and filtration to remove algal particles, the soluble sodium alginate is ... by alcohol, by calcium chloride or by mineral acid, converted to the sodium form if.
22 Alginates K. I. Draget, Norwegian University of Science and Technology
22.1
Introduction
As structural components in marine brown algae (Phaeophyceae) and as capsular polysaccharides in soil bacteria, alginates are quite abundant in nature. The industrial production is roughly 30,000 metric tons annually, being probably less than 10% of the annually bio-synthesised material in the standing macroalgae crops. As macroalgae also may be cultivated – as in mainland China – and as production by fermentation is technically possible (although not economically feasible at the moment), the sources for industrial production of alginate may be regarded as unlimited even for a steadily growing industry. As already mentioned, the biological function of alginate in brown algae is generally believed to be as a structure-forming component. The intercellular alginate gel matrix gives the plants both mechanical strength and flexibility.1 This relation between structure and function is reflected in the compositional difference of alginates in different algae or even between different tissues from the same plant (see Section 22.3.2). In Laminaria hyperborea, an algae which grows in very exposed coastal areas, the stipe and holdfast have a very high content of guluronic acid, giving high mechanical rigidity (see Section 22.6.1). The leaves of the same algae which float in the streaming water have an alginate characterised by a lower G-content giving a more flexible texture. The biological function of alginate in bacteria is not fully understood. It has been shown2 that alginate production is required for cyst formation in Azotobacter vinelandii. Cysts are metabolic dormant cells, characterised by having several layers of polysaccharide material around the cell. This polysaccharide coating protects the cells from desiccation and mechanical stress. Under favourable conditions, including the presence of water, the polysaccharide coating will swell and the cysts germinate, divide and regenerate to vegetative cells.2 The structural significance of alginates in the formation of microcysts by A. vinelandii does not explain the abundant production of exopolymer by vegetative cells under conditions not favouring cyst formation, neither does it explain the role of alginate in Pseudomonades.3 It is therefore reasonable to believe that alginate (as with other microbial exo-polysaccharides) has no single function
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for the vegetative cells itself, but rather provides the cells with a multitude of protective properties under various environmental conditions. Pharmaceutical, food and technical applications (such as in print paste for the textile industry) are the quantitative main market areas for alginates. There is also a large and growing potential for alginate in biotechnological applications. The latter is mostly connected to high value applications such as encapsulation of living cells for in vitro or in vivo use.4, 5 This type of application has been a driving force for research aimed at understanding structure-function relationships in alginates at an increasingly detailed level. Basic knowledge gained from biotechnological activities has made alginate one of the best characterised and well understood gelling polysaccharides. Focus in this chapter will be put on giving an overview of the understanding of structure-function relationships of the alginate system with emphasis on existent and potential gelling methodology and how to control these. As any potential application will have to fit within a framework with boundaries given by alginate functionality, some focus has also been put on chemical and physical limitations of alginates such as solubility and stability.
22.2
Manufacture
Alginate was first described by the British chemist E. C. C. Stanford in 1881,6 and exists as the most abundant polysaccharide in the brown algae comprising up to 40% of the dry matter. It is located in the intercellular matrix as a gel containing sodium, calcium, magnesium, strontium and barium ions.7 It is because of its ability to retain water, and its gelling, viscosifying and stabilising properties, that alginate is widely used industrially. Several bacteria also produce alginate exocellularly,3, 8, 9 and Azotobacter vinelandii has been evaluated as a source for industrial production. But at present, all commercial alginates are extracted from algal sources. The extraction of alginate from algal material is schematically illustrated in Fig. 22.1. Because alginate is insoluble within the algae with a counterion composition determined by the ion exchange equilibrium with seawater, the first step in alginate production is an ion-exchange with protons by extracting the milled algal tissue with 0.1–0.2 M mineral acid. In the second step, the alginic acid is brought into solution by neutralisation with alkali such as sodium carbonate or sodium hydroxide to form the water soluble sodium alginate. After extensive separation procedures such as sifting, floatation, centrifugation and filtration to remove algal particles, the soluble sodium alginate is precipitated directly by alcohol, by calcium chloride or by mineral acid, converted to the sodium form if needed and finally dried and milled. Besides Na-alginate, other soluble alginates are produced such as the potassium and ammonium salts. The only derivative of alginates today having a commercial value, is the propylene glycol alginate (PGA). This product is processed by an esterification of alginate with propylene oxide. PGA is used in beers and salad dressings due to its higher solubility at low pH. Following the increased popularity of alginate as an immobilisation matrix, Pronova Biomedical A/S now commercially manufactures ultrapure alginates highly compatible with mammalian biological systems. These qualities are low in pyrogens, and facilitate sterilisation of the alginate solution by filtration due to low content of aggregates.
Alginates
Fig. 22.1
22.3
381
Principal scheme for the isolation of alginate from seaweeds.
Chemical and physical properties
22.3.1 Composition and sequence Alginate is a family of unbranched binary copolymers of (1!4) linked -D-mannuronic acid (M) and -L-guluronic acid (G) residues (see Fig. 22.2(a) and (b)) of widely varying composition and sequence. The first information about the sequential structure of alginates came from the work by Haug et al.7, 10–13 By partial acidic hydrolysis and fractionation, they were able to separate alginate into three fractions of widely differing composition. Two of these contained almost homopolymeric molecules of guluronic and mannuronic acid, respectively, while a third fraction consisted of nearly equal proportions of both monomers, and was shown to contain a large number of MG dimer residues. It was concluded that alginate was a true block copolymer composed of homopolymeric regions of M and G, termed M- and G-blocks, respectively, interspersed with regions of alternating structure (MG-blocks; see Fig. 22.2(c)). In a series of papers14–16 it was shown that alginates have no regular repeating unit. Furthermore, the distribution of the monomers along the polymer chain cannot be described by Bernoullian statistics. Hence, knowledge of the monomeric composition is not sufficient to determine the sequential structure of alginates. By simulating random depolymerisation and comparing the oligomer distribution with experimental data,15 the results indicated that a second order Markov model seems to be required for a general description of monomer sequence in alginates. More detailed information about the structure became available following the introduction of high resolution 1H and 13C NMR-spectroscopy17–20 in the sequential analysis of alginate. These powerful techniques have made it possible to determine the monad frequencies FM and FG, the four nearest neighbouring (diad) frequencies FGG, FMG, FGM, FMM, and the eight next nearest neighbouring (triad) frequencies. Knowledge
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Fig. 22.2
Structural characteristics of alginates: (a) alginate monomers, (b) chain conformation, (c) block distribution.
of these frequencies enable, for example, the calculation of the average G-block length larger than 1: NG > 1 = ( FG FMGM) / FGGM. This value has been shown to correlate well with gelling properties (see Section 22.6.1). It is important to realise that in an alginate chain population, neither the composition nor the sequence of each chain will be alike. This results in a composition distribution of a certain width.
22.3.2 Source dependence Commercial alginates are produced mainly from Laminaria hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllum nodosum, Laminaria japonica, Eclonia maxima, Lessonia nigrescens, Durvillea antarctica and Sargassum spp. Table 22.1 gives some sequential parameters (determined by high field NMR-spectroscopy) for samples of these alginates. The composition and sequential structure may, however, vary according to seasonal and growth conditions.7, 21 Generally, a high content of -L-guluronic acid is found in alginate prepared from stipes of old Laminaria hyperborea plants. Alginates from A. nodosum, L. japonica and Macrocystis pyrifera are characterised by a low content of G-blocks and a low gelstrength (see Section 22.6.1). It is interesting to see how nature can tailor-make alginate to give different strengths and required flexibility to different plants and tissues.22 Alginates with more extreme compositions can be isolated from bacteria23 which can contain up to 100% mannuronate. Bacterial alginates are also commonly acetylated. Alginate with a very high content of guluronic acid can be prepared from special algal tissues such as the outer cortex of old stipes of L. hyperborea (see Table 22.1), by chemical fractionation13, 24 or by enzymatic modification in vitro using mannuronan C-5 epimerases from A. vinelandii.23 This family of enzymes is able to epimerise M-units into G-units in different patterns from almost strictly alternating to very long G-blocks. The
Alginates Table 22.1
383
Composition and some sequential parameters of algal alginates
Source
FG
FM
FGG
FMM
FGM,MG
Laminaria japonica L. digitata L. hyperborea, leaf L. hyperborea, stipe L. hyperborea, outer cortex Lessonia nigrescens Ecklonia maxima Macrocystis pyrifera Durvillea antarctica Ascophyllum nodosum, fruiting body Ascophyllum nodosum, old tissue
0.35 0.41 0.55 0.68 0.75 0.38 0.45 0.39 0.29 0.10 0.36
0.65 0.59 0.45 0.32 0.25 0.62 0.55 0.61 0.71 0.90 0.64
0.18 0.25 0.38 0.56 0.66 0.19 0.22 0.16 0.15 0.04 0.16
0.48 0.43 0.28 0.20 0.16 0.43 0.32 0.38 0.57 0.84 0.44
0.17 0.16 0.17 0.12 0.09 0.19 0.32 0.23 0.14 0.06 0.20
epimerases from A. vinelandii have been cloned and expressed, and they represent at present a powerful new tool for tailoring of alginates. It is also obvious that commercial alginates with less molecular heterogeneity, with respect to chemical composition and sequence, can be obtained by a treatment with one of the C-5 epimerases.23
22.3.3 Molecular weight Alginates, like polysaccharides in general, are polydisperse with respect to molecular weight. In this aspect they resemble more synthetic polymers than other biopolymers like proteins and nucleic acids. This may result from two different causes: (a) polysaccharides are not coded for in the DNA of the organism, but are synthesised by polymerase enzymes, and (b) during extraction there is a substantial depolymerisation of the polymer. Due to this polydispersity, the ‘molecular weight’ of an alginate becomes an average over the whole distribution of molecular weights. There are several methods for averaging the molecular weight, the two most common are the number-average, M n (which weighs the polymer molecules according to the number of molecules in a population having a specific molecular weight), and the weightaverage, M w (which weighs the polymer molecules in a population according to the weight of molecules having a specific molecular weight). The fraction M w =M n is called the polydispersity index (P.I.). A P.I. of less than 2.0 suggests that some fractionation has occurred during the production process. Precipitation, solubilisation, filtration, washing or other separating procedures may have caused loss of the high or the low molecular weight tail of the distribution. A P.I. of more than 2.0 indicates a wider distribution. This suggests mixing of products of different molecular weights to obtain a sample of a certain average molecular weight (viscosity) or that a non-random degradation of the polymer has occurred during the production process or in the raw material prior to extraction. Mixing, or more precisely ‘blending’, is a common method for alginate (and polysaccharides in general) manufacturers to reach a viscosity targeted product. In extreme cases, this implies that virtually no molecules in an alginate blend have the average molecular weight obtained from viscosity experiments; only higher and lower. The molecular weight distribution can have implications for the uses of alginates, as lowmolecular weight fragments containing only short G-blocks may not take part in gel network formation and consequently not contribute to the gel strength. Also, in some high-tech applications, the leakage of mannuronate-rich fragments from alginate gels
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may cause problems,25, 26 and a narrow molecular weight distribution is advantageous (see Section 22.5).
22.3.4 Selective binding of ions The ion-binding properties of alginates represent the basis for their gelling properties. Alginates show characteristic ion-binding properties in that their affinity for multivalent cations depends on their composition.7 The characteristic affinities are a property exclusive to polyguluronate; polymannuronate is almost without selectivity. The affinity of alginates for alkaline earth metals increase in the order Mg 1) give the highest moduli.69 But in contrast to ionic gels, also polymannuronate sequences support acid gel formation. Poly-alternating sequences seem to perturb gel formation in both cases. The obvious demand for homopolymeric sequences in acid gel formation suggests cooperative processes to be involved just as in the case of
Alginates
393
ionic gels. A broad molecular weight dependence has been observed, and this dependence becomes more pronounced with increasing content of guluronic acid residues.69 The equilibrium properties of the alginic acid gels were confirmed in a study of the swelling and partial solubilisation at pH4.70 By comparing the chemical composition and molecular weight of the alginate material leaching out from the acid gels with the same data for the original alginate, an enrichment in mannuronic acid residues was found, a reduction in the average length of G-blocks and a lowering of the molecular weight.
22.7
Regulatory status
The safety of alginic acid and its ammonium, calcium, potassium, and sodium salts were last evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) at its thirty-ninth meeting in 1992. An ADI ‘not specified’ was allocated. JECFA allocated an ADI of 0–25mg/kg bw to propylene glycol alginate at its seventeenth meeting. In the US, ammonium, calcium, potassium, and sodium alginate are included in a list of stabilisers that are generally recognised as safe (GRAS). Propylene glycol alginate is approved as a food additive (used as an emulsifier, stabiliser or thickener) and in several industrial applications (coating of fresh citrus fruit, as an inert pesticide adjuvant, and as a component of paper and paperboard in contact with aqueous and fatty foods). In Europe, alginic acid and its salts and propylene glycol are all listed as EC approved additives other than colours and sweeteners. Alginates are inscribed in Annex I of the Directive 95/2 of 1995 and as such can be used in all foodstuffs (except those cited in Annex II and those described in art. II of the Directive) under the Quantum Satis principle in the EU.
22.8 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
References
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of degradation rates of different polymers by viscosity measurements’ Carbohydr Res, 1967 5 482–5. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘The influence of reducing substances on the rate of degradation of alginates’ Acta Chem Scand, 1963 17 1473–4. SMIDSRØD, O., HAUG, A. and LARSEN, B. ‘Degradation of alginate in the presence of reducing compounds’ Acta Chem Scand, 1963 17 2628–37. ˚ K-BRÆK, G., MURANO, E. and PAOLETTI, S. ‘Alginate as immobilization material. II: Determination of SKJA polyphenol contaminants by fluorescence spectroscopy, and evaluation of methods for their removal’ Biotechnol Bioeng, 1989 33 90–4. LEO, W.J., MCLOUGHLIN, A. J. and MALONE, D. M. ‘Effects of sterilization treatments on some properties of alginate solution and gels’ Biotechnol Prog, 1990 6 51–3. ˚ K-BRÆK, G. and ØSTGAARD, K. ‘Regeneration, cultivation and differentiation DRAGET, K.I., MYHRE, S., SKJA of plant protoplasts immobilized in Ca-alginate beads’ J Plant Physiol, 1988 132 552–6. PARSONS, B. J., PHILLIPS, G. O., THOMAS, B., WEDLOCK, D. J. and CLARK-STURMAN, A. J. ‘Depolymerization of xanthan by iron-catalysed free radical reactions’ Int J Biol Macromol, 1985 7 187–92. ˚ K-BRÆK, G., SMIDSRØD, O., HEINTZ, R., LANZA, R. P. and ESPEVIK, T. ‘An SOON-SHIONG, P., OTTERLEI, M., SKJA immunologic basis for the fibrotic reaction to implanted microcapsules’ Transplant Proc, 1991 23 758–9. ˚ KSOON-SHIONG, P., FELDMAN, E., NELSON, R., HEINTZ, R., YAO, Q., YAO, T., ZHENG, N., MERIDETH, G., SKJA BRÆK, G., ESPEVIK, T., SMIDSRØD, O. and SANDFORD, P. ‘Long-term reversal of diabetes by the injection of
immunoprotected islets’ Proc Natl Acad Sci, 1993 90 5843–7. ˚ K-BRÆK, G. ‘Distribution of uronate residues STOKKE, B. T., SMIDSRØD, O., ZANETTI, F., STRAND, W. and SKJA in alginate chains in relation to gelling properties 2: Enrichment of -D-mannuronic acid and depletion of -L-guluronic acid in the sol fraction’ Carbohydr Polym, 1993 21 39–46. ˚ K-BRÆK, G. ‘Application of alginate gels in biotechnology and biomedicine’ ESPEVIK, T. and SKJA Carbohydr Eur, 1996 14 19–25. NEISER, S., DRAGET, K. and SMIDSRØD, O. ‘Gel formation in heat-treated bovine serum albumin – sodium alginate systems’ Food Hydrocolloids, 1998 12 127–32. NEISER, S., DRAGET, K. I. and SMIDSRØD, O. ‘Interactions in bovine serum albumin – calcium alginate gel systems’ Food Hydrocolloids, 1999 in press. ˚ G, K., ONSØYEN, E. and SMIDSRØD, O. ‘Na- and K-alginate; effect on Ca2+-gelation’ DRAGET, K. I., STEINSVA Carbohydr Polym, 1998 35 1–6. DRAGET, K. I., ONSØYEN, E., FJÆREIDE, T., SIMENSEN, M. K., HJELLAND, F. and SMIDSRØD, O. ‘Procedure for producing uronic acid blocks from alginate’ Intl Pat Appl no. PCT/NO98/00142, 1998. DRAGET, K. I., ONSØYEN, E., FJÆREIDE, T., SIMENSEN, M. K. and SMIDSRØD, O. ‘Use of G-block polysaccharides’ Intl Pat Appl no. PCT/NO97/00176, 1997. ONSØYEN, E. ‘Commercial applications of alginates’ Carbohydr Eur, 1996 14 26–31. ˚ K-BRÆK, G., GRASDALEN, H. and SMIDSRØD, O. ‘Inhomogeneous polysaccharide ionic gels’ Carbohydr SKJA Polym, 1989 10 31–54. MIKKELSEN, A. and ELGSÆTER, A. ‘Density distribution of calcium-induced alginate gels – a numerical study’ Biopolymers, 1995 36 17–41. ˚ K-BRÆK, G., GRASDALEN, H., DRAGET, K. I. and SMIDSRØD, O. ‘Inhomogeneous calcium alginate beads’ SKJA in Biomedical and biotechnological advances in industrial polysaccharides New York, Gordon and Breach, 1989, pp. 385–98. ˚ K-BRÆK, G. and SMIDSRØD, O. ‘Alginate as immobilization material: I. Correlation MARTINSEN, A., SKJA between chemical and physical properties of alginate gel beads’ Biotechnol Bioeng, 1989 33 79–89. DRAGET, K. I., ØSTGAARD, K. and SMIDSRØD, O. ‘Homogeneous alginate gels: a technical approach’ Carbohydr Polym, 1991 14 159–78. DRAGET, K. I., SIMENSEN, M. K., ONSØYEN, E. and SMIDSRØD, O. ‘Gel strength of Ca-limited alginate gels made in situ’ Hydrobiologia, 1993 260/261 563–5. ˚ K-BRÆK, G. and SMIDSRØD, O. ‘Alginic acid gels; the effect of alginate chemical DRAGET, K. I., SKJA composition and molecular weight’ Carbohydr Polym, 1994 25 31–8. ˚ K-BRÆK, G., CHRISTENSEN, B. E., GA ˚ SERØD, O. and SMIDSRØD, O. ‘Swelling and partial DRAGET, K. I., SKJA solubilization of alginic acid gel beads in acidic buffer’ Carbohydr Polym, 1996 29 209–15.
