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Synergistic interaction between κ-carrageenan isolated from Hypnea charoides Lamouroux and galactomannan on its gelation
Food Research International, Vol. 31, No. 8, pp. 543±548, 1998 # 1999 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0963-9969(99)00022-8 0963-9969/99/$ - see front matter
Synergistic interaction between -carrageenan isolated from Hypnea charoides LAMOUROUX and galactomannan on its gelation Masakuni Tako,* Zhi-Qing Qi, Eriko Yoza & Seizen Toyama Department of Bioscience and Biotechnology, University of the Ryukyus, Nishihara, Okinawa 903-0123, Japan The synergistic eects on rheological properties for a series of aqueous solution of -carrageenan isolated from Hypnea charoides Lamouroux and galactomannan (locust-bean gum) were investigated. At a concentration of 0.4% of total gums, gelation did not occur at room temperature, but it did at a low temperature (0 C). The maximum dynamic modulus was obtained with a series of the samples composed of K-salt of -carrageenan and locust-bean gum in the mixing ratio of 1:1 at low temperature (0 C). The less synergistic eect on the dynamic modulus was obtained in mixture solutions with Na-salt of -carrageenan and locust-bean gum. At about 25 C, gel-sol transition was observed in the mixing ratio of -carrageenan (K-salt) to locust-bean gum of 3:1 and 4:1. A possible association site between K-salt of -carrageenan and locust-bean gum was proposed. # 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: synergistic interaction, gelation mechanism, -carrageenan and galactomannan, theology.
in Scheme 1, the side chains of the xanthan molecule are inserted into the adjacent, unsubstituted segments of the mannan backbone, which is extended into a two-fold, ribbon-like structure, similar to the lock-and-key interaction. On the other hand, the synergistic interaction between -carrageenan and galactomannan (locust-bean gum) in aqueous solution for gelation is well known (Dea et al., 1972; Fernandes et al., 1991; Rees, 1972; Rochas et al., 1990). Dea et al. (1972) proposed a mechanism for the interaction between the double stranded helix of -carrageenan and unbranched smooth segments of the dmannose backbone of locust-bean gum molecule. Carrageenan has alternating disaccharide units of (1±3) linked -d-galactose-4-sulfate and (1±4) linked 3,6-anhydro- -d-galactose, and is well known for its gel forming property (Snoeren and Payens, 1976; Norton et al., 1983; Morris and Chilvers, 1983; Rochas and Rinaudo, 1984). We have proposed that the -carrageenan molecule may form intramolecular cation-bridges between the sulfate group of the d-galactose-4-sulfate residue and the ring oxygen group of an adjacent anhydro-d-galactose residue with large cations, such as K+, Rb+, and
INTRODUCTION A novel and useful property of xanthan in the food industry is not only its curious viscosity and dynamic viscoelasticity (Jeanes et al., 1961; Tako et al., 1977) where a sigmoid curve is observed with increasing temperature but also its reactivity with galactomannan, such as locust-bean gum (Pettitt, 1982; Tako et al., 1984), guar gum (Tako & Nakamura, 1985, 1986a), and tara-bean gum (Tako, 1991a). We have proposed possible binding sites for d-mannose-speci®c interaction between xanthan and galactomannan (locust-bean gum) involving the side chains of the former and backbone of the latter molecules, as illustrated in Scheme 1 (Tako, 1991b, 1992, 1993). Hydrogen bonding may take place between the hemiacetal oxygen atom of the inner d-mannose side-chain of xanthan and the hydroxyl group at C-2. As the mannan backbone of the locust-bean gum molecule has a rigidity due to an intramolecular hydrogen bonding, O(5). . .C(3) 0 (Zugenmaier, 1974) as illustrated *To whom correspondence should be addressed. Tel.: +8198-895-8814; fax: +81-98-895-8814. 543
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Scheme 1. Possible binding site for d-mannose-speci®c interaction between xanthan and galactomannan (locust-bean gum) in aqueous solution: (- - - -), hydrogen bonding; ( ), van der Waals forces of attraction. The xanthan molecule retains its ®ve-fold single-stranded helix and its side chains are inserted into adjacent unsubstituted segments of the backbone of the galactomannan molecule. This gives a lock and key mode of interaction. A molecule of xanthan may combine with two or more molecules of galactomannan, the ratio depending on the preferred conformation in aqueous solution. (L), locust-bean gum: (X), xanthan scale.
Cs+, but not with small cations, Li+ or Na+, as illustrated in Scheme 2 (Tako and Nakamura, 1986b). This model could expand to the gelling mechanism of carrageenan molcules in aqueous solution at low temperatures. Intermolecular cation-bridges might also occur after formation of a very large number of intramolecular cations-bridges which may be caused by the decrease of Brownian motion and kinetic energy of the solvent and polymer molecules (Scheme 2) (Tako and Nakamura, 1986c). Hypnea charoides Lamouroux which belongs to the red seaweed (Rhodophyceae) group is grown in the northern part of Okinawa Island (Japan). In Okinawa, Hypnea charoides Lamouroux has been used for gelling additives of a health food called ``Moi-tofu'' for over 200 years (Tohma, 1988). Annual production of Hypena charoides Lamouroux in Okinawa has been reported to be approximately 5 tons (Tohma, 1988). We have previously isolated a -carrageenan from Hypena charoides Lamouroux (Qi et al., 1997a) and its non-Newtonian behavior and dynamic viscoelasticity were also analyzed with respect to the association characteristics comparison with those of commercial -carrageenan (Qi et al., 1997b). In this investigation, we studied the rheological behavior of a mixture system of -carrageenan isolated from Hypnea charoides Lamouroux and locust-bean gum in solution, and its rheological properties have been analyzed with respect to its association characteristics in more detail so as to propose a possible model of association-site.
MATERIALS AND METHODS Material -Carrageenan used in the present study was extracted from Hypnea charoides Lamouroux which was collected in March 1995 at Nago City in Okinawa Island (Japan). An air-dried Ibaranori (6 g) was suspended in water and heated at 100 C for 1 h for extraction of -carrageenan. The extract was centrifuged at 12 000 g for 20 min and the supernatant was ®ltered through Celite 545, then KCl (100 mg) was added to the ®ltrate. The gelatinous precipitate was separated by centrifugation at 12 000 g for 20 min. The precipitate was then dialyzed while running the water overnight to complete the dissolution. The solution was diluted with water to 0.5 l, and ®ltered through Celite 545 again. In the presence of KCl (0.2%; 20 mL), ethanol (2 vol) was added to the solution, and the precipitate was collected by centrifugation at 12 000 g and dried in vacuo (2.9 g). The crude -carrageenan (2 g) was dissolved in 300 ml of distilled water and ®ltered through Celite 545. The ®ltrate was dialyzed against distilled water until free from chloride, and then ethanol (2 vol) was added to the dialyzate. The precipitate was dried in vacuo (1.8 g). Puri®ed K+ salt of -carrageenan was redissolved in water, and the solution deionized by passing through a column of Amberlite IR 120 (H+), and made neutral with 50 mM NaOH. The solution was ®ltered through Celite 545. Ethanol (2 vol) was added to the ®ltrate in
Synergistic interaction between -carrageenan and galactomannan on its gelation
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collected at a ¯ow rate of 0.2 ml minÿ1. The fractions were treated by the phenol-sulfuric acid reagent, and the color developement was measured at 490 nm. Standard dextrans, T10 (molecular mass, 10 000), T110 (110 000), and T500 (500 000): Pharmacia Chemicals, having de®nite molecular weight, were used for calibration. The molecular mass of locust-bean gum was determined by a viscometric method according to the relationship (Robinson et al., 1982) 3:8 10ÿ4 Mr0:723 for guar galactomannan. Intrinsic viscosity [Z] was determined by measuring the speci®c viscosity with an Ostwald-type viscometer at 25 C. The ¯ow time for water was 42 s. Scheme 2. Possible gelation mechanism of -carrageenan in aqueous solution: (.-.-.-), ionic bonding; (///), electrostatic forces of attraction. The arrows refer to the orientation of the conformation angles, and . The model corresponds to a double stranded helix, though the sulfate groups may be on the inside of the helix.
the presence of 0.1% NaCl, and the precipitate was dried in vacuo. Locust-bean gum (Taiyo Kagaku Co., Ltd., Yokkaichi, Japan) was the same as used in our preceding papers (Tako et al., 1984; Tako and Nakamura, 1986a,c; Tako, 1991b,1992,1993). A solution of 0.3% (w/v) locust-bean gum in hot water (85 C) was ®ltered through Celite 545, ethanol (2 vols) was added and the precipitate was dried in vacuo. Various mixed solutions of -carrageenan and locustbean gum having a total concentration of 0.4% were prepared by dissoloving locust-bean gum at hot water (85 C) and adding -carrageenan (K- and Na-salt). d-Mannose and d-galactose ratio of locust-bean gum A solution of locust-bean gum (50 mg) in 2 M H2SO4 (20 ml) was heated at 100 C for 3 h. After being cooled in an ice bath, the hydrolyzate was neutralized with BaCO3 and ®ltered through Celite 545 into a 10-mM volumetric ¯ask. Liquid chromatography was performed with a Hitachi L-6200 chromatograph (Hitachi Co., Ltd., Naka, Japan), equipped with a column of #3013-N using a mobile phase of 0.3 M boric acid (temperature 60 C; pressure, 28 Kg cmÿ2; ¯ow rate 0.5 cm/min). Molecular mass. The molecular mass of the -carrageenan was determined by high-performance liquid chromatoraphy (HPLC) (Shimadzu CRB-6A; Shimadzu Seisakusho Co., Ltd., Kyoto, Japan) on a Superose 12 column (Pharmacia; 10300 mm) with a sample loop of 200 l. The HPLC operation was performed at room temperature. The column was developed with 50 mg phosphoric buer, and the same buer was supplemented with 150 mM sodium chloride, and fractions (each, 0.6 ml) were
Viscosity and dynamic viscoelasticity measurements. Steady ¯ow viscosity at various shear rates (1.19±95.05 secÿ1) and dynamic shear viscoelasticity at a picked frequency (3.77 rad sÿ1) were measured with a rheogoniometer consisting of a coaxial cylinder (1.8 cm dia.) and rotating outer cylinder (2.2 cm dia.), 6 cm length (IR 103, Iwamoto Seisakusho, Co., Ltd., Kyoto, Japan). The temperature of the sample was controlled by circulating oil from a thermo-cool (LCH-130F, Toyo Co., Ltd., Tokyo, Japan) over the temperature range of 0± 60 and raised at a rate of 1 minÿ1 by steps. Shear rate (D), shear stress (S), and apparent viscosity () were calculated with the equation of Margules (Harris, 1977). Dynamic rigidity (G0 ) was calculated by a modi®cation of Markovitz's equation (Markovitz, 1952). Values reported are the mean of at least two determinations. RESULTS AND DISCUSSION The -carrageenan was obtained about 42.1% from the dried seaweed. The -carrageenan was composed of dgalactose, 3,6-anhydro-d-galactose, and ester sulfate in a molar ratio of 1.2:0.9:1.2. Molecular mass of the polysaccharide was estimated to be about 230 000, by liquid chromatography. As the intermolecular interaction between -carrageenan and galactomannan molecules is closely correlated with the degree of substitution of the mannan chain, the degree of substitution of locust-bean gum was determined by liquid chromatography and the ratio of d-mannose to d-galactose was calculated to be 4:1. The molecular mass determined from viscosity measurement was 226 000. The ¯ow curves, at 25 C, of a mixture of K salt of carrageenan with locust-bean gum as the ratio of the two gums was altered, keeping the total concentration constant at 0.4% approximated to shear-thinning behavior and were shifted over the higher shear stress than that of carrageenan or locust-bean gum alone, as illustrated in Fig. 1. This indicates that there is less interaction in the mixture solution, because the yield value was not observed, possibly due to the dissociation of intramolecular
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K+-bridge at 25 C (see Scheme 2). The ¯ow curves, however, of a mixture of Na-salt of -carrageenan with locust-bean gum showed Newtonian behavior and shifted to lower shear-stress than those of the mixture of K-salt of -carrageenan (not shown in the ®gure). The eect of the ratio of -carrageenan (K- and Nasalt) to locust-bean gum in the solution on the dynamic modulus at 0.4% total gums at 0 C is shown in Fig. 2. There was a synergistic interaction in the mixture with K-salt of -carrageenan solution. The very large dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean gum was 1:1±3:1. The maximum dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean gum was 1:1 which was as same as that in the case of commercial K-salt of -carrageenan with locust-bean gum (Tako and Nakamura, 1986c) where, however, a synergistic gelation occurred at 0.6% total gums. This indicates that a synergistic interaction between a mixture of locust-bean gum with the -carrageenan isolated from Hypnea charoides Lamouroux is stronger than that with commercial -carrageenan which was prepared from Eucheuma cottonii (Tako and Nakamura, 1986b).The results suggest that there are equal numbers of junction sites on both molecular chains in aqueous solution. However, a very large dynamic modulus also occurred in the mixing ratio of -carrageenan to locust-bean gum of 2:1 and 3:1. This result suggests that self-association within the -carrageenan molecules, as shown in Scheme 2, also takes place in the solutions. The synergistic increase in the dynamic modulus did not occur in a mixture with Na-salt of -carrageenan. This may be
Fig. 1. Flow curves, at 25 C, of the mixed solution of -carrageenan and locust-bean gum at a total concentration of 0.4%. Ratio of k-carrageenan to locust-bean gum: j , 4:1; , 3:1; , 2:1; , 1:1; , 1:2; , 1:3; , 1:4; *, K-salt of k-carrageenan alone; *, locust-bean gum alone.
caused by the radius of the cation Na+ being too small to associate with the ring oxygen atom of anhydro-dgalactose residue and by the hydration of Na+ being too large to take place an electrostatic forces of attraction
Fig. 2. Dynamic modulus (at 3.77 rad sÿ1 and 25 C) of a 0.4% -carrageenan±locust-bean gum solution as a function of the ratio of components: *, K salt; , Na salt. K, -carrageenan; L, locust-bean gum.
Fig. 3. Dynamic modulus at 3.77 rad sÿ1 at various temperatures for the 0.4% k-carrageenan (K-salt)-locust-bean gum. Temperature ( C): *, 0; , 5; , 10; j, 15; , 20; , 25; , 30; *, 35. K, -carrageenan; L, locust-bean gum.
Synergistic interaction between -carrageenan and galactomannan on its gelation which may be prevented by a large number of water molecules (Tako and Nakamura, 1986a,b). The result indicates that a synergistic interaction between -carrageenan and locust-bean gum solutions depends on the type of cation as does the -carrageenan alone. Though the maximum dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean
Fig. 4. Eects of temperature on the dynamic modulus of kcarrageenan- locust-bean gum solution at 0.4% total gums and 3.77 rad sÿ1. Combination ratio of k-carrageenan to locust-bean gum: j, 4:1; , 3:1; ; 2:1; , 1:1; , 1:2; , 1:3; , 1:4; *, K-salt of -carrageenan alone; (*), locust-bean gum alone.
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gum was 1:1, large values were also achieved at mixing ratios of 4:1, 3:1, and 2:1, and at low temperature range (0±15 C), as shown in Fig. 3. A very strong dynamic modulus was maintained when the mixing ratio was 3:1 over a wide range of temperature 0±25 C. The result suggests that self-association within -carrageenan molecules also takes place in addition to the synergistic interaction with locust-bean gum, as discussed above. Figure 4 shows the eects of temperature on dynamic modulus of a mixed solution of K-salt of -carrageenan and locust-bean gum at 0.4% total gums. The dynamic modulus increased greatly at a low temperature (0 C) where the maximum dynamic modulus was observed in a mixing ratio of 1:1. Large dynamic moduli were also observed in the solutions of -carrageenan to locustbean gum in the ratios of 3:1, 2:1, 4:1, at 0 C. The dynamic moduli decreased gradually with increasing temperature in the 1:1, 2:1, 1:2, and 1:3 solutions. However, in the 3:1 and 4:1 solutions, the dynamic moduli remained constant with increasing temperature up to 25 C, which was estimated to be a transition temperature, then they decreased rapidly. A constant dynamic modulus may be attributed to the self-association of Ksalt of -carrageenan molecules. A synergistic interaction also occurred even at 0.2% total gums in the 3:1 ratio of -carrageenan to locust-bean gum in the presence of 0.1% KCl (not shown in the ®gure). A transition temperature was observed when the temperature reached 25 C. This indicates that intermolecular association between -carrageenan and locust-bean gum dissociates rapidly above the transition temperature. Urea is known as a hydrogen bonding breaker so that the dynamic viscoelasticity of the mixture with locust-bean
Fig. 5. Possible association site between -carrageenan and locust-bean gum in aqueous solution: (- - - -), hydrogen bonding; (.-.-.), ionic bonding; (////), electrostatic forces of attraction. (L), locust-bean gum; (K), -carrageenan scale.
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gum was measured in the presence of urea (1 or 4 M). The dynamic viscoelasticity of the both solutions were lower than that of a mixture solution alone. The lowest dynamic viscoelasticity was observed, at 4 M urea and 13.5 mM KCl, indicating that urea prevented formation of not only intramolecular K+-bridging but also intermolecular interaction (not shown in the ®gure). The result suggests that hydrogen bonding may also take part in the interaction in co-operation with ionic bonding and electrostatic forces of attraction (see Scheme 2). CONCLUSIONS Though a mixed solution of xanthan and locust-bean gum, and konjac glucomannan gelled at 0.2 and 0.1% total gums at room temperatuire (Tako et al., 1984; Tako and Nakamura, 1986a; Tako, 1991b,1992,1993), a mixture of -carrageenan (K-salt) and locust-bean gum solution did not gel even at 0.4% total gums at room temperature, but gelled at low temperature (0 C). Less interaction was observed in a mixed solution with Na salt of -carrageenan. As proposed in our preceding papers (Tako, 1991b,1992,1993), the OH-2 of d-mannopyranosyl residue of locust-bean gum molecule might take part in hydrogen bonding with the hemiacetal oxygen atom of the inner d-mannopyranosyl side-chain of xanthan molecule (see Scheme 2). Thus, we conclude that an intermolecular interaction between -carrageenan (K-salt) and locust-bean gum at a low concentration (0.4% total gums, 1:1) may occur between the ring oxygen atom of the former and OH-2 of the d-mannopyranosyl residue with hydrogen bonding, at which the intramolecular K+-bridge is inserted into the adjacent unsubstituted segments of the backbone of the locust-bean gum molecule, as illustrated in Fig. 5. This may correspond to a single stranded helix for the -carrageenan molecule. However, when in the ratios of 3:1 and 4:1, self-association in the -carrageenan molecules seems also to take place on the dierent molecular chains, as illustrated in Scheme 2. REFERENCES Dea, I. C. M., McKinnon, A. A. and Rees, D. A. (1972) Tertiary and quaternary structure in aqueous polysacchride systems which model cell wall cohesion: reversible changes in conformation and association of agarose, carrageenan and galacto-mannan. J. Mol. Biol. 68, 153±172. Fernandes, P. B., Goncalves, M. P. and Doublier, J. L. (1991) A rheological characterization of kappa-carrageenan/galactomannan mixed gels: A comparison of locust bean gum samples. Carbohydr. Polym. 16, 253±274. Harris, J. (1977). Rheology and non-Newtonian ¯ow. New York: Longman, pp. 28±33. Jeanes, A., Pittsley, J. E. and Senti, F. R. (1961) A new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. J. Appl. Polym. Sci. 5, 519±526.
Markovitz, H. (1952) A property of bessel function and its application to the theory of two rheometers. J. Appl. Phys. 23, 1070±1077. Morris, V. J. and Chilvers, G. R. (1983) Rheological studies of speci®c cation forms of kappa carrageenan gels. Carbohydr. Polym. 3, 129±141. Norton, I. T., Goodall, D. M., Morris, E. D. and Rees, D. A. (1983) Equilibrium and dynamic studies of the disorderorder transition of kappa carrageenan. J. Chem. Soc. Faraday Trans. 1(79), 2489±2500. Pettitt, D. (1982) Xanthan gum. In Food Hydrocolloids ed. M. Glicksman, Vol. 1, pp 127±149. CRC Press, Boca Raton. Qi, Z.- Q., Tako, M. and Toyama, S. (1997) Chemical characterization of -carrageenan of Ibaranori (Hypnea charoides Lamouroux). Oro Toshitsu Kagaku 44, 137±142. Qi, Z.- Q., Tako, M. and Toyama, S. (1997) Molecular origin for the rheological characteristics of k-carrageenan isolated from Ibaranori (Hypnea charoides Lamouroux). Orotoshitsu Kagaku 44, 531±536. Rees, D. A. (1972) Shapely polysaccharides. Biochem. J. 126, 257±273. Robinson, G., Ross-Murphy, S. B. and Morris, E. R. (1982) Viscosity-molecular weight relationship, intrinsic chain ¯exibility, and dynamic solution properties of guar galactomannan. Carbohydr. Res. 107, 17±32. Rochas, C. and Rinaudo, M. (1984) Mechanism of gel formation in -carrageenan. Biopolymers 23, 735±745. Rochas, C., Taravel, F.-R. and Turquois (1990) N.M.R. studies of synergistic kappa carrageenan-carob galactomannan. Int. Biol. Macromol. 12, 353±358. Snoeren, T. H. M. and Payens, T. A. J. (1976) On the sol-gel transition in solution of kappa-carrageenan. Biochim Biophys. Acta 437, 264±272. Tako, M. (1991a) Synergisic interaction between xanthan and tara-bean gum. Carbohydr. Polym. 15, 227±239. Tako, M. (1991b) Synergistic interaction between deacylated xanthan and galactomannan. J. Carbohydr. Chem. 10, 619±633. Tako, M. (1992) Molecular origin for rheological characteristics of xanthan gum. ACS Symp. Ser. 489, 268±281. Tako, M. (1993) Binding sites for d-mannose-speci®c interaction between xanthan and galactomannan, and glucomannan. Colloids Surfaces B Biointerfaces 1, 125±131. Tako, M., Asato, A. and Nakamura, S. (1984) Rheological aspects of the intermolecular interaction between xanthan and locust bean gum in aqueous media. Agric. Biol. Chem. 48, 2995±3000. Tako, M., Nagahama, T. and Nomura, D. (1977) non-Newtonian ¯ow and dynamic viscoelasticity of xanthan gum. Nippon Nogeikagaku Kaishi 51, 513±518. Tako, M. and Nakamura, S. (1985) Synergistic interaction between xanthan and guar gum. Carbohydr. Res. 138, 206±213. Tako, M. and Nakamura, S. (1986a) d-Mannose-speci®c interaction between xanthan and d-galacto-d-mannan. FEBS Lett. 204, 33±36. Tako, M. and Nakamura, S. (1986b) Indicative evidence for a conformational transition in -carrageenan from studies of viscosity-shear rate dependence. Carbohydr. Res. 155, 200±205. Tako, M. and Nakamura, S. (1986c) Synergistic interaction between kappa carrageenan and locust bean gum in aqueous media. Agric. Biol. Chem. 50, 2817±2822. Tohma, T. (1988) Kaisourui no zouyoushyoku. In Sangoshyou no zouyoushyoku ed., S. Shokita (pp. 83±88). Midori Shobou, Tokyo Zugenmaier, P. (1974) Conformation and Packing analysis of polysaccharide and derivatives. 1: Mannan. Biopolymers 13, 1127±1139.
(Received 3 July 1998; accepted 17 January 1999)
Synergistic interaction between -carrageenan isolated from Hypnea charoides LAMOUROUX and galactomannan on its gelation Masakuni Tako,* Zhi-Qing Qi, Eriko Yoza & Seizen Toyama Department of Bioscience and Biotechnology, University of the Ryukyus, Nishihara, Okinawa 903-0123, Japan The synergistic eects on rheological properties for a series of aqueous solution of -carrageenan isolated from Hypnea charoides Lamouroux and galactomannan (locust-bean gum) were investigated. At a concentration of 0.4% of total gums, gelation did not occur at room temperature, but it did at a low temperature (0 C). The maximum dynamic modulus was obtained with a series of the samples composed of K-salt of -carrageenan and locust-bean gum in the mixing ratio of 1:1 at low temperature (0 C). The less synergistic eect on the dynamic modulus was obtained in mixture solutions with Na-salt of -carrageenan and locust-bean gum. At about 25 C, gel-sol transition was observed in the mixing ratio of -carrageenan (K-salt) to locust-bean gum of 3:1 and 4:1. A possible association site between K-salt of -carrageenan and locust-bean gum was proposed. # 1999 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: synergistic interaction, gelation mechanism, -carrageenan and galactomannan, theology.
in Scheme 1, the side chains of the xanthan molecule are inserted into the adjacent, unsubstituted segments of the mannan backbone, which is extended into a two-fold, ribbon-like structure, similar to the lock-and-key interaction. On the other hand, the synergistic interaction between -carrageenan and galactomannan (locust-bean gum) in aqueous solution for gelation is well known (Dea et al., 1972; Fernandes et al., 1991; Rees, 1972; Rochas et al., 1990). Dea et al. (1972) proposed a mechanism for the interaction between the double stranded helix of -carrageenan and unbranched smooth segments of the dmannose backbone of locust-bean gum molecule. Carrageenan has alternating disaccharide units of (1±3) linked -d-galactose-4-sulfate and (1±4) linked 3,6-anhydro- -d-galactose, and is well known for its gel forming property (Snoeren and Payens, 1976; Norton et al., 1983; Morris and Chilvers, 1983; Rochas and Rinaudo, 1984). We have proposed that the -carrageenan molecule may form intramolecular cation-bridges between the sulfate group of the d-galactose-4-sulfate residue and the ring oxygen group of an adjacent anhydro-d-galactose residue with large cations, such as K+, Rb+, and
INTRODUCTION A novel and useful property of xanthan in the food industry is not only its curious viscosity and dynamic viscoelasticity (Jeanes et al., 1961; Tako et al., 1977) where a sigmoid curve is observed with increasing temperature but also its reactivity with galactomannan, such as locust-bean gum (Pettitt, 1982; Tako et al., 1984), guar gum (Tako & Nakamura, 1985, 1986a), and tara-bean gum (Tako, 1991a). We have proposed possible binding sites for d-mannose-speci®c interaction between xanthan and galactomannan (locust-bean gum) involving the side chains of the former and backbone of the latter molecules, as illustrated in Scheme 1 (Tako, 1991b, 1992, 1993). Hydrogen bonding may take place between the hemiacetal oxygen atom of the inner d-mannose side-chain of xanthan and the hydroxyl group at C-2. As the mannan backbone of the locust-bean gum molecule has a rigidity due to an intramolecular hydrogen bonding, O(5). . .C(3) 0 (Zugenmaier, 1974) as illustrated *To whom correspondence should be addressed. Tel.: +8198-895-8814; fax: +81-98-895-8814. 543
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Scheme 1. Possible binding site for d-mannose-speci®c interaction between xanthan and galactomannan (locust-bean gum) in aqueous solution: (- - - -), hydrogen bonding; ( ), van der Waals forces of attraction. The xanthan molecule retains its ®ve-fold single-stranded helix and its side chains are inserted into adjacent unsubstituted segments of the backbone of the galactomannan molecule. This gives a lock and key mode of interaction. A molecule of xanthan may combine with two or more molecules of galactomannan, the ratio depending on the preferred conformation in aqueous solution. (L), locust-bean gum: (X), xanthan scale.
Cs+, but not with small cations, Li+ or Na+, as illustrated in Scheme 2 (Tako and Nakamura, 1986b). This model could expand to the gelling mechanism of carrageenan molcules in aqueous solution at low temperatures. Intermolecular cation-bridges might also occur after formation of a very large number of intramolecular cations-bridges which may be caused by the decrease of Brownian motion and kinetic energy of the solvent and polymer molecules (Scheme 2) (Tako and Nakamura, 1986c). Hypnea charoides Lamouroux which belongs to the red seaweed (Rhodophyceae) group is grown in the northern part of Okinawa Island (Japan). In Okinawa, Hypnea charoides Lamouroux has been used for gelling additives of a health food called ``Moi-tofu'' for over 200 years (Tohma, 1988). Annual production of Hypena charoides Lamouroux in Okinawa has been reported to be approximately 5 tons (Tohma, 1988). We have previously isolated a -carrageenan from Hypena charoides Lamouroux (Qi et al., 1997a) and its non-Newtonian behavior and dynamic viscoelasticity were also analyzed with respect to the association characteristics comparison with those of commercial -carrageenan (Qi et al., 1997b). In this investigation, we studied the rheological behavior of a mixture system of -carrageenan isolated from Hypnea charoides Lamouroux and locust-bean gum in solution, and its rheological properties have been analyzed with respect to its association characteristics in more detail so as to propose a possible model of association-site.
MATERIALS AND METHODS Material -Carrageenan used in the present study was extracted from Hypnea charoides Lamouroux which was collected in March 1995 at Nago City in Okinawa Island (Japan). An air-dried Ibaranori (6 g) was suspended in water and heated at 100 C for 1 h for extraction of -carrageenan. The extract was centrifuged at 12 000 g for 20 min and the supernatant was ®ltered through Celite 545, then KCl (100 mg) was added to the ®ltrate. The gelatinous precipitate was separated by centrifugation at 12 000 g for 20 min. The precipitate was then dialyzed while running the water overnight to complete the dissolution. The solution was diluted with water to 0.5 l, and ®ltered through Celite 545 again. In the presence of KCl (0.2%; 20 mL), ethanol (2 vol) was added to the solution, and the precipitate was collected by centrifugation at 12 000 g and dried in vacuo (2.9 g). The crude -carrageenan (2 g) was dissolved in 300 ml of distilled water and ®ltered through Celite 545. The ®ltrate was dialyzed against distilled water until free from chloride, and then ethanol (2 vol) was added to the dialyzate. The precipitate was dried in vacuo (1.8 g). Puri®ed K+ salt of -carrageenan was redissolved in water, and the solution deionized by passing through a column of Amberlite IR 120 (H+), and made neutral with 50 mM NaOH. The solution was ®ltered through Celite 545. Ethanol (2 vol) was added to the ®ltrate in
Synergistic interaction between -carrageenan and galactomannan on its gelation
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collected at a ¯ow rate of 0.2 ml minÿ1. The fractions were treated by the phenol-sulfuric acid reagent, and the color developement was measured at 490 nm. Standard dextrans, T10 (molecular mass, 10 000), T110 (110 000), and T500 (500 000): Pharmacia Chemicals, having de®nite molecular weight, were used for calibration. The molecular mass of locust-bean gum was determined by a viscometric method according to the relationship (Robinson et al., 1982) 3:8 10ÿ4 Mr0:723 for guar galactomannan. Intrinsic viscosity [Z] was determined by measuring the speci®c viscosity with an Ostwald-type viscometer at 25 C. The ¯ow time for water was 42 s. Scheme 2. Possible gelation mechanism of -carrageenan in aqueous solution: (.-.-.-), ionic bonding; (///), electrostatic forces of attraction. The arrows refer to the orientation of the conformation angles, and . The model corresponds to a double stranded helix, though the sulfate groups may be on the inside of the helix.
the presence of 0.1% NaCl, and the precipitate was dried in vacuo. Locust-bean gum (Taiyo Kagaku Co., Ltd., Yokkaichi, Japan) was the same as used in our preceding papers (Tako et al., 1984; Tako and Nakamura, 1986a,c; Tako, 1991b,1992,1993). A solution of 0.3% (w/v) locust-bean gum in hot water (85 C) was ®ltered through Celite 545, ethanol (2 vols) was added and the precipitate was dried in vacuo. Various mixed solutions of -carrageenan and locustbean gum having a total concentration of 0.4% were prepared by dissoloving locust-bean gum at hot water (85 C) and adding -carrageenan (K- and Na-salt). d-Mannose and d-galactose ratio of locust-bean gum A solution of locust-bean gum (50 mg) in 2 M H2SO4 (20 ml) was heated at 100 C for 3 h. After being cooled in an ice bath, the hydrolyzate was neutralized with BaCO3 and ®ltered through Celite 545 into a 10-mM volumetric ¯ask. Liquid chromatography was performed with a Hitachi L-6200 chromatograph (Hitachi Co., Ltd., Naka, Japan), equipped with a column of #3013-N using a mobile phase of 0.3 M boric acid (temperature 60 C; pressure, 28 Kg cmÿ2; ¯ow rate 0.5 cm/min). Molecular mass. The molecular mass of the -carrageenan was determined by high-performance liquid chromatoraphy (HPLC) (Shimadzu CRB-6A; Shimadzu Seisakusho Co., Ltd., Kyoto, Japan) on a Superose 12 column (Pharmacia; 10300 mm) with a sample loop of 200 l. The HPLC operation was performed at room temperature. The column was developed with 50 mg phosphoric buer, and the same buer was supplemented with 150 mM sodium chloride, and fractions (each, 0.6 ml) were
Viscosity and dynamic viscoelasticity measurements. Steady ¯ow viscosity at various shear rates (1.19±95.05 secÿ1) and dynamic shear viscoelasticity at a picked frequency (3.77 rad sÿ1) were measured with a rheogoniometer consisting of a coaxial cylinder (1.8 cm dia.) and rotating outer cylinder (2.2 cm dia.), 6 cm length (IR 103, Iwamoto Seisakusho, Co., Ltd., Kyoto, Japan). The temperature of the sample was controlled by circulating oil from a thermo-cool (LCH-130F, Toyo Co., Ltd., Tokyo, Japan) over the temperature range of 0± 60 and raised at a rate of 1 minÿ1 by steps. Shear rate (D), shear stress (S), and apparent viscosity () were calculated with the equation of Margules (Harris, 1977). Dynamic rigidity (G0 ) was calculated by a modi®cation of Markovitz's equation (Markovitz, 1952). Values reported are the mean of at least two determinations. RESULTS AND DISCUSSION The -carrageenan was obtained about 42.1% from the dried seaweed. The -carrageenan was composed of dgalactose, 3,6-anhydro-d-galactose, and ester sulfate in a molar ratio of 1.2:0.9:1.2. Molecular mass of the polysaccharide was estimated to be about 230 000, by liquid chromatography. As the intermolecular interaction between -carrageenan and galactomannan molecules is closely correlated with the degree of substitution of the mannan chain, the degree of substitution of locust-bean gum was determined by liquid chromatography and the ratio of d-mannose to d-galactose was calculated to be 4:1. The molecular mass determined from viscosity measurement was 226 000. The ¯ow curves, at 25 C, of a mixture of K salt of carrageenan with locust-bean gum as the ratio of the two gums was altered, keeping the total concentration constant at 0.4% approximated to shear-thinning behavior and were shifted over the higher shear stress than that of carrageenan or locust-bean gum alone, as illustrated in Fig. 1. This indicates that there is less interaction in the mixture solution, because the yield value was not observed, possibly due to the dissociation of intramolecular
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K+-bridge at 25 C (see Scheme 2). The ¯ow curves, however, of a mixture of Na-salt of -carrageenan with locust-bean gum showed Newtonian behavior and shifted to lower shear-stress than those of the mixture of K-salt of -carrageenan (not shown in the ®gure). The eect of the ratio of -carrageenan (K- and Nasalt) to locust-bean gum in the solution on the dynamic modulus at 0.4% total gums at 0 C is shown in Fig. 2. There was a synergistic interaction in the mixture with K-salt of -carrageenan solution. The very large dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean gum was 1:1±3:1. The maximum dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean gum was 1:1 which was as same as that in the case of commercial K-salt of -carrageenan with locust-bean gum (Tako and Nakamura, 1986c) where, however, a synergistic gelation occurred at 0.6% total gums. This indicates that a synergistic interaction between a mixture of locust-bean gum with the -carrageenan isolated from Hypnea charoides Lamouroux is stronger than that with commercial -carrageenan which was prepared from Eucheuma cottonii (Tako and Nakamura, 1986b).The results suggest that there are equal numbers of junction sites on both molecular chains in aqueous solution. However, a very large dynamic modulus also occurred in the mixing ratio of -carrageenan to locust-bean gum of 2:1 and 3:1. This result suggests that self-association within the -carrageenan molecules, as shown in Scheme 2, also takes place in the solutions. The synergistic increase in the dynamic modulus did not occur in a mixture with Na-salt of -carrageenan. This may be
Fig. 1. Flow curves, at 25 C, of the mixed solution of -carrageenan and locust-bean gum at a total concentration of 0.4%. Ratio of k-carrageenan to locust-bean gum: j , 4:1; , 3:1; , 2:1; , 1:1; , 1:2; , 1:3; , 1:4; *, K-salt of k-carrageenan alone; *, locust-bean gum alone.
caused by the radius of the cation Na+ being too small to associate with the ring oxygen atom of anhydro-dgalactose residue and by the hydration of Na+ being too large to take place an electrostatic forces of attraction
Fig. 2. Dynamic modulus (at 3.77 rad sÿ1 and 25 C) of a 0.4% -carrageenan±locust-bean gum solution as a function of the ratio of components: *, K salt; , Na salt. K, -carrageenan; L, locust-bean gum.
Fig. 3. Dynamic modulus at 3.77 rad sÿ1 at various temperatures for the 0.4% k-carrageenan (K-salt)-locust-bean gum. Temperature ( C): *, 0; , 5; , 10; j, 15; , 20; , 25; , 30; *, 35. K, -carrageenan; L, locust-bean gum.
Synergistic interaction between -carrageenan and galactomannan on its gelation which may be prevented by a large number of water molecules (Tako and Nakamura, 1986a,b). The result indicates that a synergistic interaction between -carrageenan and locust-bean gum solutions depends on the type of cation as does the -carrageenan alone. Though the maximum dynamic modulus was achieved when the mixing ratio of -carrageenan to locust-bean
Fig. 4. Eects of temperature on the dynamic modulus of kcarrageenan- locust-bean gum solution at 0.4% total gums and 3.77 rad sÿ1. Combination ratio of k-carrageenan to locust-bean gum: j, 4:1; , 3:1; ; 2:1; , 1:1; , 1:2; , 1:3; , 1:4; *, K-salt of -carrageenan alone; (*), locust-bean gum alone.
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gum was 1:1, large values were also achieved at mixing ratios of 4:1, 3:1, and 2:1, and at low temperature range (0±15 C), as shown in Fig. 3. A very strong dynamic modulus was maintained when the mixing ratio was 3:1 over a wide range of temperature 0±25 C. The result suggests that self-association within -carrageenan molecules also takes place in addition to the synergistic interaction with locust-bean gum, as discussed above. Figure 4 shows the eects of temperature on dynamic modulus of a mixed solution of K-salt of -carrageenan and locust-bean gum at 0.4% total gums. The dynamic modulus increased greatly at a low temperature (0 C) where the maximum dynamic modulus was observed in a mixing ratio of 1:1. Large dynamic moduli were also observed in the solutions of -carrageenan to locustbean gum in the ratios of 3:1, 2:1, 4:1, at 0 C. The dynamic moduli decreased gradually with increasing temperature in the 1:1, 2:1, 1:2, and 1:3 solutions. However, in the 3:1 and 4:1 solutions, the dynamic moduli remained constant with increasing temperature up to 25 C, which was estimated to be a transition temperature, then they decreased rapidly. A constant dynamic modulus may be attributed to the self-association of Ksalt of -carrageenan molecules. A synergistic interaction also occurred even at 0.2% total gums in the 3:1 ratio of -carrageenan to locust-bean gum in the presence of 0.1% KCl (not shown in the ®gure). A transition temperature was observed when the temperature reached 25 C. This indicates that intermolecular association between -carrageenan and locust-bean gum dissociates rapidly above the transition temperature. Urea is known as a hydrogen bonding breaker so that the dynamic viscoelasticity of the mixture with locust-bean
Fig. 5. Possible association site between -carrageenan and locust-bean gum in aqueous solution: (- - - -), hydrogen bonding; (.-.-.), ionic bonding; (////), electrostatic forces of attraction. (L), locust-bean gum; (K), -carrageenan scale.
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gum was measured in the presence of urea (1 or 4 M). The dynamic viscoelasticity of the both solutions were lower than that of a mixture solution alone. The lowest dynamic viscoelasticity was observed, at 4 M urea and 13.5 mM KCl, indicating that urea prevented formation of not only intramolecular K+-bridging but also intermolecular interaction (not shown in the ®gure). The result suggests that hydrogen bonding may also take part in the interaction in co-operation with ionic bonding and electrostatic forces of attraction (see Scheme 2). CONCLUSIONS Though a mixed solution of xanthan and locust-bean gum, and konjac glucomannan gelled at 0.2 and 0.1% total gums at room temperatuire (Tako et al., 1984; Tako and Nakamura, 1986a; Tako, 1991b,1992,1993), a mixture of -carrageenan (K-salt) and locust-bean gum solution did not gel even at 0.4% total gums at room temperature, but gelled at low temperature (0 C). Less interaction was observed in a mixed solution with Na salt of -carrageenan. As proposed in our preceding papers (Tako, 1991b,1992,1993), the OH-2 of d-mannopyranosyl residue of locust-bean gum molecule might take part in hydrogen bonding with the hemiacetal oxygen atom of the inner d-mannopyranosyl side-chain of xanthan molecule (see Scheme 2). Thus, we conclude that an intermolecular interaction between -carrageenan (K-salt) and locust-bean gum at a low concentration (0.4% total gums, 1:1) may occur between the ring oxygen atom of the former and OH-2 of the d-mannopyranosyl residue with hydrogen bonding, at which the intramolecular K+-bridge is inserted into the adjacent unsubstituted segments of the backbone of the locust-bean gum molecule, as illustrated in Fig. 5. This may correspond to a single stranded helix for the -carrageenan molecule. However, when in the ratios of 3:1 and 4:1, self-association in the -carrageenan molecules seems also to take place on the dierent molecular chains, as illustrated in Scheme 2. REFERENCES Dea, I. C. M., McKinnon, A. A. and Rees, D. A. (1972) Tertiary and quaternary structure in aqueous polysacchride systems which model cell wall cohesion: reversible changes in conformation and association of agarose, carrageenan and galacto-mannan. J. Mol. Biol. 68, 153±172. Fernandes, P. B., Goncalves, M. P. and Doublier, J. L. (1991) A rheological characterization of kappa-carrageenan/galactomannan mixed gels: A comparison of locust bean gum samples. Carbohydr. Polym. 16, 253±274. Harris, J. (1977). Rheology and non-Newtonian ¯ow. New York: Longman, pp. 28±33. Jeanes, A., Pittsley, J. E. and Senti, F. R. (1961) A new hydrocolloid polyelectrolyte produced from glucose by bacterial fermentation. J. Appl. Polym. Sci. 5, 519±526.
Markovitz, H. (1952) A property of bessel function and its application to the theory of two rheometers. J. Appl. Phys. 23, 1070±1077. Morris, V. J. and Chilvers, G. R. (1983) Rheological studies of speci®c cation forms of kappa carrageenan gels. Carbohydr. Polym. 3, 129±141. Norton, I. T., Goodall, D. M., Morris, E. D. and Rees, D. A. (1983) Equilibrium and dynamic studies of the disorderorder transition of kappa carrageenan. J. Chem. Soc. Faraday Trans. 1(79), 2489±2500. Pettitt, D. (1982) Xanthan gum. In Food Hydrocolloids ed. M. Glicksman, Vol. 1, pp 127±149. CRC Press, Boca Raton. Qi, Z.- Q., Tako, M. and Toyama, S. (1997) Chemical characterization of -carrageenan of Ibaranori (Hypnea charoides Lamouroux). Oro Toshitsu Kagaku 44, 137±142. Qi, Z.- Q., Tako, M. and Toyama, S. (1997) Molecular origin for the rheological characteristics of k-carrageenan isolated from Ibaranori (Hypnea charoides Lamouroux). Orotoshitsu Kagaku 44, 531±536. Rees, D. A. (1972) Shapely polysaccharides. Biochem. J. 126, 257±273. Robinson, G., Ross-Murphy, S. B. and Morris, E. R. (1982) Viscosity-molecular weight relationship, intrinsic chain ¯exibility, and dynamic solution properties of guar galactomannan. Carbohydr. Res. 107, 17±32. Rochas, C. and Rinaudo, M. (1984) Mechanism of gel formation in -carrageenan. Biopolymers 23, 735±745. Rochas, C., Taravel, F.-R. and Turquois (1990) N.M.R. studies of synergistic kappa carrageenan-carob galactomannan. Int. Biol. Macromol. 12, 353±358. Snoeren, T. H. M. and Payens, T. A. J. (1976) On the sol-gel transition in solution of kappa-carrageenan. Biochim Biophys. Acta 437, 264±272. Tako, M. (1991a) Synergisic interaction between xanthan and tara-bean gum. Carbohydr. Polym. 15, 227±239. Tako, M. (1991b) Synergistic interaction between deacylated xanthan and galactomannan. J. Carbohydr. Chem. 10, 619±633. Tako, M. (1992) Molecular origin for rheological characteristics of xanthan gum. ACS Symp. Ser. 489, 268±281. Tako, M. (1993) Binding sites for d-mannose-speci®c interaction between xanthan and galactomannan, and glucomannan. Colloids Surfaces B Biointerfaces 1, 125±131. Tako, M., Asato, A. and Nakamura, S. (1984) Rheological aspects of the intermolecular interaction between xanthan and locust bean gum in aqueous media. Agric. Biol. Chem. 48, 2995±3000. Tako, M., Nagahama, T. and Nomura, D. (1977) non-Newtonian ¯ow and dynamic viscoelasticity of xanthan gum. Nippon Nogeikagaku Kaishi 51, 513±518. Tako, M. and Nakamura, S. (1985) Synergistic interaction between xanthan and guar gum. Carbohydr. Res. 138, 206±213. Tako, M. and Nakamura, S. (1986a) d-Mannose-speci®c interaction between xanthan and d-galacto-d-mannan. FEBS Lett. 204, 33±36. Tako, M. and Nakamura, S. (1986b) Indicative evidence for a conformational transition in -carrageenan from studies of viscosity-shear rate dependence. Carbohydr. Res. 155, 200±205. Tako, M. and Nakamura, S. (1986c) Synergistic interaction between kappa carrageenan and locust bean gum in aqueous media. Agric. Biol. Chem. 50, 2817±2822. Tohma, T. (1988) Kaisourui no zouyoushyoku. In Sangoshyou no zouyoushyoku ed., S. Shokita (pp. 83±88). Midori Shobou, Tokyo Zugenmaier, P. (1974) Conformation and Packing analysis of polysaccharide and derivatives. 1: Mannan. Biopolymers 13, 1127±1139.
(Received 3 July 1998; accepted 17 January 1999)