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X-ray diffraction studies of kappa carrageenan-tara gum mixed gels
X-ray diffraction studies of kappa carrageenan-tara gum mixed gels P. Cairns, M. J. Miles and V. J. Morris 4FRC Food Research Institute (Norwich), Co/hey Lane, Norwich NR4 7UA. UK {Receiced 14 October 1985)
X-ray ,fibre diflkaction has been used to probe the molecular structure of tara gum kappa carrageenan mixed gels. The patterns obtained for all mixed gels studied are qualitatively and quantitatively the same as the patterns obtained .]'or pure kappa carrageenan. The results obtained show no evidence for a discrete molecular interaction between the two polysaccharides. Fluorescence microscopy qf mixed gels containing tara gum tagged with fluorescein revealed no evidence for gross phase separation at resolutions of ~ I #m. Keywords: Kappa carrageenan: tara gum; X-ray fibre diffraction: gels; gclation
The rheology of certain polysaccharides may be drastically modified by the addition of certain galactomannans ~'2. Such mixtures may gel under conditions for which neither pure component alone will gel. The mechanical properties of the mixed gel may differ significantly from the properties of gels which may be formed by either of the pure components. These effects are examples of what has been termed synergistic interactions between polysaccharides. Such synergisms are attractive commercially and have been exploited technologically 3. Synergistic interactions have been cited as examples of the development of quaternary structures by polysaccharides and have been proposed as models for complex cellular structures'* or specific recognition processes such as hostpathogen interactions 5'6. The kappa carrageenan-carob (locust bean) gum mixture is one of the most studied of the mixed gel systems. The currently accepted model for gelation of the mixture is usually illustrated ~'2'~ as shown in Figure 1. A specific molecular interaction between regions of the carrageenan helix and bare mannan regions of the galactomannan backbone has been proposed ~.2..~ although the stoichiometry and the total number of polymer molecules within the junction zones of the gel are vague and unspecified. There is no direct experimental evidence which can confirm the proposed interrnolecular interaction. X-ray fibre diffraction studies of mixed gels should yield, on the basis of Figure I, an entirely new pattern characteristic of the mixed molecular interaction. However, recent X-ray fibre diffraction studies 7 ofcarob kappa carrageenan mixed gels failed to reveal any evidence in support of the proposed molecular interaction. The mixed gels yielded fibre diffraction patterns identical to those obtained for pure kappa carrageenan. The article describes an extension of the previous X-ray fibre studies ~' on kappa carrageenan-carob gum gels to investigate kappa carrageenan-tara gum mixed gels. The sample of kappa carrageenan, extracted from Etwheuma cottonii, was purchased from the Sigma Chemical 0141 8130'86,,020124 04$t)3.00 c 1986 Butterworth & Co. [Publishers) Ltd
124
hat. J. Biol. Macromol., 1986, Vol 8, April
Company. The material was a mixed salt form containing 2.75% K ~, 0.53°J;Na ~ and 1 6°~,Ca 2+ and was used without further purification. The sample of tara gum, extracted from the seeds of Caesalpinia spinosa, was a gift from Dr C. Blood. The mannose-galactose ratio was found to be 3: 1. Gels were prepared in the following way: the dry powders were mixed in the appropriate quantities, wetted with alcohol and dispersed in water. The dispersions were heated to about 9 2 C and thoroughly mixed. The hot mixes were poured into plastic moulds, allowed to set at room temperature and stored for about 24 h. Kappa carrageenan yields rigid, brittle gels. Addition of tara gum produces softer, tougher gels which are able to withstand substantial mechanical deformation before fracture. Details of the mechanical properties of the mixed gels will be described elsewhere. The tara-kappa carrageenan mixed gels are qualitatively similar in properties to carob-kappa carrageenan mixed gels. Samples for X-ray fibre diffraction studies were prepared in the following manner. For kappa carrageenan and tara-kappa carrageenan mixtures the gels were set by pouring the hot sample on to a glass slide. The thin layer of gel was cut into thin strips and stretched under controlled relative humidity (RH). Elongations of about 5000 were obtained for kappa carrageenan and of about 100'!i, for tara-kappa carrageenan mixtures at RH of about 98%. Under the conditions used to prepare pure kappa carrageenan gels and tara-kappa carrageenan mixed gels, the pure tara gum samples did not gel. Tara gum fibres were prepared by stretching thin strips cut from a partially dried film of tara gum. Typical elongations of around 20000 were obtained at RH around 98";. The wavelength used was 0.154 nm and fibres were dusted with calcite for calibration. The interior of the diffraction camera was maintained at a controlled RH and was flushed continually with helium to reduce scattering. Fibre diffraction data were recorded photographically. The X-ray fibre patterns obtained for tara gum and kappa carrageenan at RH around 980; are shown in Figure 2 and Figure 3a, respectively. The data obtained for pure tara gum and pure kappa carrageenan are identical to these reported by other workers on pure tara 8'9 and pure carrageenan L°. Figure 4a shows the fibre patterns obtained for a range of mixed tara-carrageenan gels. Within the limits of experimental accuracy the patterns obtained for the mixed gels are qualitatively and quantitatively identical to those obtained for pure kappa carrageenan. The nature of the
lb%2 of,oro Carrageenan 'double helix'
Figure I Proposed ]'2'~ model for the molecular interaction between galactomannans and kappa carrageenan. Bare regions of the mannan backbone, unsubstituted with galactose side chains are pictured as binding to a carrageenan double helical structure
Notes to the Editor unit cell and the unit cell dimensions are unchanged and there is no evidence for a new pattern or for a tara pattern superimposed on the carrageenan pattern. The tara gum is clearly not incorporated in the carrageenan crystalline junction zones of the gels. Thus, there is no experimental evidence for co-crystallization of tara and kappa carrageenan. The lack of any tara pattern superimposed on the carrageenan pattern suggests that the tara molecules are not oriented upon stretching the gel and again argues against co-crystallization of the two polymers or tara forming an active part of the network structure. The absence of reflections attributable to tara gum is perhaps surprising in view of the high quality of the data obtained for tara gum alone (Figure 2). The tara samples
Figure 2 X-ray fibre pattern obtained for tara gum
3a
were partially dried and partial dehydration may be essential to induce crystallization of tara gum. When mixed gel fibres were dehydrated under tension and measured under dehydration an additional equatorial reflection appeared in the patterns (Figure 4b) corresponding to a spacing of 0.78 nm. This reflection does not correspond to any of the reflections of pure tara gum and disappears upon rehydration of the fibres. The unit cell dimensions of the galactomannans vary with relative humidity ~. However, the additional reflection lies outside the range 1.1-1.4nm reported 1~ for the d2o o spacing measured over extremes of relative humidity. We have been unable to explain the origin of this additional reflection. However, it is unlikely to represent a molecular interaction between tara and the carrageenan as the remaining pattern is unchanged. Further, the additional reflection may also be induced in pure carrageenan fibres on dehydration (Figure 3b). Thus the present measurements show no evidence for the proposed molecular interaction illustrated in Figure I. The present measurements cannot discount the possibility of random surface attachment of short regions of the mannan backbone of the galactomannan to carrageenan crystallites. The alternative explanation of polymer-polymer incompatibility might suggest the possibility of gross phase separation within the gels. To visualize the spatial distribution of the tara gum within the mixed gels fluorescent derivatives of tara gum were prepared as described elsewhere ~2. The fluoresceintagged tara gum was monitored using a fluorescence microscope. At resolutions of about 1 pm no gross phase separation was observed in the mixed gels. Thus, the present data suggest a carrageenan gel containing an even distribution of tara gum molecules. The presence of tara gum may compete for water and thus influence the intra- and intermolecular interactions of the carrageenan. The possibility of some degree of attachment of tara gum to the surface of carrageenan crystallites cannot be discounted.
3b
Figure 3 X-ray fibre patterns obtained for kappa carrageenan: (a) hydrated RH around 98°o; (b) dehydrated
Int. J. Biol. Macromol., 1986, Vol 8, April
125
Notes to the Editor 4a(1)
4a(2)
4a(3)
4a(4)
4a(5)
4b
Figure 4 X-ray fibre diffraction patterns for mixed tara gum kappa carrageenan gels. (a) Hydrated (RH about 98'!01.The percentage of kappa carrageenan present in the mixed gels was (1) 75°/. (2) 66%, (3) 50°/,. (4) 45% and (5) 350J~.(bl A 50j"/,,mixture is shown to illustrate the appearance of the additional reflection. These extra equatorial reflections disappeared upon rehydration
126
Int. J. Biol. Macromol., 1986, Vol 8, April
N o t e s to the Editor
References 1 2 3
4 5 6
Dea, 1. C. M. in 'Polysaccharides in Food', (Eds J. M. V. Blanshard and J. R. Mitchell), Butterworths, London, 1979, p. 299 Dea, 1. C. M. and Morrison, A. Adv. Carbohydr. Chem. Biochem. 1975, 31,241 Glicksman, M. 'Gum Technology in the Food Industry', Academic Press, New York, 1968, p. 43 Dea, l.C.M.,McKinnon, A.A.andRees, D.A.J. MoI. Biol. 1972. 68. 153 Dea, I. C. M., Morris, E. R., Rees, D. A., Welsh, E. J., Barnes, H. A. and Price, J. Carbohydr. Res. 1977, 57, 249 Powell, D. A. in 'Microbial Polysaccharides and
7 8
9 10 I1 12
Polysaccharases', (Eds R. C. W. Berkeley, G. W. Gooday and D. C. Ellwood), Academic Press, London, 1979, p. 117 Miles, M. J., Morris, V. J. and Carroll, V. Macromoleeules 1984, 17, 2443 Winter, W. T., Chien, Y. Y. and Bouckris, H. in 'Gums and Stabilisers for the Food Industry. 2. Applications of Hydrocolloids', (Eds G. O. Phillips, D. J. Wedlock and P. A. Williams), Pergamon Press, Oxford, 1984, p. 535 Chien, Y. Y. and Winter, W. T. Personal communication Elloway, H. F. PhD Thesis, University of Bristol, UK, 1977 Marchessault, R. H., Buleon, A., Deslandes, Y. and Goto, T. J. Coll. Inte(/ace Sci. 1979, 71,375 Kitamura, S., Yunokawa, H. and Kuge, T. Polym. J. 1982, 14, 85
Int. J. Biol. M a c r o m o l . , 1986, V o l 8, A p r i l
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X-ray ,fibre diflkaction has been used to probe the molecular structure of tara gum kappa carrageenan mixed gels. The patterns obtained for all mixed gels studied are qualitatively and quantitatively the same as the patterns obtained .]'or pure kappa carrageenan. The results obtained show no evidence for a discrete molecular interaction between the two polysaccharides. Fluorescence microscopy qf mixed gels containing tara gum tagged with fluorescein revealed no evidence for gross phase separation at resolutions of ~ I #m. Keywords: Kappa carrageenan: tara gum; X-ray fibre diffraction: gels; gclation
The rheology of certain polysaccharides may be drastically modified by the addition of certain galactomannans ~'2. Such mixtures may gel under conditions for which neither pure component alone will gel. The mechanical properties of the mixed gel may differ significantly from the properties of gels which may be formed by either of the pure components. These effects are examples of what has been termed synergistic interactions between polysaccharides. Such synergisms are attractive commercially and have been exploited technologically 3. Synergistic interactions have been cited as examples of the development of quaternary structures by polysaccharides and have been proposed as models for complex cellular structures'* or specific recognition processes such as hostpathogen interactions 5'6. The kappa carrageenan-carob (locust bean) gum mixture is one of the most studied of the mixed gel systems. The currently accepted model for gelation of the mixture is usually illustrated ~'2'~ as shown in Figure 1. A specific molecular interaction between regions of the carrageenan helix and bare mannan regions of the galactomannan backbone has been proposed ~.2..~ although the stoichiometry and the total number of polymer molecules within the junction zones of the gel are vague and unspecified. There is no direct experimental evidence which can confirm the proposed interrnolecular interaction. X-ray fibre diffraction studies of mixed gels should yield, on the basis of Figure I, an entirely new pattern characteristic of the mixed molecular interaction. However, recent X-ray fibre diffraction studies 7 ofcarob kappa carrageenan mixed gels failed to reveal any evidence in support of the proposed molecular interaction. The mixed gels yielded fibre diffraction patterns identical to those obtained for pure kappa carrageenan. The article describes an extension of the previous X-ray fibre studies ~' on kappa carrageenan-carob gum gels to investigate kappa carrageenan-tara gum mixed gels. The sample of kappa carrageenan, extracted from Etwheuma cottonii, was purchased from the Sigma Chemical 0141 8130'86,,020124 04$t)3.00 c 1986 Butterworth & Co. [Publishers) Ltd
124
hat. J. Biol. Macromol., 1986, Vol 8, April
Company. The material was a mixed salt form containing 2.75% K ~, 0.53°J;Na ~ and 1 6°~,Ca 2+ and was used without further purification. The sample of tara gum, extracted from the seeds of Caesalpinia spinosa, was a gift from Dr C. Blood. The mannose-galactose ratio was found to be 3: 1. Gels were prepared in the following way: the dry powders were mixed in the appropriate quantities, wetted with alcohol and dispersed in water. The dispersions were heated to about 9 2 C and thoroughly mixed. The hot mixes were poured into plastic moulds, allowed to set at room temperature and stored for about 24 h. Kappa carrageenan yields rigid, brittle gels. Addition of tara gum produces softer, tougher gels which are able to withstand substantial mechanical deformation before fracture. Details of the mechanical properties of the mixed gels will be described elsewhere. The tara-kappa carrageenan mixed gels are qualitatively similar in properties to carob-kappa carrageenan mixed gels. Samples for X-ray fibre diffraction studies were prepared in the following manner. For kappa carrageenan and tara-kappa carrageenan mixtures the gels were set by pouring the hot sample on to a glass slide. The thin layer of gel was cut into thin strips and stretched under controlled relative humidity (RH). Elongations of about 5000 were obtained for kappa carrageenan and of about 100'!i, for tara-kappa carrageenan mixtures at RH of about 98%. Under the conditions used to prepare pure kappa carrageenan gels and tara-kappa carrageenan mixed gels, the pure tara gum samples did not gel. Tara gum fibres were prepared by stretching thin strips cut from a partially dried film of tara gum. Typical elongations of around 20000 were obtained at RH around 98";. The wavelength used was 0.154 nm and fibres were dusted with calcite for calibration. The interior of the diffraction camera was maintained at a controlled RH and was flushed continually with helium to reduce scattering. Fibre diffraction data were recorded photographically. The X-ray fibre patterns obtained for tara gum and kappa carrageenan at RH around 980; are shown in Figure 2 and Figure 3a, respectively. The data obtained for pure tara gum and pure kappa carrageenan are identical to these reported by other workers on pure tara 8'9 and pure carrageenan L°. Figure 4a shows the fibre patterns obtained for a range of mixed tara-carrageenan gels. Within the limits of experimental accuracy the patterns obtained for the mixed gels are qualitatively and quantitatively identical to those obtained for pure kappa carrageenan. The nature of the
lb%2 of,oro Carrageenan 'double helix'
Figure I Proposed ]'2'~ model for the molecular interaction between galactomannans and kappa carrageenan. Bare regions of the mannan backbone, unsubstituted with galactose side chains are pictured as binding to a carrageenan double helical structure
Notes to the Editor unit cell and the unit cell dimensions are unchanged and there is no evidence for a new pattern or for a tara pattern superimposed on the carrageenan pattern. The tara gum is clearly not incorporated in the carrageenan crystalline junction zones of the gels. Thus, there is no experimental evidence for co-crystallization of tara and kappa carrageenan. The lack of any tara pattern superimposed on the carrageenan pattern suggests that the tara molecules are not oriented upon stretching the gel and again argues against co-crystallization of the two polymers or tara forming an active part of the network structure. The absence of reflections attributable to tara gum is perhaps surprising in view of the high quality of the data obtained for tara gum alone (Figure 2). The tara samples
Figure 2 X-ray fibre pattern obtained for tara gum
3a
were partially dried and partial dehydration may be essential to induce crystallization of tara gum. When mixed gel fibres were dehydrated under tension and measured under dehydration an additional equatorial reflection appeared in the patterns (Figure 4b) corresponding to a spacing of 0.78 nm. This reflection does not correspond to any of the reflections of pure tara gum and disappears upon rehydration of the fibres. The unit cell dimensions of the galactomannans vary with relative humidity ~. However, the additional reflection lies outside the range 1.1-1.4nm reported 1~ for the d2o o spacing measured over extremes of relative humidity. We have been unable to explain the origin of this additional reflection. However, it is unlikely to represent a molecular interaction between tara and the carrageenan as the remaining pattern is unchanged. Further, the additional reflection may also be induced in pure carrageenan fibres on dehydration (Figure 3b). Thus the present measurements show no evidence for the proposed molecular interaction illustrated in Figure I. The present measurements cannot discount the possibility of random surface attachment of short regions of the mannan backbone of the galactomannan to carrageenan crystallites. The alternative explanation of polymer-polymer incompatibility might suggest the possibility of gross phase separation within the gels. To visualize the spatial distribution of the tara gum within the mixed gels fluorescent derivatives of tara gum were prepared as described elsewhere ~2. The fluoresceintagged tara gum was monitored using a fluorescence microscope. At resolutions of about 1 pm no gross phase separation was observed in the mixed gels. Thus, the present data suggest a carrageenan gel containing an even distribution of tara gum molecules. The presence of tara gum may compete for water and thus influence the intra- and intermolecular interactions of the carrageenan. The possibility of some degree of attachment of tara gum to the surface of carrageenan crystallites cannot be discounted.
3b
Figure 3 X-ray fibre patterns obtained for kappa carrageenan: (a) hydrated RH around 98°o; (b) dehydrated
Int. J. Biol. Macromol., 1986, Vol 8, April
125
Notes to the Editor 4a(1)
4a(2)
4a(3)
4a(4)
4a(5)
4b
Figure 4 X-ray fibre diffraction patterns for mixed tara gum kappa carrageenan gels. (a) Hydrated (RH about 98'!01.The percentage of kappa carrageenan present in the mixed gels was (1) 75°/. (2) 66%, (3) 50°/,. (4) 45% and (5) 350J~.(bl A 50j"/,,mixture is shown to illustrate the appearance of the additional reflection. These extra equatorial reflections disappeared upon rehydration
126
Int. J. Biol. Macromol., 1986, Vol 8, April
N o t e s to the Editor
References 1 2 3
4 5 6
Dea, 1. C. M. in 'Polysaccharides in Food', (Eds J. M. V. Blanshard and J. R. Mitchell), Butterworths, London, 1979, p. 299 Dea, 1. C. M. and Morrison, A. Adv. Carbohydr. Chem. Biochem. 1975, 31,241 Glicksman, M. 'Gum Technology in the Food Industry', Academic Press, New York, 1968, p. 43 Dea, l.C.M.,McKinnon, A.A.andRees, D.A.J. MoI. Biol. 1972. 68. 153 Dea, I. C. M., Morris, E. R., Rees, D. A., Welsh, E. J., Barnes, H. A. and Price, J. Carbohydr. Res. 1977, 57, 249 Powell, D. A. in 'Microbial Polysaccharides and
7 8
9 10 I1 12
Polysaccharases', (Eds R. C. W. Berkeley, G. W. Gooday and D. C. Ellwood), Academic Press, London, 1979, p. 117 Miles, M. J., Morris, V. J. and Carroll, V. Macromoleeules 1984, 17, 2443 Winter, W. T., Chien, Y. Y. and Bouckris, H. in 'Gums and Stabilisers for the Food Industry. 2. Applications of Hydrocolloids', (Eds G. O. Phillips, D. J. Wedlock and P. A. Williams), Pergamon Press, Oxford, 1984, p. 535 Chien, Y. Y. and Winter, W. T. Personal communication Elloway, H. F. PhD Thesis, University of Bristol, UK, 1977 Marchessault, R. H., Buleon, A., Deslandes, Y. and Goto, T. J. Coll. Inte(/ace Sci. 1979, 71,375 Kitamura, S., Yunokawa, H. and Kuge, T. Polym. J. 1982, 14, 85
Int. J. Biol. M a c r o m o l . , 1986, V o l 8, A p r i l
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