Biochemical Engineering Journal 25 (2005) 165–172 Preparation of chitooligosaccharides with degree of polymerization higher than 6 by acid or enzymatic degradation of chitosan Juan Carlos Cabrera a,b , Pierre Van Cutsem b,∗ a Laboratorio de Oligosacarinas, Departamento de Fisiolog´ ıa y Bioqu´ ımica Vegetal, INCA, Cuba b Unit´ e de Recherche en Biologie Cellulaire V´ eg´ etale, Facult´ es Universitaires Notre-Dame de la Paix, Belgium Received 2 December 2004; received in revised form 1 April 2005; accepted 10 April 2005 Abstract Chitosan was depolymerized either by HCl hydrolysis or enzymatic degradation with a commercial preparation Pectinex Ultra Spl. The chitooligosaccharides released by both methods were selectively precipitated in methanol solutions and characterized using MALDI-TOF mass spectrometry. Differences between the two methods were detected and concerned the degrees of polymerization of the fragments produced and their acetylation. The enzymatic method yielded shorter fragments with a higher proportion of fully deacetylated chitooligomers. Conversely, acid hydrolysis of the starting chitosan resulted in fragments with degrees of polymerization up to sixteen and more monoacetylated residues than with the enzymatic procedure. © 2005 Elsevier B.V. All rights reserved. Keywords: Chitooligosaccharides; Enzyme technology; MALDI-TOF-MS; Enzymatic hydrolysis; Acid hydrolysis; Viscosity 1. Introduction Chitosan is a linear heteropolysaccharide composed of -1,4-linked-d-glucosamine (GlcN) and N-acetyl-d- glucosamine (GlcNAc) in varying proportions. This polysac- charide is a derivative of chitin, one of the most abundant natural amino polysaccharide extracted from the exoskele- ton of crustaceans and insect, from fungal cell walls, etc. These substances have a wide variety of applications in the biomedical, pharmacological, agricultural and biotechnolog- ical industries [1,2]. Therefore, recent studies on chitosan have attracted interest in converting it to more soluble chi- tooligosaccharides, which possess a number of interesting biological activities, such as antibacterial, antifungal [3] and antitumor [4] properties as well as immunoenhancing effects [5] on animal health. Chitosan oligosaccharides also have been shown to induce various plant defense-related cellu- lar responses [6–8] and possess by themselves antimicrobial properties [9] against a wide spectrum of phytopathogens. ∗ Corresponding author. E-mail address: pierre.vancutsem@fundp.ac.be (P. Van Cutsem). The biological activity of chitooligosaccharides is known to depend on their structure [8]. Although some reports mention a size-dependent biological activity of chitosan oligomers, larger oligomers being more potent, most studies do not consider soluble chitooligosaccharides with degrees of polymerization (DPs) higher than 6 and some methods used to estimate these DPs are even ambiguous. Different protocols have been developed to prepare chitooligosaccharides, among which acid or enzymatic depolymerization of chitosan are most frequently used. Short oligosaccharides with DPs up to 6 were mainly studied, partly because of the heterogeneous composition of chitosan hydrolysates and partly because of analytical limitations to isolate and identify chitooligosaccharides with DPs higher than 6. Chitosanolysis achieved by acids can be carried out mainly using HCl [10,11] or HNO 2 [12]. These are very sim- ple methods with good yield but they do not lend themselves to easy control and the removal of strong acid and byproducts of concomitant browning reactions is difficult. A further disadvantage of HNO 2 use is the structural modification of the end products it can provoke. The mechanism of this 1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.04.025