as a function of relative time ( t - tc)/tc leads, for all the tested temperatures, to a superimposition of the plots on a common master curve. So the critical helix content can be considered as constant even for short gel times. The dependence of the critical helix content hs on the dilution c- 1 is given in Figure 8. This plot shows that the critical helix content is a linear function of the dilution c-t. Thus the critical helix concentration Chs=h~ c is constant in the tested gelatin samples. The extrapolation to 100% helices allows the evaluation of a limit dilution just over 100. Below this value, a macroscopic network cannot be formed; however, in this dilute region, the formation of microgels after a long ageing time at a low quench temperature has been observed. In addition, for each concentration, variation of gel time with quench temperature showed two behavioural domains: a higher temperature domain where the gel times increase very strongly with the quench temperature and a lower temperature domain where the gel times are short and only weakly dependent on the quench temperature over a large temperature range. As an example, in a 6% w/w solution, the gel time is 1.5 times greater for an increase of I°C around a quench temperature of 20°C whereas it is ten times greater for a similar increase around 24°C (Figure 7). At a higher temperature which

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Figure 9 Evolutionof specificoptical rotation or helixcontent as a function of quench temperature for differentageing times of an 11% w/w aqueous gelatin solution corresponds to long gel times, the specific optical rotation, or the helix content, increases slowly and progressively with time (Figure 9). Inversely, at lower temperatures, which corresponds to short gel times, the helix content increases strongly immediately after the quenching and then considerably more slowly. In Figure 9 the region helix content-temperature where the tested sample (i.e. 11% w/w aqueous gelatin solutions) exhibits a gel fraction has been delimited from the region where all the molecular species are soluble.

Conclusion This study has shown that, for each concentration tested, the evolution of the examined parameters versus time exhibits two behaviours of growth according to whether the aqueous gelatin solutions were quenched at a low or a high temperature. Furthermore, the gelation of aqueous gelatin solution under different experimental conditions (temperature, concentration) has been found to occur at a common helix concentration, In a following paper x3, some aspects of the mechanism of renaturation will be examined in relation to these observations.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Veis, A. 'The Macromolecular Chemistry of Gelation', Academic Press, New York and London, 1964 Eagland, D., Pilling, C., Suggett, A. and Wheeler, R. G. Disc. Farad. Soc. 1974, 67, 181 Djabourov, M. and Papon, P. Polymer 1983, 24, 537 Godard, P., Biebuyck, J. J., Barriat, P. A., Naveau, H. and Mercier, J. P. Makromol. Chem. 1980, 181, 2009 Te Nijenhuis, K. Colloid Polym. Sci. 1981,/,59, 522 Te Nijenhuis, K. Colloid Polym. Sci. 1981, 259, 1017 Laurent, J. L. Thesis, Toulouse, 1981 Godard, P., Biebuyck, J. J., Daumerie, M., Naveau, H. and Mercier, J. P. J. Polyra. Sci. Polym. Phys. Edn. 1978, 16, 1817 Maquet, J. Thesis, Paris, 1983 Tomka, I., Bohonek, J., Spuhler, A. and Ribeaud, M. J. Photogr. ScL 1975, 23, 97 Umed, B. and Doty, P. Adv. Protein Chem. 1961, 16, 401 Flory, P. J. and Weaver, S. E. J. Am. Chem. Soc. 1960, 82, 4518 Durand, D._, Era_cry, J. R. and Chatellier, J. Y. Int. J. Biol. Macromol. 1985, 7, 315 Lorry, D. and Vadrine, M. Congress I.A.G. - Fribtirg, Sept. 1983