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The rheology of gelatin hydrogels modified by κ-carrageenan
LWT - Food Science and Technology 63 (2015) 612e619
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The rheology of gelatin hydrogels modified by k-carrageenan Svetlana Rostislavovna Derkach a, Sergey Olegovich Ilyin b, *, Alexandra Alexandrovna Maklakova a, Valery Grigoryevich Kulichikhin b, Alexander Yakovlevich Malkin b a b
Murmansk State Technical University, Sportivnaya ul. 13, Murmansk 183010, Russia A.V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninskii prospekt 29, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 16 January 2015 Accepted 9 March 2015 Available online 17 March 2015
We studied the impact of anionic polysaccharide (k-carrageenan) as a co-gelator of gelatin at different physico-chemical and by varying the k-carrageenan/gelatin ratio. It has been shown that increasing kcarrageenan concentration accelerates the gelation and leads to significant increase in the viscoelastic parameters of the modified gels. Non-linearity of viscoelastic properties becomes evident in much lower deformations in comparison with those of native gelatin gels. The strength of gels characterized by the yield stress also increases along with k-carrageenan concentration. The deformation (but not flow) of gels happens along the rupture surfaces appearing at the apparent yield stress. The melting temperature increases until 45 C for high k-carrageenan/gelatin ratio. The suggested explanation of the observed changes in properties of modified gels relates these effects with formation of complexes of gelatin and polysaccharide. FT-IR spectroscopy data show that (bio)polyelectrolyte complex formation is due to electrostatic interaction between positively charged groups in gelatin and negatively charged sulfate groups in k-carrageenan that leads to conformation changes of gelatin macromolecules. Micrographs (scanning electron microscopy) show that addition of even a small quantity of polysaccharide leads to radical changes in supramolecular structure of modified gels. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hydrogel Gelatin Polysaccharide Carrageenan Rheology
1. Introduction Application of native gelatin has definite restrictions determined by the required gelation rate, thermal stability, and rheological properties. So, it is necessary to modify gelatin as a gelator. This issue is answered by combination of gelatin with other component. For instance, gelatin can be modified by using some chemical cross-linking agents such as chloranhydride or formaldehyde (De Carvalho & Grosso, 2004). They are effective modifiers but not acceptable in food industry. Other method consists in addition of surfactants or biopolymers compatible with gelatin. One of the promising methods is modification of gelatin properties by adding polysaccharides as co-gelators. Their compatibility with gelatin is provided by interaction of charged gelatin and polysaccharide macroions leading to formation of (bio)polyelectrolyte complexes (Marrs & Pegg, 1997; Michon, Cuvelier, Launay, Parker, & Takerkart, 1995; Morris, 1990). The latter are capable for self-organization and gelation under a variety of * Corresponding author. Tel.: þ7 495 9554191; fax: þ7 495 6338520. E-mail address: [email protected] (S.O. Ilyin). http://dx.doi.org/10.1016/j.lwt.2015.03.024 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
external conditions (Izumrudov, 2008; Kabanov, 2005). This way opens up new possibilities for modification of rheology, melting temperature, gelation rate, and microstructure of gelatin-based products in its combined application with different ionic polysaccharides. Numerous studies showed that addition of polysaccharide in gelatin-based gels affects rheological properties of compositions. So, if conditions of (bio)polyelectrolyte complexes in aqueous phase are provided, the yield stress and viscosity of gels increase with increasing polysaccharide concentration as demonstrated for sodium alginate (Boanini, Rubini, Panzavolta, & Bigi, 2010; Derkach, 2014; Derkatch, Voronko, & Izmailova, 2001). Some studies showed that the elastic modulus of gelatin mixtures with i-carrageenan or k-carrageenan exceeds the sum of moduli of the individual components (Haug, Draget, & Smidsrod, 2004; Michon, Cuvelier, Launay, & Parker, 1996). It was also shown that strength of films obtained from fish gelatin increases by addition of kcarrageenan or gellan (Pranoto, Lee, & Park, 2007). Simultaneously, the melt temperature also increases. It was supposed that these effects are due to electrostatic interactions between the biopolymers and changes in gel microstructure.
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Modification by polysaccharides can also come out as the plasticizing effect. For example, if gellan (Lau, Tang, & Paulson, 2001), sodium alginate (Panouille & Larreta-Garde, 2009) or agar (Saxena, Kaloti, & Bohidar, 2011) is used at conditions not providing polyelectrolyte complexes formation, the strength of gels decreases in comparison with that of native gelatin gels, and gelation temperature also decreases (Harrington & Morris, 2009). One of the promising polysaccharides for creating gellous products is k-carrageenan which is also a gelator (Brandrup, Immergut, & Grulke, 1999; Yermak & Khotimchenko, 2003). The study is motivated by the use of gellous gelatine based food products. A possibility of their application is determined by the rheological properties of gels. Viscometric measurements provide information concerning viscosity levels before and after gelation that is important for processing. Dynamic low-amplitude tests characterize the rigidity of a material. A new perspective method of testing is large amplitude oscillatory stain (LAOS) measurements which give information about evolution of the rheological properties in time (or strain) (Ewoldt, Hosoi, &. McKinley, 2008). Different version of treating the results of the LAOS measurements was proposed in the current literature. One of them is the presentation of experimental data in the form of the LissajouseBowditch figures which are the dependence of stress on strain. A version of LAOS method based on the analysis of the LissajouseBowditch figures was recently developed in (Ilyin, Malkin, & Kulichikhin, 2014). At low deformation, these figures are ellipses. Distortion of these figures characterizes non-linear viscoelastic properties of a material under study (Ewoldt & McKinley, 2010; Kim, Merger, Wilhelm, & Helgeson, 2014). The goal of this work is a systematic study of viscoelastic properties of gels formed from gelatin and k-carrageenan with increasing the polysaccharide/gelatin ratio. We examine the impact of kcarrageenan on rheological, thermal stability, and gelation kinetic properties of k-carrageenan/gelatin gels. Properties of gelatine based products depend on interaction between their components including formation of (bio)polyelectrolyte complexes. So, structure estimations appear the necessary part of this study. 2. Material and methods 2.1. Materials An alkaline grade gelatin Type-B from bovine skin 225 Bloom (SigmaeAldrich, Steinheim, Germany) was used. The average molecular weight of gelatin was M v ¼ 1:0$105 g=mol. pH at the isoelectric point was 4.9. Samples of k-carrageenan (SigmaeAldrich) with average molecular weight M v ¼ 6:8$105 g=mol was used without additional purification. 2.2. Methods 2.2.1. Preparation of solutions and gels Aqueous solutions of gelatin and k-carrageenan were prepared separately. Initially, samples of given weight swelled in distillated water at 20 C during 1 h. Then they were dissolved at elevated temperatures, 50 C and 80 C, respectively. This protocol allowed us to obtain homogenous sols of both components. After that, k-carrageenan solutions added to gelatin solutions in ratios corresponding to aspirational biopolymer concentrations in sampled for further study. Gels were obtained from native gelatin (gelatin gels) or from gelatin with added k-carrageenan (modified gels) solutions under cooling to 14 C. All studies were carried out for gels with gelatin concentration, CG, from 1 to 2 g/100 g of gel and k-carrageenan concentration, CK, varied in the range from 0.05 to 1 g/100 g of gel.
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2.2.2. Rheological measurements Rheological properties of gelatin and modified gels were measured at shear deformation by Physica MCR301 (Anton Paar, Graz, Austria) rheometer using the cone-and-plate working unit (diameter of a cone was 50 mm and angle between the conical surface and the plate was 1 grad). Measurements were carried out in the following deformation modes: periodic oscillations at constant temperature (14 C) with varying amplitude, g, at constant frequency, u, or varying frequency, the range of g was 0.1e1000% and u was 0.0628e628 s1; large-strain components of the complex (dynamic) modulus were calculated according to Ilyin, Kulichikhin, and Malkin (2014); temperature scanning at g ¼ 1% and u ¼ 6.28 s1 at constant temperature of 14 C for following “aging” of samples or at increasing temperature at the rate of 2 K/min; shearing of a sample _ or stress-controlled (s) mode in at 14 C in the rate controlled (g) the range of 103e104 s1 and 0.01e200 Pa, respectively. The relative error in measuring apparent viscosity and the components of the dynamic modulus did not exceed 10%. The variation of the given temperature was within ±0.1 C. Reproducibility of the results of the rheological measurements was controlled by parallel testing of two samples of the same content. 2.2.3. Fourier transform infrared (FT-IR) analysis FT-IR spectra of the sample under study were registered with IRFourier spectrometer Nicolet 700 (Thermo Scientific, Madison WI, USA) in the middle range of 400e4000 cm1 IR radiation. FT-IR spectra were measured using dried samples obtained from gelatin and modified samples. The following protocol of sample preparation was used. Hydrogels were maintained at 12 C during 12 h. Then they were frozen at 6 C, unfrozen in dark and centrifuged. The formed sediment dried in a drying cabinet at 50 C for 5 h and finally at 25 C for 20 h. Obtained dry films grinded in a ball mill till the state of highly dispersed powder and pressed this powder with KBr forming a pellet which was used for FT-IR spectroscopy measurements. 2.2.4. Microstructure observation Gel microstructure was examined by the scanning electron microscopy (SEM) using a microscope S405-A (Hitachi, Tokyo, Japan) supplied with the SEM LEO-420 software. Gel samples preliminary stored in a desiccator at 12 C for 1 day. Before examination, sample of the 3 3 3 mm size were frosted (in vacuum) and then metalized with thin (app. 20 Å) gold layer under vacuum. 3. Results and discussion 3.1. Solegel transition at constant or increasing temperature At temperatures below 30 C, gelatin macromolecules undergo a conformation coil / helix transition (rigid form). As a result, its aqueous colloidal solutions transforms to gels if the gelatin concentration exceeds some critical threshold (Izmailova et al., 2004; Ross-Murphy, 1997). Such a sol / gel transition happens due to formation of intermolecular hydrogen bonds between carboxyl oxygen and amid hydrogen in polypeptide chain (at high content of glycineeprolineehydroxyproline amino acids). Beside, gelatin macromolecules loss solubility at cooling owing to their transition into the rigid helix conformation. These processes lead to phase decomposition and formation of a spatial network structure based on a biopolymer phase with the presence water entrapped (Gilsenan & Ross-Murphy, 2000). The k-carrageenan addition to aqueous gelatin solutions affects the kinetics of the system's gelation. Fig. 1 demonstrates that addition of k-carrageenan to gelatin solution increases the rate of
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Fig. 1. Kinetics of gelation as followed by an increase of the dynamic modulus G* normalized by its limiting value, G*t/∞. CG ¼ 2 g/100 g, concentrations of k-carrageenan, CK, are shown at the figure.
gelation in the isothermal conditions as followed by changes in the time dependence of the complex elastic modulus, G*(t) for samples with different k-carrageenan content. The gelation is reversible. In this study we follow the solegel transition (or conditionally, gel “melting”) by decrease in the storage and loss moduli by temperature scanning. Fig. 2 illustrates the experimental data. The “melt transition temperature” is assumed as the crossover point of both components of the dynamic modulus, as shown in Fig. 2. The dependence of the melt temperature on the k-carrageenan concentration in modified gel is shown in Fig. 3. As seen, this dependence is not smooth and the sharp increase in the solegel transition temperature happens at the k-carrageenan concentration above 0.1 g/100 g or at the gelatin/carrageenan ratio of 10:1. So, experimental data clearly show that addition of k-carrageenan strongly affect the thermo-stability of k-carrageenan/ gelatin systems. It is reasonable to suppose that the basic reason for this effect is formation of (bio)polyelectrolyte complexes and transformation of supramolecular structure of materials under study depending on their composition. The similar increase in the melt temperature with increasing k-carrageenan (Pranoto et al.,
Fig. 2. Temperature dependencies of the storage modulus for native gelatin gel (CG ¼ 1 g/100 g) and modified gels. The k-carrageenan concentration added to the gelatin gel, С K, is at the figure. Rate of temperature increase was 2 K/min.
Fig. 3. Concentration dependence of the melt point for the k-carrageenanegelatin gel (С G ¼ 1 g/100 g).
2007) or sodium alginate concentration (Derkatch et al., 2001) was also observed for more concentrated gelatin gels. 3.2. Viscoelasticity of modified gels The measurements of the viscoelastic properties of the systems after gelation show that they are physical gels. It is seen that in the linear domain of viscoelastic behavior, the storage modulus practically does not depend on frequency that is the direct evidence of the solid-like state of a material (Fig. 4). This is true for modified gels as well for a native gelatin gel. The storage modulus (“rigidity”) of modified gels is higher than modulus of individual components, i.e. structure formation in a modified gels e system of (bio)polyelectrolyte complexes demonstrates the synergetic effect: G0 GþK > G0 G þ G0 K. Non-linear viscoelastic behavior of the modified gels was studied by the method of large amplitude oscillatory shear (Dimitriou, Ewolt, & McKinley, 2013; Hyun et al., 2011). Large-strain storage and loss moduli are presented in Fig. 5 as functions of the amplitude of deformation. It is interesting to note that native gelatin gels maintain linearity of viscoelastic behavior up to rather large deformation of app. 60%, while modified gels demonstrate non-linearity at deformations lower by a decimal order.
Fig. 4. Frequency dependencies of the storage modulus and the loss modulus kcarrageenan, gelatin and k-carrageenanegelatin complexes; gelatin and carrageenan concentrations are shown at the figure.
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Two examples of the LissajouseBowditch figures built for gelatin gel (CG ¼ 2 g/100 g) and a modified gel (CG ¼ 2 g/100 g plus CK ¼ 1 g/100 g) are presented in Fig. 6. As was said above, the form of non-linear LissajouseBowditch figures can be used for characterization of the material behavior. At the amplitude of deformation of 10%, the LissajouseBowditch figure for the gelatin gel is practically the straight line. It means that the reaction of a material is purely elastic, while the behavior of a gel modified by carrageenan is represented by the ellipse-like figure. It means that such a gel demonstrates also viscous response. An increase in the deformation amplitude leads to the increase in area of the figure that reflects growth of dissipative losses. So, generally speaking, the increase in the share of the modifying agent leads to the decrease in elasticity and increase in plasticity of a gel. Besides, the shape of the figure changes and this allows us to evaluate the behavior of a gel as pseudo-plastic (Ilyin,
Fig. 5. Amplitude dependencies of the large-strain storage (a) and loss modulus (b) at u ¼ 6.28 s1 for native gelatin gels, С G ¼ 2 g/100 g and modified gels containing kcarrageenan; CK, are at the figure.
One can see that is the decrease in the storage modulus is monotonous along with increasing deformation, then, the loss modulus passes through a maximum. This reflects changes in the material structure formed by van der Waals bonds. After destroying intermolecular bonds at some critical deformation, the orientation of macromolecular chains starts and this leads to the decrease in the loss modulus. According to the classification proposed by (Hyun, Kim, Ahn, & Lee, 2002), this type of materials is treated as media with weak strain overshoot. Addition of little quantity of k-carrageenan to a rather concentrated gel (gelatin concentration of 2 g/100 g) results in the increase in the loss modulus but not the storage modulus and the contraction of the linear domain of viscoelasticity. So, it is possible to think that little addition of k-carrageenan change one intermolecular bonds existing in the gelatin gel for others which incorporate a new polymer but do not increase significantly their common quantity. However, is the gelatin concentrations not high enough (Fig. 4), the introduction of k-carrageenan still increases the storage modulus. Moreover larger quantities of k-carrageenan, of the order of 1 g/100 g, do increase the storage modulus of the gel (Fig. 5). The gelatin/carrageenan ratio responsible for the sharp changes in the gel properties is not constant but depends on the gelatin concentration. For example, if this ratio is 10/1 for the gelatin concentration of 1 g/100 g (Fig. 3), then in double increasing the gelatin concentration, it appears to add much more carrageenan and the critical gelatin/carrageenan ratio is reduced to 2/1.
Fig. 6. The LissajouseBowditch figures for CG ¼ 2 g/100 g gel (a) and CG ¼ 2 g/100 g plus CK ¼ 1 g/100 g gel (b). Amplitudes of deformation are shown at the figures.
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Malkin, et al., 2014). So, the gelatin gel modification by carrageenan leads to the increase in plasticity at rather moderate deformations (10e100%). 3.3. Flow curves and yielding of modified gels Flow curves measured in the stress-control scanning modes contain clearly expresses domains of the maximal and minimal Newtonian viscosities, and also the yield stress, sY (Fig. 7). The addition of small amount of k-carrageenan slightly decreases the yield stress while an increase in the k-carrageenan concentration leads to the considerable increase in the yield stress and the maximal Newtonian viscosity. Meanwhile it is worthy to discuss the physical meaning of the “maximal Newtonian viscosity”. In several publications, it was proven that “observed” flow of multi-component dispersed systems at low stresses (below the yield stress) is an artifact of the transient deformation regime caused by limited time of observation insufficient for steady flow. If deformation at the given shear rate continues the yield stress but not Newtonian flow is reached. This phenomenon was observed for very different systems e gels (Malkin, Ilyin, Roumyantseva, & Kulichikhin, 2013), suspensions (Malkin, Ilyin, Semakov, & Kulichikhin, 2012; Ur'ev, Svistunov, Potapov, & Starikov, 2007), emulsions (Masalova, Taylor, Kharatiyan, & Malkin, 2005), and dispersions in a viscoelastic medium (Ilyin, Pupchenkov, Krasheninnikov, Kulichikhin, & Malkin, 2013). So, in our case discussed in this study, one should treat the “Newtonian” plateau critically especially bearing in mind solid-like behavior of gels at low deformations (Fig. 4 and related discussion). The domain of the low (minimal) Newtonian viscosity also should be treated with caution. Indeed, general understanding of this domain as homogenous flow with completely destroyed structure can be erroneous because many experimental facts show that in reality, flow in this domain occurs in a stratified (manylayered) mode as was observed for gels (Ilyin et al., 2011) or suspensions (Ilyin, Malkin, & Kulichikhin, 2012). In scanning rate-control mode, “flow curves” appear nonmonotonous that reflects the absence of steady flow and steady (homogenous) state of these materials. This seeming “strange” character of flow curves indicates the breakup of a sample and discontinuities in a bulk. Really, in this case it is rather dangerous to speak about “flow” of gels but maybe about sliding along the lines of ruptures. Quantitatively, the modifying effect of k-carrageenan on mechanical properties of gelatin gels can be characterizes by the two
Fig. 7. Flow curved obtained in the scanning stress-control mode for native gelatin С G ¼ 1 g/100 g gels and modified gels with k-carrageenan; С K, are at the figure.
connected parameters e the storage modulus and the yield stress. As is seen, both are growing along with the increase of the kcarrageenan concentration demonstrating the increase of the strength and rigidity of the gel network of (bio)polyelectrolyte complexes. As was discussed above, the significant changes in the rheological properties of gels can be reached at some definite gelatin/carrageenan ratio. However the presentation of experimental data in log scale shows that that these data are well described rather by the exponential law (Fig. 8) than has threshold. 3.4. FT-IR spectroscopy of gels Intermolecular interaction between gelatin and k-carrageenan is confirmed by the FT-IR spectroscopy data. Corresponding experimental data are presented in Fig. 9, where spectra for native gelatin gels and modified (by k-carrageenan) gels are compared. Attribution of lines in the IR-spectrum of k-carrageenan (Fig. 9a) is based on the data of publication (Sen & Erboz, 2010). Main bands correspond to ester sulfate group (1263 cm1), 3,6-anhydridegalactose group (928 cm1), and D-galactose-4-sulfate group (848 cm1). Wide adsorption band with maximum at 3420 cm1 corresponds to vibration of the hydroxyl group in k-carrageenan. The main lines in the native gelatin spectrum (Fig. 9b, line 1) are wide band with peak at 3400 cm1 (vibration of the HN-group), characteristic adsorption at 1654 cm1 (Amide I, stretching vibrations of CO and CN groups), 1541 cm1 (Amide II, NeH and CN vibration), and 1230 cm1 (Amide III) (Prystupa & Donald, 1996; Segard & Isaksson, 2004). The most useful analytical line for characterization of the secondary structure in protein (and gelatin) is the Amide I band (Muyonga, Cole, & Duodu, 2004). As seen in Fig. 9b, addition of k-carrageenan to gelatin leads to the shift of Amide I band to the low frequency side till the frequency of 1652 cm1. The Amide II adsorption line also shifts to the low frequency side till the frequency of 1539 cm1. It is necessary to note the low-frequency shift of the adsorption band of sulfate groups of k-carrageenan till 1238 cm1. The observed shifts indicate interaction between positively charged amide groups in a polypeptide chain of gelatin and negatively charged sulfate groups in k-carrageenan with formation of (bio)polyelectrolyte complexes. Shift of the Amide I band into the low frequency domain observed for modified (by k-carrageenan) gels in comparison with a native gelatin gel suggests that the conformation state of gelatin macromolecules changes with an increase of the order structures
Fig. 8. Dependencies of the yield stress sY and the storage modulus G0 on the kcarrageenan concentration added to the gelatin gel (С G ¼ 1 g/100 g).
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Fig. 9. FT-IR spectrum for samples of (a) k-carrageenan; (b) native gelatin С G ¼ 2 g/100 g (1), and mixtures of gelatin С G ¼ 2 g/100 g with k-carrageenan, С K, g/100 g: 0.1 (2), 0.5 (3), 1 (4).
share as the result of complex formation. Evolution of the conformation state obliged to modification and an increase in intermolecular contacts might be the cause of the increase of the strength and viscoelastic parameters of modified gels. 3.5. Gel microstructure e electron microscopy patters Typical SEM microphotographs (in the same scale) of morphological patterns of 20 g/100 g native gelatin gel and the same gel with addition of only 0.5 g/100 g of k-carrageenan are compared in Fig. 10. One can say with confidence that the difference is striking. Supramolecular structure of native gelatin formed due to the self-organization process is characterized by transversal collagenlike helix with concentration dependent size (Fig. 10a). The fibrils
form a network. Gel structure also includes discrete zones (cells) distributed along fibrils inside a network, which presumably consist mainly of non-helical pieces of macromolecules. It is seen that addition of even small quantity k-carrageenan leads to radical changes in the gel structure (Fig. 10b). Polysaccharide initiates some interactions between helix zones in gelatin due to appearance of k-carrageenanegelatin polyelectrolyte complexes. Similar transformations of the morphological pattern can be observed by application of other polysaccharides, for instance, gellan (Pranoto et al., 2007) and sodium alginate (Voron'ko, Derkach, & Izmailova, 2002). The modification mechanism of k-carrageenan addition is associated with the evident hardening of the gelatin gel structure and it was quite demonstrative in the results of rheological studies.
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One can believe that contacts with k-carrageenan in a modified gels are stronger in comparison with hydrogen bonds formed by gelatin that leads to the formation of the interactions between helix zones in macromolecules. This results in the increase of viscoelastic properties and reduction of the linear domain. Also, this increases the rigidity and plasticity of a gel. The introduction of a greater quantity of carrageenan leads to the increase in mechanical properties of a material due to the increase of the hydrogen bonds. Acknowledgments The reported study was partially supported by the Russian Foundation for Basic Research, the grant number is 14-08-98811 r_North_a. References
Fig. 10. Micrographs (SEM) of inherent structure of gels; (a) native gelatin gel e CG ¼ 20 g/100 g, (b) the same gel with adding of k-carrageenan (С K ¼ 0.5 g/100 g).
4. Conclusion Modification of gelatin by adding k-carrageenan leads to significant changes in the gelation kinetics and properties of formed gels. Application of gelatin/k-carrageenan mixtures results in systematic increase in the gelation rate, values of viscoelastic parameters, strength of gels, and growth of the melting temperature. The combined using of gelatin and k-carrageenan creates a synergetic effect on the physico-chemical properties of modified gels. Changes in the rheological properties occur by the exponential law. Actually, it means that the significant change in properties is reached at the definite gelatin/carrageenan ratio and in increase in the gelatin concentration the larger share of carrageenan is needed. The observed impact of addition of k-carrageenan on the gel properties is explained by formation of complexes of gelatin and polysaccharide that was confirmed by FT-IR spectral studied. Besides addition of even minor quantity of k-carrageenan significantly changes the supramolecular structure of gelatin gels. A possible scheme of the replacement of gelatin by k-carrageenan we presented in the following scheme:
Boanini, E., Rubini, K., Panzavolta, S., & Bigi, A. (2010). Chemico-physical characterization of gelatin films modified with oxidized alginate. Acta Biomaterialia, 6(2), 383e388. Brandrup, I., Immergut, E. H., & Grulke, E. A. (1999). Polymer handbook (4th ed.). New York: John Wiley & Sons, Inc. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18(5), 717e726. Derkach, S. R. (2014). Interfacial layers of complex-forming ionic surfactants with gelatin. Advances in Colloid and Interface Science. http://dx.doi.org/10.1016/ j.cis.2014.05.001. Derkatch, S. R., Voronko, N. G., & Izmailova, V. N. (2001). Rheological properties of gelatin gels with sodium alginate. International Journal of Applied Mechanics and Engineering, 6(3), 659e673. Dimitriou, Ch. J., Ewolt, R. H., & McKinley, G. H. (2013). Describing and prescribing the constitutive response of yield stress fluids using large amplitude oscillatory shear stress (LAOSress). Journal of Rheology, 57(1), 27e70. Ewoldt, R. H., Hosoi, A. E., & McKinley, G. H. (2008). New measures for characterizing nonlinear viscoelasticity in large amplitude oscillatory shear. Journal of Rheology, 52(6), 1427e1458. Ewoldt, R. H., & McKinley, G. H. (2010). On secondary loops in LAOS via selfintersection of LissajouseBowditch curves. Rheologica Acta, 49(2), 213e219. Gilsenan, P. M., & Ross-Murphy, S. B. (2000). Rheological characterization of gelatins from mammalian and marine sources. Food Hydrocolloids, 14(3), 191e195. Harrington, C., & Morris, E. (2009). Conformational ordering and gelation of gelatin in mixtures with soluble polysaccharides. Food Hydrocolloids, 23(2), 327e336. Haug, I. J., Draget, K. I., & Smidsrod, O. (2004). Physical behavior of fish gelatinekcarrageenan mixtures. Carbohydrate Polymers, 56(1), 11e19. Hyun, K., Kim, S. H., Ahn, K. H., & Lee, S. J. (2002). Large amplitude oscillatory shear as a way to classify the complex fluids. Journal of Non-Newtonian Fluid Mechanics, 107(1e3), 51e65. Hyun, K., Wilhelm, M., Klein, C. O., Cho, K. S., Nam, J. G., Ahn, K. H., et al. (2011). A review of nonlinear oscillatory shear tests: analysis and application of large amplitude oscillatory shear (LAOS). Progress in Polymer Science, 36(12), 1697e1753. Ilyin, S., Kulichikhin, V., & Malkin, A. (2014). Characterization of material viscoelasticity at large deformations. Applied Rheology, 24(1), 13653. Ilyin, S. O., Malkin, A. Ya., & Kulichikhin, V. G. (2012). Rheological peculiarities of concentrated suspensions. Colloid Journal, 74(4), 472e482. Ilyin, S. O., Malkin, A. Ya., & Kulichikhin, V. G. (2014). Application of large amplitude oscillatory shear for the analysis of polymer material properties in the nonlinear mechanical behavior. Polymer Science, Series A, 56(1), 98e110. Ilyin, S. O., Pupchenkov, G. S., Krasheninnikov, A. I., Kulichikhin, V. G., & Malkin, A. Ya. (2013). Rheology of aqueous poly(ethylene oxide) solutions reinforced with bentonite clay. Colloid Journal, 75(3), 267e273.
S.R. Derkach et al. / LWT - Food Science and Technology 63 (2015) 612e619 Ilyin, S., Roumyantseva, T., Spiridonova, V., Semakov, A., Frenkin, E., Malkin, A., et al. (2011). Gels of cysteine/Ag-based dilute colloid systems and their rheological properties. Soft Matter, 7(19), 9090e9103. Izmailova, V. N., Derkach, S. R., Sakvarelidze, M. A., Levachev, S. M., Voron'ko, N. G., & Yampol'skaya, G. P. (2004). Gelation in gelatin and gelatin-containing multicomponent blends. Polymer Science, Series C, 46(1), 73e92. Izumrudov, V. A. (2008). Self-assembly and molecular “recognition” phenomena in solutions of (bio)polyelectrolyte complexes. Russian Chemical Reviews, 77(4), 401e413. Kabanov, V. A. (2005). Polyelectrolyte complexes in solution and in bulk. Russian Chemical Reviews, 74(1), 3e20. Kim, J., Merger, D., Wilhelm, M., & Helgeson, M. E. (2014). Microstructure and nonlinear signatures of yielding in a heterogeneous colloidal gel under large amplitude oscillatory shear. Journal of Rheology, 58(5), 1359e1390. Lau, M. H., Tang, J., & Paulson, A. T. (2001). Effect of polymer ratio and calcium concentration on gelation properties of gellan/gelatin mixed gels. Food Research International, 34(10), 879e886. Malkin, A., Ilyin, S., Roumyantseva, T., & Kulichikhin, V. (2013). Rheological evidence of gel formation in dilute poly(acrylonitrile) solutions. Macromolecules, 46(1), 257e266. Malkin, A., Ilyin, S., Semakov, A., & Kulichikhin, V. (2012). Viscoplasticity and stratified flow of colloid suspensions. Soft Matter, 8(9), 2607e2617. Marrs, W. M., & Pegg, A. M. (1997). Interactions in caseinateegelatin systems. The Imaging Science Journal, 45(3e4), 263e265. Masalova, I., Taylor, M., Kharatiyan, E., & Malkin, A. (2005). Rheopexy in highly concentrated emulsions. Journal of Rheology, 49(4), 839e849. Michon, C., Cuvelier, G., Launay, B., & Parker, A. (1996). Viscoelastic properties of icarrageenan/gelatin mixtures. Carbohydrate Polymers, 31(3), 161e169. Michon, C., Cuvelier, G., Launay, B., Parker, A., & Takerkart, G. (1995). Study of the compatibility/incompatibility of gelatin/iota-carrageenan/water mixtures. Carbohydrate Polymers, 28(4), 333e336. Morris, E. R. (1990). Mixed polymer gels. In P. Harris (Ed.), Food gels (pp. 291e359). London: Elsevier Applied Science Publishers.
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Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Fourier transform infrared (FTIR) spectroscopy study of acid soluble collagen and gelatin from skin and bones of young and adult Nile perch (Lates niloticus). Food Chemistry, 86(3), 325e332. Panouille, M., & Larreta-Garde, V. (2009). Gelation behavior of gelatin and alginate mixtures. Food Hydrocolloids, 23(4), 1074e1080. Pranoto, Y., Lee, C. M., & Park, H. J. (2007). Characterizations of fish gelatin films added with gellan and k-carrageenan. LWT e Food Science and Technology, 40(5), 766e774. Prystupa, D. A., & Donald, A. M. (1996). Infrared study of gelatin conformations in the gel and sol states. Polymer Gels and Networks, 4(2), 87e110. Ross-Murphy, S. B. (1997). Structure and rheology of gelatin gels. The Imaging Science Journal, 45(3e4), 205e209. Saxena, A., Kaloti, M., & Bohidar, H. B. (2011). Rheological properties of binary and ternary proteinepolysaccharide co-hydrogels and comparative release kinetics of salbutamol sulphate from their matrices. International Journal of Biological Macromolecules, 48(2), 263e270. Segard, V. H., & Isaksson, T. (2004). Temperature, sample and time dependent structural characteristics of gelatin gels studies by near infrared spectroscopy. Food Hydrocolloids, 18(1), 1e11. Sen, M., & Erboz, E. N. (2010). Determination of critical gelation conditions of kcarrageenan by viscosimetric and FT-IR analyses. Food Research International, 43(5), 1361e1364. Ur'ev, N. B., Svistunov, Yu S., Potapov, A. N., & Starikov, V. A. (2007). Increase in the effective viscosity of nanodisperse structured systems in shear deformation at extremely low rates. Doklady Physical Chemistry, 416(1), 237e239. Voron'ko, N. G., Derkach, S. R., & Izmailova, V. N. (2002). Rheological properties of gels of gelatin with sodium alginate. Russian Journal of Applied Chemistry, 75(5), 790e794. Yermak, I. M., & Khotimchenko, Y. S. (2003). Chemical properties, biological activities and applications of carrageenan from red algae. In M. Fingerman, & R. Nagabhushanam (Eds.), Recent advances in marine biotechnology (pp. 207e255). New York: Science Publisher Inc.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
The rheology of gelatin hydrogels modified by k-carrageenan Svetlana Rostislavovna Derkach a, Sergey Olegovich Ilyin b, *, Alexandra Alexandrovna Maklakova a, Valery Grigoryevich Kulichikhin b, Alexander Yakovlevich Malkin b a b
Murmansk State Technical University, Sportivnaya ul. 13, Murmansk 183010, Russia A.V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Leninskii prospekt 29, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 16 January 2015 Accepted 9 March 2015 Available online 17 March 2015
We studied the impact of anionic polysaccharide (k-carrageenan) as a co-gelator of gelatin at different physico-chemical and by varying the k-carrageenan/gelatin ratio. It has been shown that increasing kcarrageenan concentration accelerates the gelation and leads to significant increase in the viscoelastic parameters of the modified gels. Non-linearity of viscoelastic properties becomes evident in much lower deformations in comparison with those of native gelatin gels. The strength of gels characterized by the yield stress also increases along with k-carrageenan concentration. The deformation (but not flow) of gels happens along the rupture surfaces appearing at the apparent yield stress. The melting temperature increases until 45 C for high k-carrageenan/gelatin ratio. The suggested explanation of the observed changes in properties of modified gels relates these effects with formation of complexes of gelatin and polysaccharide. FT-IR spectroscopy data show that (bio)polyelectrolyte complex formation is due to electrostatic interaction between positively charged groups in gelatin and negatively charged sulfate groups in k-carrageenan that leads to conformation changes of gelatin macromolecules. Micrographs (scanning electron microscopy) show that addition of even a small quantity of polysaccharide leads to radical changes in supramolecular structure of modified gels. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hydrogel Gelatin Polysaccharide Carrageenan Rheology
1. Introduction Application of native gelatin has definite restrictions determined by the required gelation rate, thermal stability, and rheological properties. So, it is necessary to modify gelatin as a gelator. This issue is answered by combination of gelatin with other component. For instance, gelatin can be modified by using some chemical cross-linking agents such as chloranhydride or formaldehyde (De Carvalho & Grosso, 2004). They are effective modifiers but not acceptable in food industry. Other method consists in addition of surfactants or biopolymers compatible with gelatin. One of the promising methods is modification of gelatin properties by adding polysaccharides as co-gelators. Their compatibility with gelatin is provided by interaction of charged gelatin and polysaccharide macroions leading to formation of (bio)polyelectrolyte complexes (Marrs & Pegg, 1997; Michon, Cuvelier, Launay, Parker, & Takerkart, 1995; Morris, 1990). The latter are capable for self-organization and gelation under a variety of * Corresponding author. Tel.: þ7 495 9554191; fax: þ7 495 6338520. E-mail address: [email protected] (S.O. Ilyin). http://dx.doi.org/10.1016/j.lwt.2015.03.024 0023-6438/© 2015 Elsevier Ltd. All rights reserved.
external conditions (Izumrudov, 2008; Kabanov, 2005). This way opens up new possibilities for modification of rheology, melting temperature, gelation rate, and microstructure of gelatin-based products in its combined application with different ionic polysaccharides. Numerous studies showed that addition of polysaccharide in gelatin-based gels affects rheological properties of compositions. So, if conditions of (bio)polyelectrolyte complexes in aqueous phase are provided, the yield stress and viscosity of gels increase with increasing polysaccharide concentration as demonstrated for sodium alginate (Boanini, Rubini, Panzavolta, & Bigi, 2010; Derkach, 2014; Derkatch, Voronko, & Izmailova, 2001). Some studies showed that the elastic modulus of gelatin mixtures with i-carrageenan or k-carrageenan exceeds the sum of moduli of the individual components (Haug, Draget, & Smidsrod, 2004; Michon, Cuvelier, Launay, & Parker, 1996). It was also shown that strength of films obtained from fish gelatin increases by addition of kcarrageenan or gellan (Pranoto, Lee, & Park, 2007). Simultaneously, the melt temperature also increases. It was supposed that these effects are due to electrostatic interactions between the biopolymers and changes in gel microstructure.
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Modification by polysaccharides can also come out as the plasticizing effect. For example, if gellan (Lau, Tang, & Paulson, 2001), sodium alginate (Panouille & Larreta-Garde, 2009) or agar (Saxena, Kaloti, & Bohidar, 2011) is used at conditions not providing polyelectrolyte complexes formation, the strength of gels decreases in comparison with that of native gelatin gels, and gelation temperature also decreases (Harrington & Morris, 2009). One of the promising polysaccharides for creating gellous products is k-carrageenan which is also a gelator (Brandrup, Immergut, & Grulke, 1999; Yermak & Khotimchenko, 2003). The study is motivated by the use of gellous gelatine based food products. A possibility of their application is determined by the rheological properties of gels. Viscometric measurements provide information concerning viscosity levels before and after gelation that is important for processing. Dynamic low-amplitude tests characterize the rigidity of a material. A new perspective method of testing is large amplitude oscillatory stain (LAOS) measurements which give information about evolution of the rheological properties in time (or strain) (Ewoldt, Hosoi, &. McKinley, 2008). Different version of treating the results of the LAOS measurements was proposed in the current literature. One of them is the presentation of experimental data in the form of the LissajouseBowditch figures which are the dependence of stress on strain. A version of LAOS method based on the analysis of the LissajouseBowditch figures was recently developed in (Ilyin, Malkin, & Kulichikhin, 2014). At low deformation, these figures are ellipses. Distortion of these figures characterizes non-linear viscoelastic properties of a material under study (Ewoldt & McKinley, 2010; Kim, Merger, Wilhelm, & Helgeson, 2014). The goal of this work is a systematic study of viscoelastic properties of gels formed from gelatin and k-carrageenan with increasing the polysaccharide/gelatin ratio. We examine the impact of kcarrageenan on rheological, thermal stability, and gelation kinetic properties of k-carrageenan/gelatin gels. Properties of gelatine based products depend on interaction between their components including formation of (bio)polyelectrolyte complexes. So, structure estimations appear the necessary part of this study. 2. Material and methods 2.1. Materials An alkaline grade gelatin Type-B from bovine skin 225 Bloom (SigmaeAldrich, Steinheim, Germany) was used. The average molecular weight of gelatin was M v ¼ 1:0$105 g=mol. pH at the isoelectric point was 4.9. Samples of k-carrageenan (SigmaeAldrich) with average molecular weight M v ¼ 6:8$105 g=mol was used without additional purification. 2.2. Methods 2.2.1. Preparation of solutions and gels Aqueous solutions of gelatin and k-carrageenan were prepared separately. Initially, samples of given weight swelled in distillated water at 20 C during 1 h. Then they were dissolved at elevated temperatures, 50 C and 80 C, respectively. This protocol allowed us to obtain homogenous sols of both components. After that, k-carrageenan solutions added to gelatin solutions in ratios corresponding to aspirational biopolymer concentrations in sampled for further study. Gels were obtained from native gelatin (gelatin gels) or from gelatin with added k-carrageenan (modified gels) solutions under cooling to 14 C. All studies were carried out for gels with gelatin concentration, CG, from 1 to 2 g/100 g of gel and k-carrageenan concentration, CK, varied in the range from 0.05 to 1 g/100 g of gel.
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2.2.2. Rheological measurements Rheological properties of gelatin and modified gels were measured at shear deformation by Physica MCR301 (Anton Paar, Graz, Austria) rheometer using the cone-and-plate working unit (diameter of a cone was 50 mm and angle between the conical surface and the plate was 1 grad). Measurements were carried out in the following deformation modes: periodic oscillations at constant temperature (14 C) with varying amplitude, g, at constant frequency, u, or varying frequency, the range of g was 0.1e1000% and u was 0.0628e628 s1; large-strain components of the complex (dynamic) modulus were calculated according to Ilyin, Kulichikhin, and Malkin (2014); temperature scanning at g ¼ 1% and u ¼ 6.28 s1 at constant temperature of 14 C for following “aging” of samples or at increasing temperature at the rate of 2 K/min; shearing of a sample _ or stress-controlled (s) mode in at 14 C in the rate controlled (g) the range of 103e104 s1 and 0.01e200 Pa, respectively. The relative error in measuring apparent viscosity and the components of the dynamic modulus did not exceed 10%. The variation of the given temperature was within ±0.1 C. Reproducibility of the results of the rheological measurements was controlled by parallel testing of two samples of the same content. 2.2.3. Fourier transform infrared (FT-IR) analysis FT-IR spectra of the sample under study were registered with IRFourier spectrometer Nicolet 700 (Thermo Scientific, Madison WI, USA) in the middle range of 400e4000 cm1 IR radiation. FT-IR spectra were measured using dried samples obtained from gelatin and modified samples. The following protocol of sample preparation was used. Hydrogels were maintained at 12 C during 12 h. Then they were frozen at 6 C, unfrozen in dark and centrifuged. The formed sediment dried in a drying cabinet at 50 C for 5 h and finally at 25 C for 20 h. Obtained dry films grinded in a ball mill till the state of highly dispersed powder and pressed this powder with KBr forming a pellet which was used for FT-IR spectroscopy measurements. 2.2.4. Microstructure observation Gel microstructure was examined by the scanning electron microscopy (SEM) using a microscope S405-A (Hitachi, Tokyo, Japan) supplied with the SEM LEO-420 software. Gel samples preliminary stored in a desiccator at 12 C for 1 day. Before examination, sample of the 3 3 3 mm size were frosted (in vacuum) and then metalized with thin (app. 20 Å) gold layer under vacuum. 3. Results and discussion 3.1. Solegel transition at constant or increasing temperature At temperatures below 30 C, gelatin macromolecules undergo a conformation coil / helix transition (rigid form). As a result, its aqueous colloidal solutions transforms to gels if the gelatin concentration exceeds some critical threshold (Izmailova et al., 2004; Ross-Murphy, 1997). Such a sol / gel transition happens due to formation of intermolecular hydrogen bonds between carboxyl oxygen and amid hydrogen in polypeptide chain (at high content of glycineeprolineehydroxyproline amino acids). Beside, gelatin macromolecules loss solubility at cooling owing to their transition into the rigid helix conformation. These processes lead to phase decomposition and formation of a spatial network structure based on a biopolymer phase with the presence water entrapped (Gilsenan & Ross-Murphy, 2000). The k-carrageenan addition to aqueous gelatin solutions affects the kinetics of the system's gelation. Fig. 1 demonstrates that addition of k-carrageenan to gelatin solution increases the rate of
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Fig. 1. Kinetics of gelation as followed by an increase of the dynamic modulus G* normalized by its limiting value, G*t/∞. CG ¼ 2 g/100 g, concentrations of k-carrageenan, CK, are shown at the figure.
gelation in the isothermal conditions as followed by changes in the time dependence of the complex elastic modulus, G*(t) for samples with different k-carrageenan content. The gelation is reversible. In this study we follow the solegel transition (or conditionally, gel “melting”) by decrease in the storage and loss moduli by temperature scanning. Fig. 2 illustrates the experimental data. The “melt transition temperature” is assumed as the crossover point of both components of the dynamic modulus, as shown in Fig. 2. The dependence of the melt temperature on the k-carrageenan concentration in modified gel is shown in Fig. 3. As seen, this dependence is not smooth and the sharp increase in the solegel transition temperature happens at the k-carrageenan concentration above 0.1 g/100 g or at the gelatin/carrageenan ratio of 10:1. So, experimental data clearly show that addition of k-carrageenan strongly affect the thermo-stability of k-carrageenan/ gelatin systems. It is reasonable to suppose that the basic reason for this effect is formation of (bio)polyelectrolyte complexes and transformation of supramolecular structure of materials under study depending on their composition. The similar increase in the melt temperature with increasing k-carrageenan (Pranoto et al.,
Fig. 2. Temperature dependencies of the storage modulus for native gelatin gel (CG ¼ 1 g/100 g) and modified gels. The k-carrageenan concentration added to the gelatin gel, С K, is at the figure. Rate of temperature increase was 2 K/min.
Fig. 3. Concentration dependence of the melt point for the k-carrageenanegelatin gel (С G ¼ 1 g/100 g).
2007) or sodium alginate concentration (Derkatch et al., 2001) was also observed for more concentrated gelatin gels. 3.2. Viscoelasticity of modified gels The measurements of the viscoelastic properties of the systems after gelation show that they are physical gels. It is seen that in the linear domain of viscoelastic behavior, the storage modulus practically does not depend on frequency that is the direct evidence of the solid-like state of a material (Fig. 4). This is true for modified gels as well for a native gelatin gel. The storage modulus (“rigidity”) of modified gels is higher than modulus of individual components, i.e. structure formation in a modified gels e system of (bio)polyelectrolyte complexes demonstrates the synergetic effect: G0 GþK > G0 G þ G0 K. Non-linear viscoelastic behavior of the modified gels was studied by the method of large amplitude oscillatory shear (Dimitriou, Ewolt, & McKinley, 2013; Hyun et al., 2011). Large-strain storage and loss moduli are presented in Fig. 5 as functions of the amplitude of deformation. It is interesting to note that native gelatin gels maintain linearity of viscoelastic behavior up to rather large deformation of app. 60%, while modified gels demonstrate non-linearity at deformations lower by a decimal order.
Fig. 4. Frequency dependencies of the storage modulus and the loss modulus kcarrageenan, gelatin and k-carrageenanegelatin complexes; gelatin and carrageenan concentrations are shown at the figure.
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Two examples of the LissajouseBowditch figures built for gelatin gel (CG ¼ 2 g/100 g) and a modified gel (CG ¼ 2 g/100 g plus CK ¼ 1 g/100 g) are presented in Fig. 6. As was said above, the form of non-linear LissajouseBowditch figures can be used for characterization of the material behavior. At the amplitude of deformation of 10%, the LissajouseBowditch figure for the gelatin gel is practically the straight line. It means that the reaction of a material is purely elastic, while the behavior of a gel modified by carrageenan is represented by the ellipse-like figure. It means that such a gel demonstrates also viscous response. An increase in the deformation amplitude leads to the increase in area of the figure that reflects growth of dissipative losses. So, generally speaking, the increase in the share of the modifying agent leads to the decrease in elasticity and increase in plasticity of a gel. Besides, the shape of the figure changes and this allows us to evaluate the behavior of a gel as pseudo-plastic (Ilyin,
Fig. 5. Amplitude dependencies of the large-strain storage (a) and loss modulus (b) at u ¼ 6.28 s1 for native gelatin gels, С G ¼ 2 g/100 g and modified gels containing kcarrageenan; CK, are at the figure.
One can see that is the decrease in the storage modulus is monotonous along with increasing deformation, then, the loss modulus passes through a maximum. This reflects changes in the material structure formed by van der Waals bonds. After destroying intermolecular bonds at some critical deformation, the orientation of macromolecular chains starts and this leads to the decrease in the loss modulus. According to the classification proposed by (Hyun, Kim, Ahn, & Lee, 2002), this type of materials is treated as media with weak strain overshoot. Addition of little quantity of k-carrageenan to a rather concentrated gel (gelatin concentration of 2 g/100 g) results in the increase in the loss modulus but not the storage modulus and the contraction of the linear domain of viscoelasticity. So, it is possible to think that little addition of k-carrageenan change one intermolecular bonds existing in the gelatin gel for others which incorporate a new polymer but do not increase significantly their common quantity. However, is the gelatin concentrations not high enough (Fig. 4), the introduction of k-carrageenan still increases the storage modulus. Moreover larger quantities of k-carrageenan, of the order of 1 g/100 g, do increase the storage modulus of the gel (Fig. 5). The gelatin/carrageenan ratio responsible for the sharp changes in the gel properties is not constant but depends on the gelatin concentration. For example, if this ratio is 10/1 for the gelatin concentration of 1 g/100 g (Fig. 3), then in double increasing the gelatin concentration, it appears to add much more carrageenan and the critical gelatin/carrageenan ratio is reduced to 2/1.
Fig. 6. The LissajouseBowditch figures for CG ¼ 2 g/100 g gel (a) and CG ¼ 2 g/100 g plus CK ¼ 1 g/100 g gel (b). Amplitudes of deformation are shown at the figures.
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Malkin, et al., 2014). So, the gelatin gel modification by carrageenan leads to the increase in plasticity at rather moderate deformations (10e100%). 3.3. Flow curves and yielding of modified gels Flow curves measured in the stress-control scanning modes contain clearly expresses domains of the maximal and minimal Newtonian viscosities, and also the yield stress, sY (Fig. 7). The addition of small amount of k-carrageenan slightly decreases the yield stress while an increase in the k-carrageenan concentration leads to the considerable increase in the yield stress and the maximal Newtonian viscosity. Meanwhile it is worthy to discuss the physical meaning of the “maximal Newtonian viscosity”. In several publications, it was proven that “observed” flow of multi-component dispersed systems at low stresses (below the yield stress) is an artifact of the transient deformation regime caused by limited time of observation insufficient for steady flow. If deformation at the given shear rate continues the yield stress but not Newtonian flow is reached. This phenomenon was observed for very different systems e gels (Malkin, Ilyin, Roumyantseva, & Kulichikhin, 2013), suspensions (Malkin, Ilyin, Semakov, & Kulichikhin, 2012; Ur'ev, Svistunov, Potapov, & Starikov, 2007), emulsions (Masalova, Taylor, Kharatiyan, & Malkin, 2005), and dispersions in a viscoelastic medium (Ilyin, Pupchenkov, Krasheninnikov, Kulichikhin, & Malkin, 2013). So, in our case discussed in this study, one should treat the “Newtonian” plateau critically especially bearing in mind solid-like behavior of gels at low deformations (Fig. 4 and related discussion). The domain of the low (minimal) Newtonian viscosity also should be treated with caution. Indeed, general understanding of this domain as homogenous flow with completely destroyed structure can be erroneous because many experimental facts show that in reality, flow in this domain occurs in a stratified (manylayered) mode as was observed for gels (Ilyin et al., 2011) or suspensions (Ilyin, Malkin, & Kulichikhin, 2012). In scanning rate-control mode, “flow curves” appear nonmonotonous that reflects the absence of steady flow and steady (homogenous) state of these materials. This seeming “strange” character of flow curves indicates the breakup of a sample and discontinuities in a bulk. Really, in this case it is rather dangerous to speak about “flow” of gels but maybe about sliding along the lines of ruptures. Quantitatively, the modifying effect of k-carrageenan on mechanical properties of gelatin gels can be characterizes by the two
Fig. 7. Flow curved obtained in the scanning stress-control mode for native gelatin С G ¼ 1 g/100 g gels and modified gels with k-carrageenan; С K, are at the figure.
connected parameters e the storage modulus and the yield stress. As is seen, both are growing along with the increase of the kcarrageenan concentration demonstrating the increase of the strength and rigidity of the gel network of (bio)polyelectrolyte complexes. As was discussed above, the significant changes in the rheological properties of gels can be reached at some definite gelatin/carrageenan ratio. However the presentation of experimental data in log scale shows that that these data are well described rather by the exponential law (Fig. 8) than has threshold. 3.4. FT-IR spectroscopy of gels Intermolecular interaction between gelatin and k-carrageenan is confirmed by the FT-IR spectroscopy data. Corresponding experimental data are presented in Fig. 9, where spectra for native gelatin gels and modified (by k-carrageenan) gels are compared. Attribution of lines in the IR-spectrum of k-carrageenan (Fig. 9a) is based on the data of publication (Sen & Erboz, 2010). Main bands correspond to ester sulfate group (1263 cm1), 3,6-anhydridegalactose group (928 cm1), and D-galactose-4-sulfate group (848 cm1). Wide adsorption band with maximum at 3420 cm1 corresponds to vibration of the hydroxyl group in k-carrageenan. The main lines in the native gelatin spectrum (Fig. 9b, line 1) are wide band with peak at 3400 cm1 (vibration of the HN-group), characteristic adsorption at 1654 cm1 (Amide I, stretching vibrations of CO and CN groups), 1541 cm1 (Amide II, NeH and CN vibration), and 1230 cm1 (Amide III) (Prystupa & Donald, 1996; Segard & Isaksson, 2004). The most useful analytical line for characterization of the secondary structure in protein (and gelatin) is the Amide I band (Muyonga, Cole, & Duodu, 2004). As seen in Fig. 9b, addition of k-carrageenan to gelatin leads to the shift of Amide I band to the low frequency side till the frequency of 1652 cm1. The Amide II adsorption line also shifts to the low frequency side till the frequency of 1539 cm1. It is necessary to note the low-frequency shift of the adsorption band of sulfate groups of k-carrageenan till 1238 cm1. The observed shifts indicate interaction between positively charged amide groups in a polypeptide chain of gelatin and negatively charged sulfate groups in k-carrageenan with formation of (bio)polyelectrolyte complexes. Shift of the Amide I band into the low frequency domain observed for modified (by k-carrageenan) gels in comparison with a native gelatin gel suggests that the conformation state of gelatin macromolecules changes with an increase of the order structures
Fig. 8. Dependencies of the yield stress sY and the storage modulus G0 on the kcarrageenan concentration added to the gelatin gel (С G ¼ 1 g/100 g).
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Fig. 9. FT-IR spectrum for samples of (a) k-carrageenan; (b) native gelatin С G ¼ 2 g/100 g (1), and mixtures of gelatin С G ¼ 2 g/100 g with k-carrageenan, С K, g/100 g: 0.1 (2), 0.5 (3), 1 (4).
share as the result of complex formation. Evolution of the conformation state obliged to modification and an increase in intermolecular contacts might be the cause of the increase of the strength and viscoelastic parameters of modified gels. 3.5. Gel microstructure e electron microscopy patters Typical SEM microphotographs (in the same scale) of morphological patterns of 20 g/100 g native gelatin gel and the same gel with addition of only 0.5 g/100 g of k-carrageenan are compared in Fig. 10. One can say with confidence that the difference is striking. Supramolecular structure of native gelatin formed due to the self-organization process is characterized by transversal collagenlike helix with concentration dependent size (Fig. 10a). The fibrils
form a network. Gel structure also includes discrete zones (cells) distributed along fibrils inside a network, which presumably consist mainly of non-helical pieces of macromolecules. It is seen that addition of even small quantity k-carrageenan leads to radical changes in the gel structure (Fig. 10b). Polysaccharide initiates some interactions between helix zones in gelatin due to appearance of k-carrageenanegelatin polyelectrolyte complexes. Similar transformations of the morphological pattern can be observed by application of other polysaccharides, for instance, gellan (Pranoto et al., 2007) and sodium alginate (Voron'ko, Derkach, & Izmailova, 2002). The modification mechanism of k-carrageenan addition is associated with the evident hardening of the gelatin gel structure and it was quite demonstrative in the results of rheological studies.
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One can believe that contacts with k-carrageenan in a modified gels are stronger in comparison with hydrogen bonds formed by gelatin that leads to the formation of the interactions between helix zones in macromolecules. This results in the increase of viscoelastic properties and reduction of the linear domain. Also, this increases the rigidity and plasticity of a gel. The introduction of a greater quantity of carrageenan leads to the increase in mechanical properties of a material due to the increase of the hydrogen bonds. Acknowledgments The reported study was partially supported by the Russian Foundation for Basic Research, the grant number is 14-08-98811 r_North_a. References
Fig. 10. Micrographs (SEM) of inherent structure of gels; (a) native gelatin gel e CG ¼ 20 g/100 g, (b) the same gel with adding of k-carrageenan (С K ¼ 0.5 g/100 g).
4. Conclusion Modification of gelatin by adding k-carrageenan leads to significant changes in the gelation kinetics and properties of formed gels. Application of gelatin/k-carrageenan mixtures results in systematic increase in the gelation rate, values of viscoelastic parameters, strength of gels, and growth of the melting temperature. The combined using of gelatin and k-carrageenan creates a synergetic effect on the physico-chemical properties of modified gels. Changes in the rheological properties occur by the exponential law. Actually, it means that the significant change in properties is reached at the definite gelatin/carrageenan ratio and in increase in the gelatin concentration the larger share of carrageenan is needed. The observed impact of addition of k-carrageenan on the gel properties is explained by formation of complexes of gelatin and polysaccharide that was confirmed by FT-IR spectral studied. Besides addition of even minor quantity of k-carrageenan significantly changes the supramolecular structure of gelatin gels. A possible scheme of the replacement of gelatin by k-carrageenan we presented in the following scheme:
Boanini, E., Rubini, K., Panzavolta, S., & Bigi, A. (2010). Chemico-physical characterization of gelatin films modified with oxidized alginate. Acta Biomaterialia, 6(2), 383e388. Brandrup, I., Immergut, E. H., & Grulke, E. A. (1999). Polymer handbook (4th ed.). New York: John Wiley & Sons, Inc. De Carvalho, R. A., & Grosso, C. R. F. (2004). Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18(5), 717e726. Derkach, S. R. (2014). Interfacial layers of complex-forming ionic surfactants with gelatin. Advances in Colloid and Interface Science. http://dx.doi.org/10.1016/ j.cis.2014.05.001. Derkatch, S. R., Voronko, N. G., & Izmailova, V. N. (2001). Rheological properties of gelatin gels with sodium alginate. International Journal of Applied Mechanics and Engineering, 6(3), 659e673. Dimitriou, Ch. J., Ewolt, R. H., & McKinley, G. H. (2013). Describing and prescribing the constitutive response of yield stress fluids using large amplitude oscillatory shear stress (LAOSress). Journal of Rheology, 57(1), 27e70. Ewoldt, R. H., Hosoi, A. E., & McKinley, G. H. (2008). New measures for characterizing nonlinear viscoelasticity in large amplitude oscillatory shear. Journal of Rheology, 52(6), 1427e1458. Ewoldt, R. H., & McKinley, G. H. (2010). On secondary loops in LAOS via selfintersection of LissajouseBowditch curves. Rheologica Acta, 49(2), 213e219. Gilsenan, P. M., & Ross-Murphy, S. B. (2000). Rheological characterization of gelatins from mammalian and marine sources. Food Hydrocolloids, 14(3), 191e195. Harrington, C., & Morris, E. (2009). Conformational ordering and gelation of gelatin in mixtures with soluble polysaccharides. Food Hydrocolloids, 23(2), 327e336. Haug, I. J., Draget, K. I., & Smidsrod, O. (2004). Physical behavior of fish gelatinekcarrageenan mixtures. Carbohydrate Polymers, 56(1), 11e19. Hyun, K., Kim, S. H., Ahn, K. H., & Lee, S. J. (2002). Large amplitude oscillatory shear as a way to classify the complex fluids. Journal of Non-Newtonian Fluid Mechanics, 107(1e3), 51e65. Hyun, K., Wilhelm, M., Klein, C. O., Cho, K. S., Nam, J. G., Ahn, K. H., et al. (2011). A review of nonlinear oscillatory shear tests: analysis and application of large amplitude oscillatory shear (LAOS). Progress in Polymer Science, 36(12), 1697e1753. Ilyin, S., Kulichikhin, V., & Malkin, A. (2014). Characterization of material viscoelasticity at large deformations. Applied Rheology, 24(1), 13653. Ilyin, S. O., Malkin, A. Ya., & Kulichikhin, V. G. (2012). Rheological peculiarities of concentrated suspensions. Colloid Journal, 74(4), 472e482. Ilyin, S. O., Malkin, A. Ya., & Kulichikhin, V. G. (2014). Application of large amplitude oscillatory shear for the analysis of polymer material properties in the nonlinear mechanical behavior. Polymer Science, Series A, 56(1), 98e110. Ilyin, S. O., Pupchenkov, G. S., Krasheninnikov, A. I., Kulichikhin, V. G., & Malkin, A. Ya. (2013). Rheology of aqueous poly(ethylene oxide) solutions reinforced with bentonite clay. Colloid Journal, 75(3), 267e273.
S.R. Derkach et al. / LWT - Food Science and Technology 63 (2015) 612e619 Ilyin, S., Roumyantseva, T., Spiridonova, V., Semakov, A., Frenkin, E., Malkin, A., et al. (2011). Gels of cysteine/Ag-based dilute colloid systems and their rheological properties. Soft Matter, 7(19), 9090e9103. Izmailova, V. N., Derkach, S. R., Sakvarelidze, M. A., Levachev, S. M., Voron'ko, N. G., & Yampol'skaya, G. P. (2004). Gelation in gelatin and gelatin-containing multicomponent blends. Polymer Science, Series C, 46(1), 73e92. Izumrudov, V. A. (2008). Self-assembly and molecular “recognition” phenomena in solutions of (bio)polyelectrolyte complexes. Russian Chemical Reviews, 77(4), 401e413. Kabanov, V. A. (2005). Polyelectrolyte complexes in solution and in bulk. Russian Chemical Reviews, 74(1), 3e20. Kim, J., Merger, D., Wilhelm, M., & Helgeson, M. E. (2014). Microstructure and nonlinear signatures of yielding in a heterogeneous colloidal gel under large amplitude oscillatory shear. Journal of Rheology, 58(5), 1359e1390. Lau, M. H., Tang, J., & Paulson, A. T. (2001). Effect of polymer ratio and calcium concentration on gelation properties of gellan/gelatin mixed gels. Food Research International, 34(10), 879e886. Malkin, A., Ilyin, S., Roumyantseva, T., & Kulichikhin, V. (2013). Rheological evidence of gel formation in dilute poly(acrylonitrile) solutions. Macromolecules, 46(1), 257e266. Malkin, A., Ilyin, S., Semakov, A., & Kulichikhin, V. (2012). Viscoplasticity and stratified flow of colloid suspensions. Soft Matter, 8(9), 2607e2617. Marrs, W. M., & Pegg, A. M. (1997). Interactions in caseinateegelatin systems. The Imaging Science Journal, 45(3e4), 263e265. Masalova, I., Taylor, M., Kharatiyan, E., & Malkin, A. (2005). Rheopexy in highly concentrated emulsions. Journal of Rheology, 49(4), 839e849. Michon, C., Cuvelier, G., Launay, B., & Parker, A. (1996). Viscoelastic properties of icarrageenan/gelatin mixtures. Carbohydrate Polymers, 31(3), 161e169. Michon, C., Cuvelier, G., Launay, B., Parker, A., & Takerkart, G. (1995). Study of the compatibility/incompatibility of gelatin/iota-carrageenan/water mixtures. Carbohydrate Polymers, 28(4), 333e336. Morris, E. R. (1990). Mixed polymer gels. In P. Harris (Ed.), Food gels (pp. 291e359). London: Elsevier Applied Science Publishers.
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Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Fourier transform infrared (FTIR) spectroscopy study of acid soluble collagen and gelatin from skin and bones of young and adult Nile perch (Lates niloticus). Food Chemistry, 86(3), 325e332. Panouille, M., & Larreta-Garde, V. (2009). Gelation behavior of gelatin and alginate mixtures. Food Hydrocolloids, 23(4), 1074e1080. Pranoto, Y., Lee, C. M., & Park, H. J. (2007). Characterizations of fish gelatin films added with gellan and k-carrageenan. LWT e Food Science and Technology, 40(5), 766e774. Prystupa, D. A., & Donald, A. M. (1996). Infrared study of gelatin conformations in the gel and sol states. Polymer Gels and Networks, 4(2), 87e110. Ross-Murphy, S. B. (1997). Structure and rheology of gelatin gels. The Imaging Science Journal, 45(3e4), 205e209. Saxena, A., Kaloti, M., & Bohidar, H. B. (2011). Rheological properties of binary and ternary proteinepolysaccharide co-hydrogels and comparative release kinetics of salbutamol sulphate from their matrices. International Journal of Biological Macromolecules, 48(2), 263e270. Segard, V. H., & Isaksson, T. (2004). Temperature, sample and time dependent structural characteristics of gelatin gels studies by near infrared spectroscopy. Food Hydrocolloids, 18(1), 1e11. Sen, M., & Erboz, E. N. (2010). Determination of critical gelation conditions of kcarrageenan by viscosimetric and FT-IR analyses. Food Research International, 43(5), 1361e1364. Ur'ev, N. B., Svistunov, Yu S., Potapov, A. N., & Starikov, V. A. (2007). Increase in the effective viscosity of nanodisperse structured systems in shear deformation at extremely low rates. Doklady Physical Chemistry, 416(1), 237e239. Voron'ko, N. G., Derkach, S. R., & Izmailova, V. N. (2002). Rheological properties of gels of gelatin with sodium alginate. Russian Journal of Applied Chemistry, 75(5), 790e794. Yermak, I. M., & Khotimchenko, Y. S. (2003). Chemical properties, biological activities and applications of carrageenan from red algae. In M. Fingerman, & R. Nagabhushanam (Eds.), Recent advances in marine biotechnology (pp. 207e255). New York: Science Publisher Inc.