Journal of Applied Phycology

, Volume 7, Issue 6, pp 565–576 | Cite as

Modified procedures for extraction and analysis of carrageenan applied to the red alga Hypnea musciformis

  • Svein Halvo Knutsen
  • Erminio Murano
  • Monica D'Amato
  • Renato Toffanin
  • Roberto Rizzo
  • Sergio Paoletti
Article

Abstract

Dried powder of Hypnea musciformis was extracted with water at pH 7 after an initial short pre-treatment with cold, diluted HCl. Carrageenans were isolated by alcohol precipitation after an amylase treatment and a filtration of the extracts. The yields at 25 and 90 °C were 25 and 75% (w/w) of the dry alga, with molecular weights (Mw) corresponding to 194 000 and 245 000, respectively. The chemical structure was dominated by G4S-DA-(kappa-carrageenan or carrageenose 4′-sulphate). A simple fractionation procedure for kappa-carrageenase hydrolysates, based on stirring in different enthanol/water mixtures, is introduced. NMR analysis showed that oligosaccharides with a repeating DA-G4S structure were the main constituents in the enzymic hydrolysates of the carrageenans from Hypnea musciformis. These oligosaccharides were solubilized in an ethanol concentration from 96 to 48% (v/v). In some enzyme resistant fractions D6S-G4S and DA2S-G4S sequences and D2S,6S unites were detected by 13C-NMR.

Key words

Rhodophyta red alga Hypnea musciformis carrageenans extraction kappa-carrageenase oligosaccharides fractionation NMR 

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References

  1. Anderson NST, Dolan CS, Rees DA (1965) Evidence for a common structural pattern in the polysaccharide sulphates of the Rhodophyceae. Nature 205: 1060–1062.Google Scholar
  2. ArmisenR, Galatas F (1987) Production, properties and uses of agar. In: McHugh DJ (ed.), Production and Utilisation of Products from Commerical Seaweeds. FAO Fish. Tech. Pap. 288: 1–57.Google Scholar
  3. Bellion C, Hamer GK, Yaphe W (1981) Analysis of kappa-iota hybrid carrageenans with kappa-carrageenase, iota-carrageenase and 13C-NMR. Proc. Int. Seaweed Symp. 10: 473–478.Google Scholar
  4. Bellion C, Brigand G, Prome JC, Welti D, Bociek S (1983) Identification et caractérisation des précurseurs biologiques des carraghenanes par spectroscopie de R.M.N. du 13C. Carbohydr. Res. 19: 31–48.Google Scholar
  5. Ciancia M, Matulewicz MC, Finch P, Cerezo A (1993) Determination of the structures of cystocarpic carrageenans from Gigartina skottsbergii by methylation analysis and NMR spectroscopy. Carb. Res. 238: 241–248.Google Scholar
  6. Craigie JS, Leigh C (1978) Carrageenans and agars. In: Hellebust JA, Craigie JS (eds), Handbook of Phycological Methods. Physiological and Biochemical methods, Cambridge University Press, Cambridge, 109–131.Google Scholar
  7. Craigie JS, Wong KF (1979) Carrageenan biosynthesis. Proc. Int. Seaweed Symp. 9: 369–377.Google Scholar
  8. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350–356.Google Scholar
  9. Greer CW, Shomer I, Goldstein ME, Yaphe W (1984) Analysis of carrageenan from Hypnea musciformis using kappa- and iota-carrageenases and 13C-NMR spectroscopy. Carbohydr. Res. 129: 189–196.Google Scholar
  10. Indergaard M, Knutsen SH (1990) Seasonal differences in ash, carbon, fibre and nitrogen components of Furcelaria lumbricalis (Gigarthinales, Rhodophyceae), Norway Bot. mar. 33: 327–334.Google Scholar
  11. King GM, Lauterbach GE (1987) Characterisation of carrageenan nitrogen content and its susceptibility to enzymatic hydrolysis. Bot. mar. 30: 33–39.Google Scholar
  12. Knutsen SH (1991a) Carrageenase production in a culture of Pseudomonas carrageenovora growing on kappa-carrageenan. In: Garcia-Reina G, Pedersen M (eds), Proceedings of a COST-48 workship at Grand Canaria, Spain, 277–281.Google Scholar
  13. Knutsen SH (1991b) Chromatographic separation and high field proton NMR of kappa-carrageenase, treated carrageenans. In: Proceedings of the European Meeting on Marine Biomass and their Studies. Oebalia 17, 2 Suppl.: 387–399.Google Scholar
  14. Knutsen SH, Grasdalen H (1987) Characterisation of water-extractable polysaccharides from Norwegian Furcellaria lumbricalis (Huds.) Lam. (Gigartinales, Rhodophyceae) by IR and NMR spectroscopy. Bot. mar. 30: 497–505.Google Scholar
  15. Knutsen SH, Grasdalen H (1992a) The use of neocarrabiose oligosaccharides with different length and sulphate substitution as model compounds for 1H-NMR spectroscopy. Carb. Res. 229: 233–244.Google Scholar
  16. Knutsen SH, Grasdalen H (1992b) Analysis of carrageenans by enzymic degradation, gel-filtration and 1H-NMR spectroscopy. Carb. Polym. 19: 199–210.Google Scholar
  17. Knutsen SH, Myslabodski DE, Grasdalen H (1990) Characterisation of carrageenan fractions from Norwegian Furcellaria lumbricalis (Huds.) Lamour. by t 1H-NMR spectroscopy. Carbohydr. Res. 206: 367–372.Google Scholar
  18. Knutsen SH, Myslabodski DE, Larsen B, Usov AI (1994) A modified system of nomenclature for red algal galactans. Bot. mar. 37: 163–169.Google Scholar
  19. Lahaye M, Rochas C, Yaphe W (1986) A new procedure for determining the heterogeneity of agar polymers in Gracilaria spp. (Gracilariaceae, Rhodophyta). Can. J. Bot. 64: 579–585.Google Scholar
  20. Lahaye M, Revol JF, Rochas C, McLachlan J, Yaphe W (1988) The chemical structure of Gracilaria crassissima (P. et H. Crouan in Scramm et Mazé) P. et H. Crouan in Scramm et Mazé) and G. tikvahiae McLachlan (Gigartinales, Rhodophyta) Cell-Wall Polysaccharides. Bot. mar. 31: 491–501.Google Scholar
  21. Lahaye M, Yaphe W, Phan-Viet MT, Rochas C (1989) 13C-NMR spectroscopic investigation of methylated and charged agarose oligosaccharides and polysaccharides. Carbohydr. Res. 190: 249–265.Google Scholar
  22. Larsen B (1973) Alginic acid. In: Hellebust JA, Craigie JS (eds), Handbook of Phycological Methods. Physiological and Biochemical methods, Cambridge University Press, Cambridge: 143–149.Google Scholar
  23. Larsen B, Hoøen K, Østgaard K (1993) Kinetics and specificity of alginate lyases. Hydrobiologia 260/61: 557–561.Google Scholar
  24. Landry S (1987) Relation entre la structure moléculaire et les propriétés mécaniques des gels de carragenanes. Ph. D. Thesis. University of Grenoble, 153 pp.Google Scholar
  25. McLean MW Williamson FB (1979) Kappa-carrageenase from Pseudomonas carrageenovora. Eur. J. Biochem. 133: 553–558.Google Scholar
  26. Miller GL (1959) Use of dinitrosalisylic acid reagent for determination of reducing sugars. Anal. Chem. 31: 426–428.Google Scholar
  27. Morrice L, McLean MW Long WF, Williamson, FB (1983) Porphyran primary structure. An investigation using β-agarase I from Pseudomonas atlantica and 13C-NMR. spectroscopy. Eur. J. Biochem. 133: 673–684.Google Scholar
  28. Murano E, Toffanin R, Zanetti F, Knutsen SH, Paoletti S, Rizzo R (1992) Chemical and macromolecular characterisation of agar polymers from Gracilaria dura (C. Agardh) J. Agardh (Gracilariaceae, Rhodophyta). Carbohydr. Polym. 18: 171–178.Google Scholar
  29. Murano E, Toffanin R, Knutsen SH, Focher B, Rizzo R, Paoletti S (1993) Evaluation of steam explosion as pretreatment in agar extraction from Gracilaria dura (C. Agardh) J. Agardh (Gracilariaceae, Rhodophyta). J. appl. Phycol. 5: 417–424.Google Scholar
  30. Østgaard K, Wangen B, Knutsen SH, Aasen IM (1992) Large scale production and purification of kappa-carrageenase, from Pseudomonas carrageenovora for applications in seaweed biotechnology. Enzyme Microbiol. Technology 15: 326–333.Google Scholar
  31. Rees DA (1969) Structural, conformational and mechanism in the formation of polysaccharide gels and networks. Adv. Carbohydr. Biochem. 24: 267–332.Google Scholar
  32. Rochas C, Heyraud A (1981) Acid and enzymic hydrolysis of kappacarrageenan. Polym. Bull.: 81–86.Google Scholar
  33. Stortz CA, Cerezo AS (1992) The 13C-NMR. spectroscopy of carrageenans: calculation of chemical shifts and computer aided structural determination. Carbohydr. Polym. 18: 237–242.Google Scholar
  34. Toffanin R, Knutsen SH, Bertocchi C, Rizzo R, Murano E (1994) Detection of cellulose in the cell wall of some red algae by 13C-NMR spectroscopy. Carb. Res. 262: 167–171.Google Scholar
  35. Usov AI (1992) Sulphated polysaccharides of red seaweeds. Food Hydrocolloids 6: 9–23.Google Scholar
  36. Welti D (1977) Carrageenans. Part 12. The 300 Mhz proton magnetic resonance spectra of methyl-β-D-galactopyranoside, methyl 3,6-anhydro-α-D-galactopyranoside, agarose, kappa-carrageenan, and segments of iota-carrageenan and agarose sulphate. J. chem. Research (S): 312–313; J. chem. Research (M): 3566–3587.Google Scholar
  37. Young G, Goring DAI (1958) The stability of carrageenin in dried Irish Moss (Chondrus crispus). J. Sci. Food Agric. 9; 539–541.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Svein Halvo Knutsen
    • 1
  • Erminio Murano
    • 2
  • Monica D'Amato
    • 3
  • Renato Toffanin
    • 2
  • Roberto Rizzo
    • 4
  • Sergio Paoletti
    • 4
  1. 1.Department of Biotechnological SciencesAgricultural University of NorwayÅsNorway
  2. 2.POLI-biòs Research Centre and POLY-techArea di Ricerca di TriestTriesteItaly
  3. 3.Consorzio C.O.S.T.E.Via Traversa Li Greci 1SiracusaItaly
  4. 4.Dipartemento di Biochimica, Biofisica e Chimica della MacromolecoleUniversità di Trieste Via L. Giorgieri 1TriesteItaly

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