Abstract
Nitrous acid deaminative depolymerisation was used to prepare three chitosan oligomer (CO) mixtures from high-molecular weight chitosan. These mixtures of chitosan oligosaccharides were analysed by electrospray ionisation mass spectroscopy, potentiometric titration and gel permeation chromatography. A method based on potentiometric titration of the amino groups of the oligomers gave an average degree of polymerisation (DP) for the three preparations of 5 (CODP5), 9 (CODP9) and 14 (CODP14). Chitosan acetate and the chitosan oligomer mixtures were assayed against Leptographium procerum, Sphaeropsis sapinea and Trichoderma harzianum on nutrient media. Leptographium procerum and S. sapinea growth was prevented by chitosan acetate and chitosan oligomers at concentrations of 0.3–0.4% (w/v), whereas T. harzianum was able to overcome the fungistatic action of these compounds. The oligomer preparation CODP14 exhibited superior specific activity to both CODP5 and chitosan acetate, suggesting an optimum molecular weight for bioactivity. All oligomer preparations were more effective at pH 4 than at pH 6. This result, in combination with the inactivity of N-acetylated CODP14, indicated that amino group protonation was an important factor for fungistatic activity. The CODP14 preparation was reduced with sodium borohydride and fractionated by alkali precipitation and ion exchange chromatography. Bioassays of these fractions pointed towards DP and degree of deacetylation (DD) as key factors in chito-oligosaccharide bioactivity. Conversely, the terminal aldehyde groups generated by depolymerisation did not contribute to the activity observed.
References
Allan, C.R., Hadwiger, L.A. (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp. Mycol.3:285–287.10.1016/S0147-5975(79)80054-7Search in Google Scholar
Allan, G.G., Peyron, M. (1995) Molecular weight manipulation of chitosan II: prediction and control of extent of depolymerisation by nitrous acid. Carbohydr. Res.277:273–282.10.1016/0008-6215(95)00208-BSearch in Google Scholar
Bell, A.A., Hubbard, J.C., Liu, L. (1998) Effects of chitin and chitosan on the incidence and severity of Fusarium yellows of celery. Plant Dis.82:322–328.10.1094/PDIS.1998.82.3.322Search in Google Scholar
Benhamou, J., Kloepper, J.W., Tuzun, S. (1998) Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response. Planta204:153–168.10.1007/s004250050242Search in Google Scholar
Cha, S.Y., Lee, J.K., Lim, B.S., Lee, T.S., Park, W.H. (2001) Conjugated vinyl derivatives of chito-oligosaccharide: synthesis and characterisation. J. Poly. Sci. A Poly. Chem.39:880–887.10.1002/1099-0518(20010315)39:6<880::AID-POLA1062>3.0.CO;2-KSearch in Google Scholar
Chittenden, C., Wakeling, R., Kreber, B. (2003) Growth of two selected sapstain fungi and one mould on chitosan amended nutrient medium. Document No. IRG/WP/03-10466. International Research Group on Wood Protection, Stockholm, Sweden.Search in Google Scholar
Cuero, R.G. (1999) Antimicrobial action of exogenous chitosan. In: Chitin and Chitinases. Eds. Jolles, P., Muzzarelli, R.A.A. Birkhäuser Verlag, Basel. pp. 315–333.10.1007/978-3-0348-8757-1_23Search in Google Scholar
Domard, A., Cartier, N. (1989) Preparation, separation and characterisation of the d-glucosamine oligomer series. In: Chitin and Chitosan. Eds. Skjåk-Braek, G., Anthonsen, T., Sandford, P. Elsevier, London. pp. 383–387.Search in Google Scholar
Fang, S.W., Li, C.F., Shih, D.Y.C. (1994) Antifungal activity of chitosan and its preservative effect on low-sugar candied kumquat. J. Food Protect.56:136–140.10.4315/0362-028X-57.2.136Search in Google Scholar
Felse, P.A., Panda, T. (1999) Studies on applications of chitin and its derivatives. Bioprocess Eng.20:505–512.10.1007/s004490050622Search in Google Scholar
Hirai, A., Odani, H., Nakajima, A. (1991) Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy. Polym. Bull.26:87–94.10.1007/BF00299352Search in Google Scholar
Hirano, S., Nagao, N. (1989) Effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens. Agric. Biol. Chem.53:3065–3066.Search in Google Scholar
Hirano, S., Kondo, Y., Fujii, K. (1985) Preparation of acetylated derivatives of modified chito-oligosaccharides by the depolymerisation of partially N-acetylated chitosan with nitrous acid. Carbohydr. Res.144:338–341.10.1016/S0008-6215(00)90682-7Search in Google Scholar
Horowitz, S.T., Roseman, S., Blumenthal, H.J. (1957) The preparation of glucosamine oligosaccharides I: Separation. J. Am. Chem. Soc.79:5046–5049.10.1021/ja01575a059Search in Google Scholar
Jeon, Y.J., Park, P.-J., Kim, S.K. (2001) Antimicrobial effect of chito-oligosaccharides produced by bioreactor. Carbohydr. Polym.44:71–76.10.1016/S0144-8617(00)00200-9Search in Google Scholar
Kendra, D.F., Hadwiger, L.A. (1984) Characterisation of the smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Exp. Mycol.8:276–281.10.1016/0147-5975(84)90013-6Search in Google Scholar
Kumagai, H., Furukawa, I., Sakuno, T., Kishimoto, J. (1990) Anti-mold activity of chitosan-treated wood. Evaluation of chitosan and chitosan/copper sulfate jointly treated wood. Res. Bull. Tottori Uni. For.19:59–65.Search in Google Scholar
Kumar, M.N.V.R. (2000) A review of chitin and chitosan applications. Reactive Funct. Polym.46:1–27.Search in Google Scholar
Leuba, J.L., Stossel, P. (1986) Chitosan and other polyamines: antifungal activity and interaction with biological membranes. In: Chitin in Nature and Technology. Eds. Muzzarelli, R., Jeuniaux, C., Gooday, G.W. Plenum Press, New York. pp. 215–222.10.1007/978-1-4613-2167-5_29Search in Google Scholar
Li, H., Yu, T. (2000) Effect of chitosan on incidence of brown rot, quality and physiological attributes of postharvest peach fruit. J. Sci. Food Agric.81:269–274.Search in Google Scholar
Maoz, M., Morrell, J.J. (2004) Ability of chitosan to limit wood decay under laboratory conditions. Document No. IRG/WP 04-30339. International Research Group on Wood Protection, Stockholm, Sweden.Search in Google Scholar
Muraki, E., Yaku, F., Kojima, H. (1993) Preparation and crystallisation of d-glucosamine oligosaccharides with DP 6-8. Carbohydr. Res. 239:227–237.Search in Google Scholar
Muzzarelli, C., Muzzarelli, R.A.A. (2003) Chitin related food science today (and two centuries ago). AgroFood Ind. Hi-Tech39:39–42.Search in Google Scholar
Nilsson, K., Bjurman, J. (1998) Chitin as an indicator of the biomass of two wood decay fungi in relation to temperature, incubation time and media composition. Can. J. Microbiol.44:575–581.10.1139/w98-039Search in Google Scholar
No, H.K., Park, N.Y., Lee S.H., Meyers, S.A. (2002) Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int. J. Food Microbiol.74:65–72.10.1016/S0168-1605(01)00717-6Search in Google Scholar
Pirttilä, A.M., Laukkanen, H., Hohtola, A. (2002) Chitinase production in pine callus (Pinus sylvestris L.): a defence reaction against endophytes?Planta214:848–852.Search in Google Scholar
Rhoades, J., Roller, S. (2000) Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods. Appl. Environ. Microbiol.66:80–86.10.1128/AEM.66.1.80-86.2000Search in Google Scholar
Roller, S., Covill, N. (1999) The antifungal properties of chitosan in laboratory media and apple juice. Int. J. Food Microbiol.47:67–77.10.1016/S0168-1605(99)00006-9Search in Google Scholar
Sagoo, S.K., Board, R., Roller, S. (2002) Chitosan potentiates the antimicrobial action of sodium benzoate on spoilage yeasts. 34:168–172.10.1046/j.1472-765x.2002.01067.xSearch in Google Scholar PubMed
Sannan, T., Kurita, K., Iwakura, Y. (1976) Studies on chitin 2: effect of deacetylation on solubility. Makromol. Chem.177:3589–3600.10.1002/macp.1976.021771210Search in Google Scholar
Sashiwa, H., Saimoto, H., Shigemasa Y., Seiichi, T. (1993) N-Acetyl group distribution in partially deacetylated chitins prepared under homogeneous conditions. Carbohydr. Res.242:167–172.10.1016/0008-6215(93)80031-9Search in Google Scholar
Shahidi, F., Arachchi, J.K.V., Jeon, Y.-J. (1999) Food applications of chitin and chitosans. Trends Food Sci. Technol.10:37–51.10.1016/S0924-2244(99)00017-5Search in Google Scholar
Stossel, P., Leuba, J.L. (1984) Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. Phytopathol. Z.111:82–90.10.1111/j.1439-0434.1984.tb04244.xSearch in Google Scholar
Sudarshan, N.R., Hoover, D.G., Knorr, D. (1992) Antibacterial action of chitosan. Food Biotechnol.6:257–272.10.1080/08905439209549838Search in Google Scholar
Tanigawa, T., Tanaka, Y., Sashiwa, H., Saimato, H., Shigemasa, Y. (1991) Various biological effects of chitin derivatives. In: Advances in Chitin and Chitosan. Eds. Brine, C.J., Sandford, P.A., Zikakis, J.P. Elsevier, London. pp. 206–215.Search in Google Scholar
Thornalley, P.J. (2003) The enzymatic defence against glycation in health, disease and therapeutics. Biochem. Soc. Trans.31:1341–1342.10.1042/bst0311341Search in Google Scholar
Tømmeraas, K., Varum, K.M., Christensen B.E., Smidsrød, O. (2001) Preparation and characterisation of oligosaccharides produced by nitrous acid depolymerisation of chitosans. Carbohydr. Res.333:137–144.10.1016/S0008-6215(01)00130-6Search in Google Scholar
Uchida, Y., Izume, M., Ohtakara, A. (1988) Preparation of chitosan oligomers with purified chitosanase and its application. In: Chitin and Chitosan. Eds. Skjåk-Braek, G., Anthonsen, T., Sandford, P. Elsevier, London. pp. 373–382.Search in Google Scholar
Wang, G.-H. (1992) Inhibition and inactivation of five species of foodborne pathogens by chitosan. J. Food Protect.55:916–919.10.4315/0362-028X-55.11.916Search in Google Scholar
Yalpani, M., Pantaleone, D. (1994) An examination of the unusual susceptibilities of aminoglycans to enzymatic hydrolysis. Carbohydr. Res.256:159–175.10.1016/0008-6215(94)84235-3Search in Google Scholar
Zhang, D., Quantick, P.C. (1998) Antifungal effects of chitosan coating on fresh strawberries and raspberries during storage. J. Hort. Sci. Biotechnol.73:763–767.10.1080/14620316.1998.11511045Search in Google Scholar
Zhang, M., Tan, T., Yuan, H., Rui, C. (2003) Insecticidal and fungicidal activities of chitosan and oligo-chitosan. J. Bioactive Compatible Polym.18:391–400.10.1177/0883911503039019Search in Google Scholar
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