Rheological properties of mixtures of κ-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

Rheological properties of mixtures of κ-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

International Journal of Biological Macromolecules 27 (2000) 349 – 353 www.elsevier.com/locate/ijbiomac Rheological properties of mixtures of k-carra...

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International Journal of Biological Macromolecules 27 (2000) 349 – 353 www.elsevier.com/locate/ijbiomac

Rheological properties of mixtures of k-carrageenan from Hypnea musciformis and galactomannan from Cassia ja6anica C.T. Andrade a,*, E.G. Azero a, L. Luciano b, M.P. Gonc¸alves b a

Instituto de Macromole´culas Professora Eloisa Mano, Uni6ersidade Federal do Rio de Janeiro, P.O. Box 68525, 21945 -970 Rio de Janeiro, RJ, Brazil b CEQUP/Departamento de Engenharia Quı´mica, Faculdade de Engenharia da Uni6ersidade do Porto, Rua dos Bragas, 4050 -123 Porto, Portugal Received 12 January 2000; accepted 21 April 2000

Abstract Mixed gels of k-carrageenan (k-car) from Hypnea musciformis and galactomannans (Gal) from Cassia ja6anica (CJ) and locust bean gum (LBG) were compared using dynamic viscoelastic measurements and compression tests. Mixed gels at 5 g/l of total polymer concentration in 0.1 M KCl showed a synergistic maximum in viscoelastic measurements for k-car/CJ and k-car/LBG at 2:1 and 4:1 ratios, respectively. The synergistic maximum obtained from compression tests carried out for mixed gels at 10 g/l of total polymer concentration in 0.25 M KCl was the same for both k-car/CJ and k-car/LBG gels. An enhancement in the storage modulus (G%) and the loss modulus (G¦) was observed in the mechanical spectra for the mixtures in relation to k-car. The proportionally higher increase in G¦ compared with G%, as indicated by the values of the loss tangent (tan d), suggests that the Gal adhere non-specifically to the k-car network. © 2000 Elsevier Science B.V. All rights reserved. Keywords: k-Carrageenan; Non-traditional galactomannans; Rheology

1. Introduction Galactomannan (Gal) is the name given to a group of neutral polysaccharides obtained from the seed endosperm of some Leguminosae. They are composed of a linear mannose (M) backbone bearing side chains of a single galactose (G) unit. Depending on the source, the M/G varies [1,2]. Gals from Ceratonia siliqua (locust bean gum, LBG), Cyamopsis tetragonolobus (guar gum, GG) and Caesalpinia spinosa (tara gum, TG) are commercially available and find a wide range of food and industrial uses due to their ability to form very viscous solutions. k-Carrageenan (k-car) is the least sulphated fraction of a great family of natural polysaccharides extracted from certain red seaweeds (i.e. Eucheuma cottonii, Abbre6iations: CJ, Cassia ja6anica; CP, Caesalpinia pulcherrima; Gal, galactomannan; G, galactose; GG, guar gum; k-car, k-carrageenan; LBG, locust bean gum; M, mannose; TG, tara gum. * Corresponding author. Tel.: + 55-21-5627220; fax: +55-212701317. E-mail address: [email protected] (C.T. Andrade).

Gigartina acicularis and Hypnea musciformis). This linear polysaccharide is composed of repeating disaccharide units of 1,3-linked b-D-galactopyranose and 1,4-linked 3,6-anhydro-D-galactopyranose. k-Car can form thermoreversible gels, influenced by the presence and concentration of certain ions. Before gelation occurs, k-car undergoes a coil-helix conformational transition. Cations such as K+, Cs+, Rb+ and NH+ 4 bind specifically to the double-helix, increasing stability and promoting chain aggregation [3–5]. Anions such as I− and SCN− bind to the chain, promoting helix formation but preventing subsequent aggregation [4,6]. The gels of k-car are brittle and prone to syneresis. The properties of these gels can be improved by the addition of Gal. A certain number of papers refer to the effect of the M/G of the Gal on the properties of the resulting gel, synergy increases in the order GGBTGBLBG, which have M/G of 2:1, 3:1 and 4:1, respectively [7–9]. Several models have been proposed over the last decades to explain the synergistic effects [7,10–12]; however, considerable controversy still exists. The great majority of these papers refer to commercial Gals.

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C.T. Andrade et al. / International Journal of Biological Macromolecules 27 (2000) 349–353

Little is known about binary aqueous mixtures with non-traditional Gals [13]. In a previous work, the solution properties of the seed Gals of Caesalpinia pulcherrima (CP) and Cassia ja6anica (CJ) were investigated [14] in comparison with LBG. No significant differences were found between the viscous properties of these Gals. The fact that CP and CJ seed gums could be used as efficient thickening agents does not mean that they would display synergistic effects with other polysaccharides, such as k-car and xanthan, as is the case for LBG. The aim of the present work was to study the rheological and mechanical behaviours of binary aqueous mixtures of CJ seed gum or LBG/k-car. 2. Materials and methods

2.1. Materials Commercial-grade LBG (HG Type M-175) was kindly provided by INDAL (Portugal). The CJ seed gum was obtained and purified as described [14]. The chemical composition and physical chemical parameters of the Gal samples were presented elsewhere [14]. Fig. 2. Cooling (a) and heating (b) curves for k-car/Gal mixtures at 5.0 g/l of total polymer concentration in 0.1 M KCl. Frequency, 6.28 rad/s. k-car ( G%,  G¦); k-car/LBG 4:1 ( G%, D G¦); k-car/CJ 3:1 ( G%, G¦).

Fig. 3. Effect of k-car/Gal ratio on G% of mixed gels at the total polymer concentration of 5.0 g/l in 0.1 M KCl at 25°C. Frequency, 6.37 rad/s. (), LBJ; ( ), CJ.

Fig. 1. Flow chart for the extraction and purification processes of k-car.

The k-car sample was extracted from Hypnea musciformis, a red seaweed growing in the north-eastern coast of Brazil. After alkaline treatment to convert the biological precursor m-carrageenan into k-car, the seaweed was washed and extracted with water at 85°C.

C.T. Andrade et al. / International Journal of Biological Macromolecules 27 (2000) 349–353

The polysaccharide was recovered by precipitation in 95% ethyl alcohol, dried and purified. Fig. 1 shows the entire process. The sulphate content, as determined by the modified Tabatabai method [15], was 24.5% w/w. The ash content, obtained according to the American Society for Testing and Materials (ASTM-D 1439-72), was 25.6% w/w. The intrinsic viscosity in 0.1 M NaCl and 25°C, from Huggins’ extrapolation, was 9.68 dl/g. The viscosity average molar mass, M( 6 =6.1 ×105, for the k-car sample was calculated using the Mark– Houwink relationship, [h]= KM( a6



where K= 3.1× 10 − 3 ml/g and a= 0.95 are constants taken from the literature [16].

2.2. Preparation of the solutions For performing rheological measurements, the required weight of Gal and k-car was first dispersed in distilled water under moderate agitation for 1 h, at room temperature, and then heated at 90°C for 30 min with stirring. Mixtures (5.0 g/l) were prepared by adding the required volume of each polymer solution. Solid KCl was then added to a final concentration of 0.1 M. For performing mechanical experiments, the required weights of Gal and k-car powders were mixed before adding the solvent (0.25 M KCl solution) to give a final concentration of 10.0 g/l. The mixture was first heated at 70°C for 1 h and then at 90°C for 15 min. The hot mixture was poured into cylindrical glass moulds (20 mm internal diameter) and cooled to room temperature. The resulting gels were cut to give 20 mm probes and immersed in the solvent for 20 h at 15°C. Before performing the mechanical experiments, the gels were left to equilibrate at 25°C for 4 h.

2.3. Rheological measurements

Fig. 4. Mechanical spectra of k-car gels and of k-car/Gal mixed gels at the maximum of synergy at total polymer concentration of 5.0 g/l in 0.1 M KCl, at 25°C. k-car, ( ) G%, () G¦, ( ) h*; k-car/LBG, () G%, (D) G¦, (9) h*; k-car/CJ, ( ) G%, ( ) G¦, (+ ) h*.

Measurements were undertaken using a controlled stress rheometer CarriMed CSL 50 fitted with a cone/ plate geometry (2° cone angle, 4.0 cm diameter, 55 mm gap). The strain amplitude was fixed at 2.0. The hot mixture was poured directly onto the plate of the rheometer, at 85°C, and covered with light oil to prevent dehydration. A temperature sweep experiment was done from 85 to 15°C, at the rate of 1°C/min and a constant frequency of 6.28 rad/s, followed by a time sweep experiment, at the same frequency, and a mechanical spectrum, both at 5°C. Then, the temperature was raised to 25°C, at a constant rate of 1°C/min, and new time sweep and mechanical spectrum experiments were performed. Finally, the temperature was increased to 85°C at the rate of 1°C/min. The sol–gel transition was taken as the temperature at which a definitive and sharp increase in G%was observed.

2.4. Mechanical measurements

Fig. 5. Effect of k-car/CJ ratio on the tan d values for 5.0 g/l gels in 0.1 M KCl, at 25°C; ( ) k-car alone; ( ) k-car/CJ 1:1; () k-car/CJ 2:1; (+) k-car/CJ 3:1; () k-car/CJ 4:1.

The mechanical properties were determined by compression experiments at 5 mm/min and 25°C, using an Instron Universal Testing Machine, model 4204, equipped with a 1 kN compression cell. The average value from a total of five probes was retained. Young’s modulus, E, was determined as

C.T. Andrade et al. / International Journal of Biological Macromolecules 27 (2000) 349–353


Table 1 Parameters obtained from compression tests in k-car and k-car/Gal gels at 10 g/l total polymer concentration in 0.25 M KCl at 25°C Mixing ratio (k-car:Gal)

5:1 4:1 3:1 2:1 1:1 1:0

smax (10−4 Pa)b

E (10−4 Pa)a LBG


4.5 5.7 6.6 8.8 8.5 3.9

4.8 4.7 4.9 4.4 3.2

(9 0.18) (90.03) (90.08) (90.08) (90.11) (90.06)

LBG (90.16) (9 0.4) (90.13) (90.29) (90.21)

14.7 20.5 24.7 49.3 43.1 7.2

omax (%)c CJ

( 9 0.36) ( 91.29) ( 9 0.49) ( 9 1.40) ( 9 0.96) (9 0.98)

15.1 17.7 18.3 23.8 10.8

LBG ( 9 1.39) ( 91.54) ( 9 0.87) ( 9 1.87) ( 9 0.29)

32.4 34.6 45.9 38.2 34.8 28.8

CJ ( 91.17) ( 91.35) ( 9 2.18) ( 9 1.31) ( 9 1.55) ( 90.10)

32.0 34.6 33.9 35.3 35.3

(9 1.28) (9 2.39) ( 9 1.38) ( 9 2.23) (9 1.70)


E, Young’s modulus. smax, stress at break. c omax, deformation at break. b




where L0 is the initial height and A0 is the initial cross-section of the probe; DF/DL is the initial slope of the stress–strain curve.

3. Results and discussion Heating/cooling cycles, at a fixed frequency of 6.28 rad/s, were performed in 0.1 M KCl solutions of k-car alone and of mixtures of k-car with CJ or LBG at a total polysaccharide concentration of 5 g/l. The cooling and heating curves obtained for k-car, k-car/CJ (3/1 w/w ratio) and k-car/LBG (4/1 w/w ratio) are shown (Fig. 2). The gelation temperature, Tg, was 55°C for k-car, 60°C for k-car/LBG and 72°C for k-car /CJ (Fig. 2a). The same trend was observed for all other mixing ratios. The addition of Gal results in an increase of Tg; the effect was more pronounced for CJ than for LBG, in the above conditions. Also, when heating the cured gels, from 25 to 85°C, the melting temperature, Tm, was of 74–75°C for k-car and higher than 85°C for the mixed systems (Fig. 2b). The thermal hysteresis observed was higher for the k-car/Gal mixtures than for k-car alone, in accordance with previous findings [17,18]. In order to compare the effects of the two different Gals on the properties of mixed k-car/Gal gels, dynamic measurements were performed at variable ratios of the two polysaccharides over a frequency range of 0.062–100 rad/s. The values of the storage modulus, G%, obtained at 6.37 rad/s from mechanical spectra at 25°C, were plotted versus the percent k-car in the respective mixtures (Fig. 3). In both cases, a synergistic maximum was observed though for different k-car/Gal ratios, 4/1 for LBG and 2/1 for CJ. The values of G %max did not differ very much for both Gals. The results for LBG are consistent with those obtained by Tako and

Nakamura [19] and Fernandes et al. [9]. Standing and Hermansson [18] reported a lower ratio (35/65) for 1% gels in 0.1 M KCl while Arnaud et al. [20] reported a higher ratio (92/8) for 3% gels without KCl. This difference may reflect the effects of concentration, KCl content and temperature. In our case, LBG and CJ displayed quite similar M/G and small differences were observed in intrinsic viscosity and molar mass [14]. The differences observed in the rheological behaviour of the gels suggest a dependence upon the fine structure of the Gal chain. Mechanical spectra for gels of k-car alone and mixtures with LBG and CJ at the maximum of synergy are presented in Fig. 4. Significant enhancement in moduli, G% and G¦, was observed for the mixtures in comparison with k-car alone. All the spectra are typical of true gels with G% much higher than G¦ and almost frequency independent. Also, the plot of log h* versus log v was linear with a slope of − 1 as expected for true gels [21].

Fig. 6. Effect of k-car/Gal ratio on the stress at break values, smax, for 10 g/l gels in 0.25 M KCl, at 25°C. ( ) k-car/LBG and () k-car/CJ mixed gels.

C.T. Andrade et al. / International Journal of Biological Macromolecules 27 (2000) 349–353

However, the values of h* were higher for the mixtures than for k-car alone. The enhancement in the moduli depended on the mixing ratio as well as the separation of G% and G¦. This can be observed in Fig. 5 for mixtures k-car/CJ; for mixtures k-car/LBG a similar pattern was obtained. In the frequency range studied, tan d values for all mixed gels were higher than those determined for k-car alone. The increase in G¦ was proportionally higher than that of G% (Fig. 5). This may be related to the nature of the mixed network and seems consistent with a structure where mixed junction zones, as defined by Rees [22], were not formed. Rather, under the conditions used in the present work, self-aggregation of k-car chain segments is promoted for the three systems. The role of the Gal in the synergistic behaviour observed would be of adhering non-specifically (not limited to the junction zones) to the k-car network [23]. Higher concentrations of the polysaccharides and KCl were used in order to obtain self-supporting gels to be used in compression tests. Similar ratios to those used in the rheological experiments were chosen. The results in Table 1 show that the mechanical properties of the mixed gels depend on the kind of Gal used (LBG or CJ) as well as on the mixing ratio. Values determined for Young’s modulus, E, and stress at break, smax, are higher in mixed systems k-car/LBG for all mixing ratios, except for the 5:1 ratio, in which they are similar to those obtained for k-car/CJ system. For both kinds of mixed systems, omax is higher than for k-car alone, which means that both mixed gels are more cohesive than k-car alone. Also, values of smax show that the addition of Gal (LBG or CJ) to k-car gives rise to gels more resistant to rupture. Fig. 6 shows a plot of smax versus k-car concentration for k-car/LBG and k-car/CJ mixed gels at a total polymer concentration of 1%. In both curves, a maximum is observed at a k-car/Gal ratio of 2:1. Similar results for LBG were obtained in compression tests by Cairns et al. [8]. Fiszmann et al. [24] reported a lower ratio (1:1) while Arnaud et al. [20] found a much higher one (92/8). The differences between these results may be attributed to the different experimental conditions.

k-car. These findings should encourage academic and technological research into the use of CJ as an interesting alternative to the traditional LBG Gal.

Acknowledgements C.T.A. and M.P.G. are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil, and Instituto de Cooperac¸a˜o Cientı´fica e Tecnolo´gica Internacional (ICCTI), Portugal, for a CNPq/ICCTI award. E.G.A. thanks Fundac¸a˜o Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Brazil, for financial assistance. L.L. thanks Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal, for a grant (PRAXIS XXI/BTI/1208/97).

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4. Conclusions CJ seed gum, a Gal with solution properties similar to LBG, was used in gelling mixtures with k-car. From the present results, obtained from viscoelastic measurements and compression tests, it can be concluded that CJ and LBG display similar synergistic effects with



[22] [23] [24]

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