Carbohydrate Polymers 64 (2006) 66–72

www.elsevier.com/locate/carbpol

Bactericidal and antifungal activities of a low molecular weight

chitosan and its N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives
Vladimir E. Tikhonov a,*, Evgeniya A. Stepnova a, Valery G. Babak a, Igor A. Yamskov a,
Javier Palma-Guerrero b, Hans-Börje Jansson b, Luis V. Lopez-Llorca b, Jesus Salinas b,
Denis V. Gerasimenko c, Inna D. Avdienko c, Valery P. Varlamov c
a
Laboratory of Physiologically active biopolymers, A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
Vavilov st.28, 119991 Moscow, Russia
b
Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, University of Alicante, Aptdo. 99 E-03080, Alicante, Spain
c
Laboratory of Enzyme Engineering, Bioengineering Center, Russian Academy of Sciences, 117312 Moscow, Russia

Received 22 March 2005; received in revised form 26 October 2005; accepted 27 October 2005
Available online 15 December 2005

Abstract
Low molecular weight chitosan (4.6 kDa) and N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives of different degrees of substitution were tested for
their antimicrobial activity against Escherichia coli, Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, Candida kruisei
and Fusarium oxysporum f. sp. radicis lycopersici. The results indicated that the chitosans show high activities against all bacteria, yeast and
filamentous fungus. In fact, they suppressed fungal colony growth and inhibited fungal spore germination at 0.01% (w/v) concentration. A higher
activity of chitosan and its derivatives was obtained with a substitution degree of 3 mol% for E. coli and B. subtilis, while a substitution degree of
9 mol% was usually preferable for increased activity against P. aureofaciens, E. agglomerans and C. kruesei.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Chitosan derivatives; Antibacterial activity; Antifungal activity

1. Introduction
Chitosan, b-(1/4)-2-acetamido-2-deoxy-D-glucan, is the
most abundant among the natural aminopolysaccharides.
Chitosan, the poly-(b-1/4)-2-amino-2-deoxy-D-glucopyra-
nose, is a collective name for a group of partially and
fully deacetylated chitin. This polysaccharide was found to
be non-toxic, biocompatible and biodegradable (Lui, Dunn,
Grandmaison, & Goosen, 1997). Chitosan has found several of
applications being employed either alone or in blends with
other natural polymers (starch, gelatin, alginates) in the food
and pharmaceutical industries mainly due to its high
biodegradability and antimicrobial properties (Arvanitoyannis,
1999; Arvanitoyannis, Nakayama, & Aiba, 1998; Hague et al.,
2005; Kim, Chen, Wang, & Rajapakse, 2005; Yamada, Akiba,
Shibuya, Kashiwada, Matsuda, & Hirata, 2005). Microbiolo-
gical activity of chitosan has been detected for many bacteria,
* Corresponding author. Tel.: C7 095 1359375; fax: C7 095 1355085.
E-mail address: tikhon@ineos.ac.ru (V.E. Tikhonov).
0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2005.10.021
filamentous fungi and yeasts (Hirano & Nagao, 1989; Kendra
& Hadwiser, 1984; Uchida, Izumi, & Ohtakara, 1989; Ueno,
Yamaguchi, Sakairi, Nishi, & Tokura, 1997).
These studies show that the biological activity of chitosan
depends significantly on its physico-chemical properties such
as molecular weight and molecular fraction of glucosamine-
units in the chitosan polymer chain (usually referred as the
degree of chitosan N-deacetylation). These factors influence
chitosan solubility and interaction with the cell walls of target
microorganisms. Chitosan is insoluble in aqueous media at
neutral and basic conditions, but is soluble in aqueous diluted
acids. However, in some fields the application of this
polysaccharide is limited by its high molecular weight resulting
in low solubility in aqueous diluted acids and high viscosity of
the solution even at low chitosan concentration. Medical
chitosan applications require, as a starting material, a low
molecular weight (LMW) chitosan with a high solubility and
low viscosity in water at physiologically acceptable pH values.
Numerous studies on bactericidal activity of chitosan have
been carried out (Badawy et al., 2004; Muzzarelli et al., 1990;
Muzzarelli et al., 2001; Rhoades & Roller, 2000) and reviewed
(Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003;
Chirkov, 2002), and some controversial evidences for
 V.E. Tikhonov et al. / Carbohydrate Polymers 64 (2006) 66–72 67

a correlation between bactericidal activity and chitosan
molecular weight have been found. Most investigators used
the uncertain term ‘low molecular weight chitosan’ for a
partially depolymerized chitosan not indicating exactly its
molecular weight. Only a few data on bactericidal activity of
low molecular weight chitosan could be compared depending
on bacteria tested, conditions of biological test and chitosan
molecular weight, but even in this case the results did not
correspond to each other. Thus, 9.3 kDa chitosan restricted
growth of Escherichia coli while 2.2 kDa chitosan promoted
growth of this bacterium (Tokura, Ueno, Miyazaki, & Nishi,
1997). Increase in chitosan molecular weight led to a decrease
in chitosan activity against E. coli in some studies (Gerasi-
menko, Avdienko, Bannikova, Zueva, & Varlamov, 2004;
Zheng & Zhu, 2003), while in the others an increased activity
for a high molecular weight (HMW) chitosan in comparison
with LMW chitosan have been found (Kyung, Thomas, Chan,
& Park, 2003). In contrast to the above mentioned results, no
differences in HMW chitosan and LMW chitosan activities
were found against E. coli (Jeon, Park, & Kim, 2001; Zhishen,
Dongfeng, & Weiliang, 2001) and Bacillus subtilis (Gerasi-
menko et al., 2004; Jeon et al., 2001). At present, it has been
commonly recognized that the biological activity of chitosan
depends on its molecular weight, deacetylation degree,
chitosan derivatization, degree of substitution, length and
position of a substituent in glucosamine units of chitosan, pH of
chitosan solution and, of course, the target organism. These
variations suggested to lead to two different mechanisms of
chitosan and target microorganism interaction: the first—
adsorption of chitosan to cell wallsleading to cell walls
covering, membrane disruption and cell leakage—is mainly
connected with HMW chitosan; the second—penetration of
chitosan into living cells leading to inhibition of various
enzymes and interference with synthesis of mRNA and
proteins (Rabea et al., 2003; Chirkov, 2002; Zheng & Zhu,
2003). Experimental conditions of chitosan bioassays can also
bias the results of such tests. Moreover, we believe that a
suitable method of LMW chitosan preparation for experiments
should be adopted. In fact, the method chosen (e.g. acidic or
enzymatic hydrolysis, oxidative depolymerization by
CH2OH
H O O H
H H
OH H H
O H
... O O
...
H NH
m n
O C COOH
CH CH2
CH2
peroxides, oxidative deamination by nitrite) will determine
the chemical structure of the reducing end of the chitosan
polysaccharide chain and may, in turn, affect the biological
activity of LMW chitosans.
In the present study, biocidal activities of chemically
prepared LMW chitosan of narrow molecular mass distribution
and some of its synthetic lipo-derivatives were tested against
important species of Gram-positive and Gram-negative
bacteria, the yeast Candida kruisei and Fusarium oxysporum
f. sp. radicis lycopersici, a common fungal pathogen of plants
distributed worldwide. The results were compared with
bactericidal activity of enzymatically prepared LMW chitosan.
2. Materials and methods
Chitosan was produced from Kamchatka-peninsula-shelf
crab shell chitin with a degree of deacetylation (DD) of 82%
and viscosity–average molecular weight of 300 kDa (BioChit,
Moscow, Russia).
2.1. Sample preparation
A series of N-/2(3)-(dodec-2-enyl)succinoyl/chitosans
(DDC-chitosans) were prepared via reaction of HMW chitosan
with 2-(dodecen-1-yl)succinic anhydride (SIGMA) at the
conditions described by Hirano, Ohe, & Ono (1976). LMW
chitosan and a series of N-/2(3)-(dodec-2-enyl)succinoyl/-
derivatives (DDC–LMW chitosans) were prepared by acidic
hydrolysis of the chitosan and DDC-chitosans following
procedure described by Domard & Carter (1989).
DDC content (n) in DDC–LMW chitosans (Fig. 1) was
determined by titration of carboxyl-groups after conversion of
DDC–LMW chitosan to the corresponding DDC–LMW
Chitins following the procedure described by Tikhonov,
Radigina, & Yamskov (1996).
Amino-group contents (p) in LMW chitosan and
DDC–LMW chitosans (Fig. 1) was determined by elemental
microanalysis of chloride ions.
Acetyl-group content (m) in LMW chitosan and
DDC–LMW chitosans (Fig. 1) was calculated as a difference:
CH2OH CH2OH
O O H O O
OH H OH H
H
H
... ...
H NHCCH3 H NH3+Cl- p
O

CH

CH
(CH2)8

CH3

Fig. 1. Chemical structure of the DDC–LMW chitosans (2-isomer is only shown in fragment m).
68 V.E. Tikhonov et al. / Carbohydrate Polymers 64 (2006) 66–72
mZ1K(nCp) since no difference in DD was found for the
starting chitosan and the prepared LMW chitosan.
2.2. Surface tension activity
The surface tension at the air–water interface of aqueous
solutions of chitosans was measured by Wilhelmy’s plate
method using tensiometer K100 Mk1 (Kruss, Germany). All
the measurements were made for a sufficiently long time
(usually longer than 10,000 s) in order to follow the effect of
ageing on the surface tension of the adsorption layers.
2.3. Cultures for antimicrobial tests
Bacteria and yeast were obtained from the Russian
collection of industrial microorganisms (VKPM) and the
American type culture collection (ATCC):
E. coli ATCC 5945, Pseudomonas aureofaciens VKPM
B-7542, Enterobacter agglomerans VKPM B-7541, (all Gram-
negative), B. subtilis VKPM B-7540 (Gram-positive), and C.
kruisei VKPM Y-2594. F. oxysporum f. sp. radicis lycopersici
(isolate 127) was from the culture collection of the Laboratory
for Plant Pathology, Department of Marine Sciences and
Applied Biology, University of Alicante, Spain.
2.4. Bacteria and yeast cultivation
E. coli, P. aureofaciens, E. agglomerans were grown on
slanted agar under aerobic conditions as described in Bergey’s
manual of Systematic Bacteriology (Sneath, 1986). C. kruisei
was cultivated on Sabouraud agar slants. B. subtilis was grown
in flasks containing LB Broth (SIGMA). The flasks were
incubated shaking (100 rpm) for 24 h at 37 8C. The cultured
cells (bacteria and yeast) were suspended into 0.1 M sodium
acetate buffer (pH 6.5) at a concentration of 109 cells/ml.
2.5. Bactericidal test
A 0.1 ml suspension of the microorganism to be tested was
added to 0.9 ml LMW chitosan or DDC–LMW chitosan
solution (0.1 or 1% w/v) in 0.1 M sodium acetate buffer (pH
6.5) under sterile conditions. The mixture was incubated
shaking for 1 h at 37 8C, and the number of viable cells was
calculated. Bactericidal activity was expressed as percentage of
cell death.
2.6. Fungistatic activity test
Fungistatic activity of LMW chitosan and DDC–LMW
chitosans was determined by a radial hyphal growth bioassay.
A sample of chitosans to be tested was dissolved in 0.25 M
HCl, and the pH was adjusted to 5.0–5.5 with 1 M NaOH.
Potato dextrose agar (PDA) media including 0.1, 0.5 or
1.0 mg/ml of a given chitosan solution was sterilized at 121 8C
for 20 min and then poured onto sterile petri dishes (9-cm
diameter). The plates were inoculated with 6-mm-diameter
plugs taken from the margins of 3–5-days old colonies of
F. oxysporum on PDA. Three replicates for each sample
concentration were used. Control plates (PDA only) were also
inoculated with the fungus. All plates were incubated in the
dark at 24 8C. The radial colony growth was measured daily for
2 weeks.
2.7. Germination of conidia of F. oxysporum
LMW chitosan or DDC–LMW chitosan were dissolved in
0.25 M HCl and the pH was adjusted to 5.6 with 1 M NaOH.
The chitosan solutions were filter-sterilized (Acrodisc, Gel-
man, 0.22 mm pore size). These experiments were performed
using multi-well microscope slides each containing 10 wells
(ICN Biomedicals, Aurora, OH USA). Each well was filled
with 25 ml of the chitosan solutions (0, 0.01, 0.001,
0.0001 mg/ml) containing 106 conidia/ml. After 24 h, the
numbers of germinated and non-germinated conidia were
recorded and the percentage germination was calculated
(Laflamme, Benhamou, Bussieres, & Dessureault, 1999).
3. Results and discussion
3.1. LMW chitosan and DDC–LMW chitosans preparation
Reactions of HMW chitosan with 2-dodecenylsuccinic
anhydride were carried out in a 1% aqueous acetic
acid/methanol (1:2 v/v) solution (pH 4.5) at 500 C led to
N-substituted chitosans bearing 2 (3)-(dodec-2-enyl)succinoyl-
groups in correspondence with the numerous studies on
reaction of chitosan with anhydrides (Hirano et al., 1976).
After acidic hydrolysis of chitosan and DDC-chitosans,
hydrochlorides of LMW chitosan and DDC–LMW chitosans
were separated by size exclusion chromatography (Domard et
al., 1989). Chemical structures of DDC–LMW chitosans are
shown in Fig. 1. Their fractional compositions determined by
chemical analysis of C/N ratio, chlorine content and titration of
the substances obtained are presented in Table 1. Carboxylic
groups (and corresponding DDC fragment contents) in DDC–
LMW chitosans were determined by their titration with 0.05 M
NaOH after conversion of DDC–LMW chitosans to corre-
sponding DDC-chitins via complete N-acetylation using acetic
acid anhydride following Hirano’s method of chitosan
acetylation (Hirano et al., 1976). The presence of a band at
1715 cm-1 in IR spectra of a DDC–LMW chitosan
Table 1
DDC-chitosans fractional composition, molecular weight (Mw) and poly-
dispersity index (PI)
Sample m (mol%) n (mol%) p (mol%) Mw (kDa)
and/PI
LMW chitosan – 20 80
DDC(3%)–LMW 3 20 77 4.6/1.6
chitosan
DDC(9%)–LMW 9 20 71
chitosan
DDC(16%)–LMW 16 20 64
chitosan
 V.E. Tikhonov et al. / Carbohydrate Polymers 64 (2006) 66–72 69

Table 2
Effect of LMW chitosan and LMW chitosan derivatives on bacteria and yeast (sample concentration: 1% w/v)

Bacteria Viable cells (CFU/mlGerror)/cell death (%) Control

LWL Chitosan DDC(3%)–LMW DDC(9%)–LMW DDC(16%)–LMW Viable cells
chitosan chitosan chitosan (CFU/mlGerror)
E. coli (4,7G0,1)10791,6% (1,3G0,1)107 98,5% (1,4G0,1)108 75,0% (1,3G0,1)108 76,8% (5,6G0,2)108
P. aureofaciens (1,7G0,1)10790,4% (3,2G0,1)107 86,5% (1,3G0,1)106 96,0% (4,5G0,1)107 92,0% (6,7G0,2)108
B. subtilis (3,6G0,1)10464,6% (1,0G0,1)102 99,9% (4,0G0,1)103 96,0% (2,0G0,1)10398,0% (1,0G0,1)105
E. agglomerans (3,0G0,1)105 99,991% (1,3G0,1)105 99,996% (1,0G0,1)105 99,997% (1,8G0,1)105 99,994% (3,5G0,3)109
Yeast
C. kruisei (2,1G0,1)102 99,9989% (3,4G0,1)102 99,9983% (1,2G0,1)102 99,9994% (2,7G0,1)102 99,9987% (2,0G0,1)107

hydrochloride confirmed the presence of carboxyl-groups in
DDC substituted chitosan hydrochloride. Gel permission
chromatography revealed that LMW chitosan and DDC–
LMW chitosans had average molecular weights of 4.6 kDa
and a narrow molecular mass distribution with polydispersity
index of 1.6 (Table 1).
3.2. Bactericidal and antifungal activities of LMW chitosan
and DDC–LMW chitosans
In our study, LMW chitosan and three LMW chitosan
derivatives bearing long aliphatic chains and differing in the
degree of N-substitution were tested for their activity against
Gram-negative, Gram-positive bacteria, yeast and filamentous
fungus. Low molecular weight chitosan appeared to better
adsorb to and penetrate cell walls of microorganisms (Zheng &
Zhu, 2003), so that chemical modification of chitosan
(i.e. N-substituted chitosan) by aliphatic chains may affect
the antimicrobial properties of DDC–LMW chitosans.
Our results indicate that after a short exposure time (1 h),
1% (w/v) LMW chitosan and its derivatives exhibited high
antimicrobial activity against the Gram-negative, Gram-
positive bacteria and the yeast tested (Table 2). Thus,
DDC(3%)–LMW chitosan reduced number of living E. coli
and P. aureofaciens cells 67 and 25-fold, respectively, while
the number of B. subtilis cells was reduced about 3 log units.
Most dramatic action of LMW chitosan and DDC(9%)–LMW
chitosan were demonstrated against E. agglomerans cells
(reduction about 4 log units). Although experimentally
detectable but practically not large differences there were
found between the action of LMW chitosan and DDC–LMW
DDC–LMW chitosans against B. subtilis (up to 99.9% cell
death) in comparison with activity against E. coli (60% cell
death) and B. subtilis (70% cell death) of enzymatically
prepared 5 kDa chitosan (Jeon et al., 2001). Although these
differences in antimicrobial activities of chemically and
enzymatically prepared low molecular weight chitosans still
remain unknown, they may be due to differences in N-acetyl
groups distribution along polymer chains of these chitosans
(this hypothesis needs to be verified).
High antimicrobial activity of LMW chitosan and
DDC–LMW chitosans was demonstrated against C. kruisei:
the number of viable cells was reduced about 5 log unit for all
synthesized chitosans. It should be mentioned that very low
activity (about of 1% cell death) was found for enzymatically
prepared 5 kDa chitosan at the same concentration
(Gerasimenko et al., 2004). Our experiments also show that
DDC–LMW chitosans caused C. kruisei cell death depending
on the contents of DDC-residues. As shown in Fig. 2, C. kruisei
cell death increased from 55.2 to 95.4% when 3 mol% of DDC
fragments were introduced to LMW chitosan. However, further
increase in DDC contents led to a decrease in activity.
Although there was no correlation between activity towards
C. kruisei and surface tension activities of DDC–LMW
chitosans with different DDC contents (Fig. 3), the presence
of a long aliphatic chain should at least facilitate absorption of
a substituted LMW chitosan onto cell walls via hydrophobic
100
90
80
70
Cell death, %

chitosans, DDC(3%)–LMW chitosan could be considered as

preferable antimicrobial agent against E. coli and B. subtilis 60
while DDC(9%)–LMW chitosan was usually more active 50
against P. aureofaciens, E. agglomerans and C. kruisei. 40
Antimicrobial activity of the chemically prepared LMW 30
chitosan and its DDC-derivatives were compared with that of
20
chitosan of 5 kDa obtained by enzymatic hydrolysis of high
10
molecular weight chitosan (Gerasimenko et al., 2004). As a
result, LMW chitosan and DDC–LMW chitosans displayed 0
0 3 6 9 12 15 18
higher activities (up to 98.5% cell death) towards E. coli than
DDC (mol%)
an enzymatically prepared 5 kDa chitosan (3.8% cell death) at
the same experimental conditions (Gerasimenko et al., 2004). Fig. 2. DDC–LMW chitosans activity against cells of the yeast C. kruisei versus
Higher activities were also found for LMW chitosan and degree of DDC contents. Sample concentration: 0.1% (1 mg/ml).
70 V.E. Tikhonov et al. / Carbohydrate Polymers 64 (2006) 66–72

75 (a)
70 50

Radius of colony (mm)

65 40
60
σ, mN/m

30
55
50 20
45
10
40
35 0
0 2 4 6 8 10 12 14
30
0 3 6 9 12 15 Time of growth (days)
(b)
DDC (mol.%) 50

Radius of colony (mm)

Fig. 3. Surface tension activity of LMW chitosan and DDC–LMW chitosan 40
versus DDC contents. Sample concentration: 0.1% (w/v). (where: s—surface
tension, milliNewton per meter). 30

interaction with cell wall proteins and thus increase their 20

activity. Also, the presence of 3O9 mol% DDC may be
considered as a preferable DDC-content for cell wall binding
and penetration into the cells in spite of that LMW chitosan and
DDC(16%)–LMW chitosan demonstrated even higher activity
against some species tested in comparison with DDC(3%)–
LMW chitosan and DDC(9%)–LMW chitosan. The observed
decrease of activity of LMW chitosans with increased DDC
contents may be explained by formation of micelle-like
structures and interpolyelectrolyte aggregates (Fig. 4) in
aqueous solution via hydrophobic interaction between long
alkyl chains (Lui, Sun, Zhang, & Yaok, 2003). These alkyl
chains hidden inside aggregates (Fig. 4(b)) could not interact
with cell wall proteins by forming complexes. Following this
hypothesis, we may conclude that the conformation of
DDC–LMW chitosans in solution may be a factor, which at
least should be taken into account for prediction of
antimicrobial activity of low molecular weight chitosan and
its hydrophobically modified derivatives. The verification of
this hypothesis requires usage of fluorescent labeled LMW
chitosan and DDC–LMW chitosans and dynamic light
scattering investigations (to be published).
Activity of LMW chitosan and DDC–LMW chitosan
containing 3 mol% of DDC residues against F. oxysporum f.
sp. radicis lycopersici was determined by measuring the
evolution of colony radius at several concentrations of
(a) (b)
Fig. 4. Schematic presentation of different conformations in aqueous solutions
for hydrophobically modified LMW chitosans with low (a) and high (b) alkyl
chain contents.
10
0
2 4 6 8 10 12
Time of growth (days)
(c)
Radius of colony(mm) 50
40
30
20
10
0
2 4 6 8 10 12
Time of growth (days)
Fig. 5. Effect of LMW chitosan and DDC(3%)–LMW chitosan on hyphal
growth of Fusarium oxysporum f.sp.radicis lycopersici: concentration:
1 mg/ml (-&-) control; (-:-) LMW chitosan; (-$-) DDC–LMW chitosan;
LMW chitosan concentration (w/v): (-&-) control; (-B-) 0.1%; (-:-) 0.5%;
(-$-) 1%; DDC(3%)–LMW chitosan concentration (w/v): &(-B-) 0.1%;
(-:-) 0.5%; (-$-) 1%. Bars, standard error.
chitosans tested. Comparative data on fungistatic activities of
the substances are shown in Fig. 5(a)–(c). Growth of F.
oxysporum hyphae was suppressed by LMW chitosan and
DDC(3%)–LMW chitosan. Fungicidal activity of
DDC(3 mol%)–LMW chitosan was more active against F.
oxysporum than that of LMW chitosan at all concentrations
(Fig. 5(b) and (c)).
The effect of antifungal compounds on fungal spore
germination was higher than that on hyphal growth. The effect
of LMW chitosan and DDC(3%)–LMW chitosan on germina-
tion of conidia of F. oxysporum was studied at a concentration
range from 0.01 to 0.0001 mg/ml and the results are shown in
Table 3. Our data demonstrate that both LMW chitosan and
DDC(3%)–LMW chitosan abolished germination of
F. oxysporum conidia at 0.01 mg/ml (0.001% solution).
When conidia were treated with any of the chitosans at
0.01 mg/ml no germination was found upon plating on
different growth media (Palma-Guerrero et al., unpublished
 V.E. Tikhonov et al. / Carbohydrate Polymers 64 (2006) 66–72
Table 3
Effect of different LMW chitosans on spore germination of F. oxysporum f.sp.
radicis lycopersici conidia
Type of chitosan Chitosan concen- Germination, % (meanGstandard
tration (mg/ml) error)
LMW 0.01 0 of the D-glucosamine oligomer series. In G. Skjak-Braek, T. Anthonsen,
0.001 84.01G2.03
0.0001 99.07G0.54
0 99.67G0.33
DDC(3%)–LMW 0.01 0 Gerasimenko, D. V., Avdienko, I. D., Bannikova, G. E., Zueva, O. Y., &
0.001 82.06G4.65
0.0001 98.98G4.94
0 99.25G0.44
results). This was found for incubation up to 30 days at 240 8C.
All the substances tested were fungistatic (i.e. reduced growth
rate of developing hyphae), but they also possessed fungicidal
activity, since they completely inhibited spore germination at a
concentration of 0.01 mg/ml. This inhibitory effect was
decreased significantly with concentration.
4. Conclusion
LMW chitosan and its DDC-derivatives showed high
activity against bacteria, yeast and fungus tested. The
preferment of chitosan derivatization was found for a
substitution degree of 3 mol% for E. coli and B. subtilis
while substitution dergree of 9 mol% was usually preferable
for activity against P. aureofaciens, E. agglomerans and C.
kruisei. DDC(3%)–LMW chitosan, in particular, inhibited
growth of F. oxysporum f. sp. radicis lycopersici.
From comparison of these results and previously published
data on bactericidal activity of enzymatically prepared 5 kDa
chitosan (Gerasimenko et al., 2004) we could conclude that in
spite of lack of knowledge on the antimicrobial action
mechanism, the origin of low molecular weight chitosan and
its derivatives as well as the method used for low molecular
weight chitosan and its derivatives preparation are the
important issue especially in biomedical applications.
The results of this study suggest new means to investigate the
mode of action of these biocidal substances of natural origin.
Acknowledgements
This work was partly supported by a grant from the Spanish
Ministry of Science and Education (AGL 2004-05808).
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