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 Fisiologı́a y Bioquı́mica Vegetal, INCA, Cuba
b Unité de Recherche en Biologie Cellulaire Végétale, Facultés 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 The biological activity of chitooligosaccharides is known

to depend on their structure [8]. Although some reports
Chitosan is a linear heteropolysaccharide composed mention a size-dependent biological activity of chitosan
of ␤-1,4-linked-d-glucosamine (GlcN) and N-acetyl-d- oligomers, larger oligomers being more potent, most studies
glucosamine (GlcNAc) in varying proportions. This polysac- do not consider soluble chitooligosaccharides with degrees of
charide is a derivative of chitin, one of the most abundant polymerization (DPs) higher than 6 and some methods used
natural amino polysaccharide extracted from the exoskele- to estimate these DPs are even ambiguous.
ton of crustaceans and insect, from fungal cell walls, etc. Different protocols have been developed to prepare
These substances have a wide variety of applications in the chitooligosaccharides, among which acid or enzymatic
biomedical, pharmacological, agricultural and biotechnolog- depolymerization of chitosan are most frequently used.
ical industries [1,2]. Therefore, recent studies on chitosan Short oligosaccharides with DPs up to 6 were mainly
have attracted interest in converting it to more soluble chi- studied, partly because of the heterogeneous composition
tooligosaccharides, which possess a number of interesting of chitosan hydrolysates and partly because of analytical
biological activities, such as antibacterial, antifungal [3] and limitations to isolate and identify chitooligosaccharides with
antitumor [4] properties as well as immunoenhancing effects DPs higher than 6.
[5] on animal health. Chitosan oligosaccharides also have Chitosanolysis achieved by acids can be carried out
been shown to induce various plant defense-related cellu- mainly using HCl [10,11] or HNO2 [12]. These are very sim-
lar responses [6–8] and possess by themselves antimicrobial ple methods with good yield but they do not lend themselves
properties [9] against a wide spectrum of phytopathogens. to easy control and the removal of strong acid and byproducts
of concomitant browning reactions is difficult. A further
∗ Corresponding author. disadvantage of HNO2 use is the structural modification
E-mail address: [email protected] (P. Van Cutsem). 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
166 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172

reaction involves a deamination of a 2-amino-2-deoxy-d- Viscosity measurements—The viscosity of an aliquot

glucopyranose forming 2,5-anhydro-d-mannose at the new (0.4 mL) of the reaction mixture was measured using a mi-
reducing end. cro falling ball viscometer (Thermo Haake Inc., USA). Time
Enzymatic methods are specific and easier to control but courses of relative (ηrel = t/t0 ) and specific ηsp = (t − t0 /t0 ) vis-
their commercial use is limited due to cost and availability of cosities were determined (t: flow time of the reaction mixture;
chitosanases. To overcome these limitations, chitosan hydrol- t0 : flow time of buffer).
ysis by food grade enzymes has been evaluated. Non-specific Reducing sugars and glucosamine analysis—At appropri-
activity of various enzymes on chitosan has been reported ate time intervals, aliquots of the reaction mixture were boiled
and includes proteases [13], hemicellulases [14–16], lipases for 10 min to terminate the reaction. Unhydrolyzed chitosan
[17] and pectinases [18,19]. These studies focused mainly on was precipitated by adjusting the pH to 9.0 with 0.1 M NaOH,
factors affecting the enzymatic depolymerization, its kinetic and the amounts of reducing sugars and glucosamine in the
and the characterization of low molecular weight chitosans supernatant were determined by Schales’ modified method
obtained. [22] and Ride and Drysdale [23] method, respectively, using
In this study, we prepared and characterized defined chi- d-glucosamine as standard.
tooligosaccharide mixtures enriched in oligomers with DPs
>6. Chitosan was degraded either by acid hydrolysis or enzy- 2.3. Acid hydrolysis of chitosan
matically using a commercial pectinase and a selective pre-
cipitation of the degradation products was evaluated using Chitosan (2 g) was dissolved in dilute acetic acid and dried
MALDI-TOF mass spectrometry. To our knowledge, this is under vacuum until the content became a gelatinous paste.
the first detailed report on the production of chitooligosaccha- 100 mL of concentrated HCl (37%) was added and the sus-
rides using a non-purified commercial enzyme preparation. pension heated for 30 min at 72 ◦ C under stirring. The reac-
tion was stopped by immersion in an ice bath. Most of the
solvent and HCl was evaporated under vacuum; the residue
2. Materials was then resuspended in water and the solution evaporated,
those last two operations being repeated twice. The residue
Chitin from Cuban lobster was supplied by Mario Muñoz was finally dissolved in water and the solution brought to pH
Pharmaceutical Laboratories (Cuba) and used to prepare 6.5 by addition of concentrated 10 M NaOH.
chitosan under heterogeneous conditions following the
methodology described by Cabrera et al. [20]. Commercially 2.4. Selective fractionation of chitosan oligosaccharides
available Pectinex Ultra Spl (Novozymes A/S, Bagsvaerd, in methanol/water
Denmark) was used without further purification.
The neutralized chitosan hydrolysates were precipitated
2.1. Charcaterization of the chitosan
with final methanol concentrations of 70, 80 and 90% (v/v).
The potentiometric determination of the degree of acety- Each precipitate was centrifuged, washed with its corre-
lation was carried out following the method given by Muz- sponding methanol solution and vacuum dried. The super-
zarelli [21]. Chitosan was dissolved in an excess of acid and natants were concentrated under reduced pressure.
titrated with 0.1 M NaOH. The volume of base used between
the two inflexion points observed on the titration curve cor- 2.5. TLC of chitooligosaccharides
responded to the acid consumed for the salification of amine
groups and allowed the determination of the degree of acety- The chitooligomers were separated by TLC on silica
lation of the chitosan. gel plates (MERCK 60. GF-254) using n-propanol: wa-
The viscosity average molecular weight (Mv ) determi- ter: concentrated ammonia 7:2:1 (v:v:v) as solvent. Spots
nation of chitosan was performed using a Ubbelohde cap- were visualised by charring with 10% H2 SO4 in ethanol
illary viscometer (Ø = 0.5 mm) at 25 ◦ C. The solvent was and each spot was identified by comparison with authentic
0.3 M acetic acid/0.2 M sodium acetate and the average vis- samples.
cometric molecular weight was calculated using the classical
Mark–Houwink equation; [␩] = Km Mva , where [η] is the in- 2.6. Mass spectrometry
trinsic viscosity Km = 3.5 × 104 , a = 0.76.
An amount of 0.5 ␮L of the sample solution was mixed
2.2. Hydrolysis of chitosan by Pectinex Ultra Spl on the target with 2 ␮L of a solution of 2,5-dihydroxybenzoic
acid as a matrix (15 mg/mL) in 30% aqueous ethanol. Mass
Chitosan at a 10.0 g/L concentration was dissolved by spectra were recorded on a Bruker Ultraflex (Bruker Dal-
overnight shaking at room temperature in 0.175 M acetate tonik, Bremen, Germany) in the positive ion mode. A ni-
buffer pH 5.5. This chitosan solution (90 mL) was mixed with trogen laser (337 nm, 3 ns pulse width, 3 Hz) was used. All
10 mL of Pectinex Ultra Spl. The reaction mixture was incu- spectra were measured in the reflector mode using external
bated at 37 ◦ C for 24 h. calibration.
 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172 167

3. Results and discussion

3.1. Chitosan oligomers obtained by acid hydrolysis

The molecular weight (Mw ) of the chitosan used was found

to be 82.9 kDa by viscosimetry as deduced from an intrinsic
viscosity [η] of 416 mL/g and its DA was 12% as determined
by titration. These parameters are in close agreement with
previously published data for other chitosan obtained by sim-
ilar ways [20]. For depolymerization, a low DA chitosan was
submitted to acid hydrolysis following essentially the proce-
dure described by Domard and Cartier [10] but the reaction
time was limited to 30 min in order to increase the proportion
of high molecular weight oligomers.
To reduce the proportion of low DP oligomers in the hy-
drolysis mixture, a selective precipitation in methanol so-
lutions was used. By MALDI-TOF analysis of the soluble
phase, it appeared that chitooligomers with DP up to 11 were
soluble in 70 and 80% methanol and up to DP 8 only in
90% methanol (Fig. 1). In the 90% methanol precipitate,
chitooligosaccharide yield amounted to 17% of the initial
chitosan under the current conditions. The higher DP chi-
tooligomers were precipitated as a white powder, leaving in
the methanol solution the brown byproducts generated during
the acid hydrolysis.
Since MALDI-TOF analysis is limited to molecular
weights higher than 500 Da due to interference of the matrix
signals, methanol soluble chitooligosaccharides were also
analysed by silica TLC. Fig. 2 shows that methanol solu-
ble chitooligosaccharides comprise oligomers with DPs up
to 6. Higher DPs could not be separated by this method.
Tenuous little spots were observed between spots cor-
responding to fully deacetylated chitosan oligosaccharides.
We speculate that these tiny spots correspond to par-
tially acetylated chitooligosaccharides. This is supported by
the MALDI-TOF-MS observation of monoacetylated chi-
tooligosaccharides in the samples.
The MALDI-TOF-MS of the chitosan fractions precipi-
tated by 90% methanol from the acid hydrolysate is shown in
Fig. 3. Oligomers of DPs up to 16 were identified, which is
clearly higher than what was found in the supernatant. Letzel
et al. [24] showed that separation of chitosan hydrolysates
by gel permeation chromatography allows the MALDI-TOF
detection of chitooligosaccharides with DPs up to 50.
This fraction contained chitooligosaccharides (GlcN-
oligomers) and several of their partial N-acetylated form
(Table 1). During acid hydrolysis, not only the O-glycosidic
linkage between residues but also the N-acetyl linkage can
be hydrolyzed, even if the rate of hydrolysis in concentrated
acid is about ten times higher than the rate of deacetyla-
tion [25]. These last authors also showed a high specificity
in the acid reaction of the different glycosidic linkages of Fig. 1. MALDI-TOF-MS of methanol soluble chitooligosaccharides ob-
partially N-acetylated chitosans: the rate of acid hydrolysis tained by acid hydrolysis of the chitosan. Identified peaks are labeled as
of GlcN–GlcN and GlcN–GlcNAc is lower than the rate of dpAc , where dp indicates degree of polymerization and Ac, the number of
cleavage of GlcNAc–GlcNAc and GlcNAc–GlcN, explain- acetyl group.
ing why new reducing ends are dominated by acetylated
168 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172

Table 1
Assigned ion composition of MALDI-TOF-MS spectra of chitooligosac-
charides prepared by 30 min of acid hydrolysis and isolated by selective
precipitation in 90% methanol
m/z Types Ion composition
524.2 [M + Na]+ (GlcN)3
540.1 [M + K]+
566.2 [M + Na]+ (GlcN)2 –GlcNAc
582.1 [M + K]+
684.9 [M + Na]+ (GlcN)4
701.2 [M + K]+
743.2 [M + K]+ (GlcN)3 –GlcNAc
846.3 [M + Na]+ (GlcN)5
862.3 [M + K]+
904.3 [M + K]+ (GlcN)4 –GlcNAc
1007.4 [M + Na]+ (GlcN)6
1023.4 [M + K]+
1065.4 [M + K]+ (GlcN)5 –GlcNAc
1168.4 [M + Na]+ (GlcN)7
1184.4 [M + K]+
1226.4 [M + K] + (GlcN)6 –GlcNAc
1329.5 [M + Na]+ (GlcN)8
1345.5 [M + K]+
1387.5 [M + K]+ (GlcN)7 –GlcNAc
1490.5 [M + Na]+ (GlcN)9
1506.5 [M + K]+
1548.5 [M + K]+ (GlcN)8 –GlcNAc
1651.6 [M + Na]+ (GlcN)10
1667.6 [M + K]+
1709.6 [M + K]+ (GlcN)9 –GlcNAc
1812.7 [M + Na]+ (GlcN)11
1828.7 [M + K]+
1870.7 [M + K]+ (GlcN)10 –GlcNAc
Fig. 2. Thin layer chromatography analysis of methanol soluble products
1973.7 [M + Na]+ (GlcN)12
resulting from chitosan hydrolysis by HCl. TLC performed on silica gel plate
1989.7 [M + K]+
in a solvent system composed of n-propanol–water–ammonia water (7:2:1,
2031.8 [M + K]+ (GlcN)11 –GlcNAc
v/v/v). The plate was developed by spraying ethanol containing 10% sulfuric
2150.8 [M + K]+ (GlcN)13
acid. To the left of the TLC, the degree of polymerization of corresponding
2192.9 [M + K]+ (GlcN)12 –GlcNAc
chitooligosaccharides is shown.
2311.9 [M + K]+ (GlcN)14
2473.0 [M + K]+
2634.1 [M + K]+ (GlcN)16

units. The analysis of our MS data show that the acid digests
contained a wide distribution of both fully deacetylated and
monoacetylated chitooligosaccharides. This pattern could be
related to a random distribution of the acetyl substituents in
the chitosan used for hydrolysis.
From these results, acid hydrolysis of chitosan combined
with a selective methanol precipitation appears to be a quick
and simple method to obtain good yields of chitooligosac-
charides with DPs up to 16 and little low molecular weight
oligomers.

3.2. Enzymatic degradation of chitosan

Chitosan was incubated with Pectinex Ultra Spl at 37 ◦ C

and pH 5.5 under constant stirring. The optimal pH and
Fig. 3. MALDI-TOF-MS of 90% methanol insoluble fraction of chi-
tooligomers obtained by a 30 min acid hydrolysis of chitosan. Identified temperature were found to be between 5.0 and 6.0 and 40
peaks are labeled as dpAc , where dp indicates degree of polymerization and and 50 ◦ C, respectively. However, the reaction was carried
Ac, the number of acetyl group. out at 37 ◦ C since the enzyme lost activity at its optimum
 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172 169

Fig. 4. Time course of enzymatic chitosan hydrolysis. Viscosity decrease

(
) of the enzyme–chitosan mixture and reducing sugars content () of the
supernatant after alkaline precipitation as a function of hydrolysis time. Fig. 5. Glucosamine content () and glucosamine/reducing sugars ratio
(䊉) in the supernatant after alkaline precipitation of the reaction mixture as
a function of enzymatic hydrolysis time.
temperature range (data not shown). The optimum pH range
for chitosanase activity of Pectinex Ultra Spl was in the range As chitosans are relatively rigid linear polysaccharides,
of reported values for its chitinolytic activity [26] and higher any change of their viscosity reflects modification of their
than those of chitosanase activity for the pectinase isozyme degree of polymerization. The enzymatic degradation was
purified from Aspergillus niger [27]. therefore followed by measuring the viscosity of the reaction

Fig. 6. MALDI-TOF-MS of the chitooligosaccharides oligomers mixtures obtained during the chitosan hydrolysis with Pectinex Ultra Spl. The proportion of
low molecular weigth oligomers was reduced by precipitation in 90% MeOH. Identified peaks are labeled as dpAc , where dp indicate degree of polymerization
and Ac, the number of acetyl group. The hydrolysis time is labeled in each spectrum.
170 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172

mixture and determining reducing sugar and glucosamine Enzymatically produced oligosaccharides with DPs >11
contents in the hydrolyzed chitosan fraction as a function of have been obtained from chitosans: Zhang and coworkers
time (Fig. 4). The enzyme treatment resulted in a fast and [15] using MALDI-TOF-MS detected chitooligosaccharides
substantial loss of viscosity of the solution during the initial with DPs between 3 and 17 released by a mixture of cellulase,
5 h. A 50% decrease of viscosity within the first 60 min was alpha amylase, and proteinase. They found that the peak in-
associated with a low reducing sugar and glucosamine con- tensities of non-acetylated fragments and of the mono-, di- or
tent in the degraded fraction, suggesting an endo mode of tri-acetylated forms of the same fragments were all similar.
action of the Pectinex Ultra Spl preparation on chitosan. It is very clear from Fig. 6 that the strongest m/z signals
The amount of degraded chitosan measured as glu- originate from oligomers of DPs 6–8 and that the m/z intensity
cosamine in the hydrolyzed fraction gradually increased profiles of these enzymatic digests do not change significantly
during the enzymatic hydrolysis (Fig. 5). However, the over time. This confirms that the Pectinex mixture constantly
glucosamine to reducing sugar ratio did not increase very releases short fragments of homogeneous size, which is to
much in solution and tended to stabilize at value of about 3 be compared to the MS spectra obtained from the acid hy-
after 1 day of enzymatic hydrolysis. This behavior suggests drolyzed chitosan precipitate (Fig. 3) that revealed the pres-
a low and relatively similar degree of polymerization ence of much longer chitooligomers. Another characteristic
of chitooligosaccharides released during the enzymatic of the Pectinex enzymes is the presence in the digestion prod-
degradation. ucts of fragments with one and/or three acetyl residues. The
In order to confirm this, the composition of the hydrolyzed MALDI-TOF analysis could not reveal the position of the
fractions produced by the enzymes was analyzed by MALDI- acetyl substituents, but in all cases the most abundant peak
TOF mass spectrometry. Since we are interested in longer did still correspond to fully desacetylated oligomers.
oligomers, most of the shorter DPs were eliminated with the From the comparison of the MS spectra, it seems that there
supernatant of a methanol selective precipitation. MALDI- were less acetylated oligomers in the enzymatically prepared
MS analysis obtained in positive-ion mode of 90% methanol samples than in the acid hydrolyzed ones. Since no chitin
insoluble chitooligosaccharides in the Pectinex Ultra Spl di- acetylesterase activity could be detected in Pectinex Ultra Spl
gests after 4, 12, 24 and 72 h are shown on Fig. 6. More infor- (unpublished results), this suggests that the enzymatic prepa-
mation about the assigned structure of each signal at different ration preferentially attacks close to acetylated residues on
times of hydrolysis is given in Table 2. Chitosan fragments chitosan chains. This is in agreement with previous obser-
were detected mainly as sodium and/or potassium adduct ions vations showing an increased degrading activity of Pectinex
and, under the conditions used, only chitooligosaccharides up enzymes with an increased degree of acetylation of the chi-
to 12 mers were observed. tosan sample [28].

Table 2
Assigned ion composition of MALDI-TOF-MS spectra of chitooligosaccharides with degree of polymerization higher than 6 prepared by enzymatic hydrolysis
for the indicated time and fractionated by selective precipitation in 90% methanol
m/z Ion composition Types Hydrolysis time (h)
2 4 8 12 16 24 48 72
1007.4 (GlcN)6 [M + Na]+ X X X X X X X X
1023.4 [M + K]+ X X X X X X X
1049.9 (GlcN)5 –GlcNAc [M + Na]+ X X X X X X X
1065.4 [M + K]+ X X
1109.9 (GlcN)3 –(GlcNAc)3 [M + H]+ X X X X X X X
1168.4 (GlcN)7 [M + Na]+ X X X X X X X X
1184.4 [M + K]+ X X X X X X X X
1210.7 (GlcN)6 –GlcNAc [M + Na]+ X X X X X X X
1226.4 [M + K]+ X X X
1270.7 (GlcN)4 –(GlcNAc)3 [M + H]+ X X X X X X X
1329.5 (GlcN)8 [M + Na]+ X X X X X X X X
1345.5 [M + K]+ X X X X X X X X
1371.8 (GlcN)7 –GlcNAc [M + Na]+ X X X X X X X
1387.5 [M + K]+ X X X X
1490.5 (GlcN)9 [M + Na]+ X X X X X X X X
1506.5 [M + K]+ X X X X X X
1532.8 (GlcN)8 –GlcNAc [M + Na]+ X X X X X X X
1548.5 [M + K]+ X X
1651.6 (GlcN)10 [M + Na]+ X X X X X X
1667.6 [M + K]+ X
1812.7 (GlcN)11 [M + Na]+ X X X X X
1973.7 (GlcN)12 [M + Na]+ X X
 J.C. Cabrera, P. Van Cutsem / Biochemical Engineering Journal 25 (2005) 165–172 171

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Acknowledgements
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rector of INCA, for supporting oligosaccharin research and
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