Carbohydrate Polymers 63 (2006) 367–374

www.elsevier.com/locate/carbpol

Water-solubility of chitosan and its antimicrobial activity

Caiqin Qin a,b,*, Huirong Li a,b, Qi Xiao b, Yi Liu b, Juncheng Zhu b, Yumin Du b
a
Laboratory for Natural Polysaccharides, Xiaogan University, Xiaogan 432100, China
b
College of Chemistry and Environmental Science, Wuhan University, Wuhan 430072, China
Received 11 August 2005; received in revised form 29 August 2005; accepted 6 September 2005
Available online 8 November 2005

Abstract

Chitosan samples with different molecular weights were prepared by depolymerization with hemicellusase, and water-soluble half N-acetylated
chitosan samples were obtained by N-acetylation with acetic anhydride. The action of chitosans with molecular weights Mw from 1.4!103 to
4.0!105 on the growth of Staphylococcus aureus, Escherichia coli and Candida albicans was explored by microcalorimetry. The water-soluble
half N-acetylated chitosans and chitooligomers had no significant antimicrobial activity. Moreover, water-soluble chitosans and chitooligomers
promoted the growth of C. albicans. In contrast, water-insoluble chitosan in acidic medium exhibited inhibitory effect against these
microorganisms. The water-insoluble chitosans with Mw around 5!104 were the optimum for antimicrobial action in these tested samples. The
antimicrobial mechanism of dissolved water-insoluble chitosan was hypothesized as forming an impervious layer around the cell. The results
suggest that optimum chitosan as food preservative should be water-insoluble chitosan from mild depolymerization of native chitosan.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Water-solubility; Microorganism; Microcalorimetry

1. Introduction activity of chitosan against different groups of microorganisms

has received considerable attention in recent years (Rabea,
Chitosan, a natural linear biopolyaminosaccharide is Badawy, Stevens, Smagghe, & Steurbaut, 2003). Chitosan,
obtained by alkaline deacetylation of chitin, which is the however, shows its antibacterial activity only in an acidic
principal component of protective cuticles of crustaceans such medium, which is usually ascribed to the poor solubility of
as crabs, shrimps, prawns, lobsters and cell walls of some fungi chitosan at high pH (Liu, Du, Wang, & Sun, 2004; No, Lee,
such as aspergillus and mucor. Chitosan is a weak base and is Park, & Meyers, 2003). These reported antimicrobial activities
insoluble in water and organic solvent. However, it is soluble in might be the effect of dissolved chitosan in acidic media such
dilute aqueous acidic solution (pH!6.5), which can convert as acetic acid (Devlieghere, Vermeulen, & Debevere, 2004),
glucosamine units into soluble form R–NHC 3 (Kumar,
lactic acid (Papineau, Hoover, Knorr, & Farkas, 1991),
Muzzarelli, Muzzarelli, Sashiwa, & Domb, 2004). It gets glutamic acid (Roller, & Covill, 1999; Sudharshan, Hoover,
precipitated in alkaline solution or with polyanions and forms & Knorr, 1992) and hydrochloric acid (Chung, Wang, Chen, &
gel at lower pH. Li, 2003). The antimicrobial activity of chitosan was reported
Chitosan is inexpensive, biodegradable, and nontoxic for to be dependent on its molecular weight and degree of
mammals. This makes it suitable for use as an additive in the deacetylation DD (Jeon, Park, & Kim, 2001; Yoshihiko,
food industry (Koide, 1998; Shahidi, Arachchi, & Jeon, 1999), Mayumi, Takahiro, Hiroyuki, Yoshihiro and Ichiro, 2003). The
as a hydrating agent in cosmetics, and more recently as a antibacterial effect of chitosan oligomers was also investigated,
pharmaceutical agent in biomedicine (Dodane & Vilivalam, but the media is still acidic (Choi, Kim, Yoo, Oh, Choi and
1998; Illum, 2003; Khor & Lim, 2003). The antimicrobial Kim, 2001; Gerasimenko, Avdienko, Bannikova, Zueva, &
Varlamov, 2004; No, Park, Lee, & Meyers, 2002).
Chitosan oligomers, which can be achieved by degradation
* Corresponding author. Address: Laboratory for Natural Polysaccharides, of chitosan polymer chain, are water-soluble. In addition,
Xiaogan University, Xiaogan 432100, China. Tel.: C86 712 2345697; fax: water-soluble chitosan can be obtained through a chemical
C86 712 2345265. modification in which the degree of substitution is controlled.
E-mail address: qincaiqin@yahoo.com (C. Qin). For instance, it is known that water-soluble chitosan with about
0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. 50% DD can be obtained from chitin by hydrolysis with alkali
doi:10.1016/j.carbpol.2005.09.023 (Kurita, Sannan, & Iwakura, 1977) or from chitosan by
368 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374

N-acetylation with acetic anhydride (Kubota, Tatsumoto, Sano, CS2, CS3 and CS4 were collected after drying at 50 8C in
& Toya, 2000). vacuum for 48 h, respectively.
But up to the present, special information on the correlation The left mixture continued to react for 4 h. Membrane of
of water-solubility and antimicrobial activity of chitosan is 10 kDa was used to remove enzyme, and the ultrafiltrate was
lacking. Very few attempts have been made to date to assess further separated by membrane of 1 kDa. The filtrate was
the antibacterial activity of chitosan with free –NH2 (not NHC3) neutralized with NaOH to pH 10. The precipitate was washed
in detail, although it is necessary for understanding correctly thoroughly with distilled water to obtain sample CS5 after
the antimicrobial effect of chitosan. drying over phosphorus pentoxide in vacuum. The filtrate was
The main objective of this study was to investigate whether concentrated, precipitated by adding ethanol, and washed
the chitosan with free –NH2 itself has antimicrobial effect, and thoroughly with ethanol to obtain water-soluble sample CS6
to explore preliminarily the relationship between the anti- after drying over phosphorus pentoxide in vacuum.
microbial activity and water-solubility of chitosan. Chitosans
with different molecular weights and water-solubility were 2.3. Half-N-acetylation of chitosan
prepared. Microcalorimetry method was employed to deter-
mine the action of chitosans on growth of three typical human 2.2 g chitosan was dissolved in 3% acetic acid solution
pathogenic microorganisms in vitro. (50 ml), and acetic anhydride (0.56 g) in 50 ml ethanol was
added. After stirring at 40 8C for 12 h, the solution was
neutralized with NaOH to pH 9, precipitated by adding ethanol,
2. Experimental and washed thoroughly with ethanol to obtain half-acetylated
sample (HCS1, HCS2 and HCS3) after drying in vacuum.
2.1. Materials and equipment
2.4. Potentioniometric determination of DD
Native chitosan CS0 and hemicellusase solution were
obtained from Hubei Yufeng Biology Engineering Co., Ltd The chitosan (0.3 g) was dissolved in a known excess of
(China). Peptone and yeast extract were biological reagents 0.1 M HCl acid (20 ml). From the titration of this solution with
from Oxoid, all other chemicals were of analytical grade and a 0.1 M NaOH solution, a curve with two inflexion points was
were used without further purification. obtained. The amount of the acid consumed between these two
The UF membranes (OMEGA) were purchased from points was considered to correspond to the amount of the free
PallFiltron Corporation (USA). FT-IR spectra were recorded amino groups in the solution (Tolaimate, Desbrieres, Rhazi,
with KBr pellets on a Perkin–Elmer Spectrum One B Alagui, Vincendon and Vottero, 2000). The titration was
spectrophotometer. X-ray diffraction patterns of samples performed with a DELTA-320-S pH meter.
were measured by a Shimadzu XRD-6000 diffractrometer
and used a Cu Ka target at 40 kV and 30 mA at 23 8C. 2.5. Molecular weight determination
Staphylococcus aureus (CCTCC AB910393) and Escher-
ichia coli (CCTCC AB91112) were provided by Chinese Weight-average molecular weight (Mw), number-average
Center of Type Culture Collection, Wuhan University, China. molecular weight (Mn) and molecular weight dispersion
Candida albicans was provided by the Hospital of Hubei (Mw/Mn) were measured by a gel permeation chromatography
Province, China. The three strains were grown to the stationary (GPC). GPC system incorporated a TSP P100 instrument
phase in nutrient broth at 37 8C for bacteria or at 30 8C for (USA). The connected columns (TSK G5000-PW and TSK
C. albicans, and these suspensions were used as the inocula for G3000-PW) were used. 0.2 M CH3COOH/0.1 M CH3COONa
the microbiological assay. was used as the eluent. The flow rate was maintained at
TAM air (an eight-channel isothermal batch calorimeter for 1.0 ml/min. The eluent was monitored by RI 150 refractive
heat flow measurements, whose limit of detectibility is 2 mW) index detector. The sample concentration was approximately
manufactured by Thermometric AB of Sweden was used to 0.4% (w/v). The standards used to calibrate the column were
obtain the metabolic growth power-time curves of microor- TOSOH pullulan (Japan). All data provided by the GPC system
ganisms, and PicoLog software (Pico Technology Ltd, UK) were collected and analysed using the Jiangshen Workstation
was used to treat the data (Wadsö, 2002). software package (Dalian, China).

2.2. Preparation of samples by enzymatic hydrolysis 2.6. Estimation of water-solubility

CS0 (200 g) was completely dissolved in 3000 ml 5% (w/v) The pH dependence of water solubility of chitosan was
acetic acid with a water bath at 50 8C, and 25 ml of evaluated from turbidity. The sample (100 mg) was dissolved
hemicellusase solution was added. At time intervals of 10, in 1% (w/v) HAc (100 ml). Following stepwise addition of
20, 30 and 60 min, 300 ml mixture was respectively taken out, concentrated NaOH, the transmittance of the solution was
boiled for 10 min, and filtered to remove the enzyme. The recorded on a UV-1601 Shimadzu spectrophotometer (Japan)
filtrate was neutralized with NaOH to pH 10. The precipitate using a quartz cell with an optical path length of 1 cm at
was washed thoroughly with distilled water. The samples CS1, 600 nm.
 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374 369

2.7. Assays for action of chitosan on microorganisms

by microcalorimetry

LB medium (NaCl 5 g, peptone 10 g, yeast extract 5 g/l, pH

7.0) was sterilized by autoclaving for 20 min at 120 8C.
The inocula were homogeneously distributed into 50 ml of
LB medium at a concentration 106 CFU/ml for bacteria and
105 CFU/ml for C. albicans by gentle shaking. A liquor of 5 ml
of the suspensions were added into 20 ml sterilized ampoules
containing test samples and sealed tightly. Sterilized water was
used as a control instead of sample. The ampoules were placed
in the calorimeter and signals obtained during growth were
detected. The experiments were run at 37.00 8C for bacteria or
30.00 8C for C. albicans. Each batch experiments were carried
out at least twice, the standard error of the mean usually did not
exceed 2%.

3. Results and discussion

3.1. Preparation of chitosan samples

Fig. 1. IR spectra of CS3 and HCS1.

A series of chitosan samples CS1, CS2, CS3, CS4, CS5 and amide II band, also indicating that HCS1 had lower DD. These
CS6 were prepared by depolymerization of CS0 with catalysis data coincided well with the data of potentioniometric
of hemicellulase. HCS1, HCS2 and HCS3 were prepared by determination of DD.
N-acetylation of degraded chitosan with acetic anhydride in The X-ray diffraction patterns of CS3 and half N-acetylated
acetic acid–water–ethanol complex solvent. The calculated chitosans were shown in Fig. 2. The WAXD pattern of CS3
values of Mw and Mw/Mn obtained by GPC are given in Table 1. exhibited its two characteristic peaks at 2qZ10.48, 19.88 and
In this paper, CS0, CS1, CS2, CS3 and CS4 were called as 21.88, the ‘tendon hydrate polymorph’ (Belamie, Domard, &
‘water-insoluble chitosan’, which were not soluble in pure Giraud-Guille, 1997; Qin et al., 2003). HCS1 and HCS2 only
water (Qin, Du, Zong, Zeng, Liu and Zhou, 2003); HCS1 and displayed a sharply decreased wide peak at 2qZ19.88, which
HCS2 with about 50% DD were called as ‘water-soluble suggests that they were amorphous and had good water-
chitosan’, which were soluble in pure water (Kubota et al., solubility (Kurita et al., 1977).
2000); CS6 and HCS3 with a low degree of polymerization
(DP!10) were called as chitooligomers, which were very easy
to dissolve in pure water. 3.2. Effect of pH on solubility of chitosan samples
The IR spectra of degraded chitosan product CS3 and the
half N-acetylated product HCS1 were shown in Fig. 1. The The pH dependence of transmittance of chitosan solution
absorption bands at 1650 and 1600 cmK1 in CS3 were, was shown in Fig. 3. All tested samples had good solubility at
respectively, referenced as amide I band and N–H bending
mode of –NH2 (Dong, Xu, & Wang, 2001). HCS1 had the
lower relative absorption intensity of –NH2, and higher
absorption intensity of amide III band at 1316 cm K1,
suggesting that HCS1 had lower DD. The absorption band at a. CS3
1586 cmK1 in HCS1 was considered as the contribution of b. HCS1
c. HCS2
Table 1
I (cps)

Parameters of chitosan samples

Sample Mw!10K3 Mw/Mn DD (%) a

CS0 400 4.12 85.5
CS1 130 3.69 85.6
b
CS2 78 3.44 86.1
CS3 48 3.32 86.2
CS4 17 2.91 86.4 c
CS5 2.8 1.61 87.5
CS6 1.4 1.52 88.0 10 20 30 40
HCS1 53 3.56 52.1
HCS2 18 2.27 55.6 Theta-2Theta (deg)
HCS3 1.4 1.49 54.5
Fig. 2. X-ray diffraction patterns of CS3, HCS1 and HCS2.
370 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374

100 1999). Moreover, it allows the rapid measurement of microbial

activity on line, and is intact on the sample (Rège & Sand,
1998). Because of the possibility of testing batch-wise or in
80 continuous culture, almost all conditions, which influence the
growth of the cells, can rapidly be simulated. Thus,
60 CS0 microcalorimetry can be used for measurements of metabolism
T%

CS1 of microorganism.
CS2 The power-time curves for growth of microorganisms are
CS3
40 CS4 continuously recorded by computer. The calorimetric power,
CS5 P, which reflects the multiplication of the cells, can be used as a
CS6 parameter to characterize the growth of the cells. Since, PZ
20 HCS1
HCS2 dQ/dt, the area under the curve records the heat output Q
released during the experimental period t (Wadsö, 2002).
0
4 5 6 7 8 9 10 11
3.3.2. Effect of water-solubility of chitosan
pH The optimal pH for growth of S. aureus and E. coli is 7.2–
Fig. 3. pH dependence of solubility of chitosan. 7.6, and of C. albicans is 5.5 (Shen, 2000). Acid has also an
inhibitory effect on the growth of S. aureus and E. coli (Chung
et al., 2003). The power-time curves for growth of C. albicans,
pH!6. The dissolved macromolecular CS0 was easily S. aureus and E. coli were shown in Figs. 4–6, respectively.
transferred to floc with addition of NaOH whereas the 10 mM HAc alone stimulated growth of C. albicans by
chitooligomer CS6 were water-soluble over a wide pH range. comparison with the controls containing no additions.
The water solubility obviously depended on the molecular However, addition of HCS2 at concentration of 1.4 g/L greatly
weight of chitosan. As Mw of chitosan with 85–88% DD promoted growth of C. albicans (Fig. 4), although chitosan
decreased, its solubility increased at pH 7. When chitosan was with free –NH2 is an alkaline substance. Similarly, HCS1 at
firstly dissolved in aqueous acetic acid, the solubility at neutral concentration of 2.8 g/L exhibited no inhibitory effect on
pH appeared to be higher than that in pure water. The ionic S. aureus (Fig. 5) and E. coli (Fig. 6). The results suggest that
strength might be a cause for this phenomenon. The half-N- the water-soluble chitosans themselves had no antimicrobial
acetylated chitosans HCS1 and HCS2 had higher water- activity on yeast C. albicans and bacteria S. aureus and E. coli.
solubility than the corresponding chitosans with the similar Acid might be very important for the antimicrobial effect of
molecular weight. water-insoluble chitosan. In absence of acid, 5 g/L CS0, CS3 or
CS4 alone had no inhibitory effect on development of these
3.3. Effect of chitosan on microorganisms microorganisms in neutral LB media (data not shown) due to
its indissolubility. However, in the presence of 10 mM HAc,
0.8 g/L CS4 completely inhibited growth of C. albicans
3.3.1. Verification of method
whereas 1.4 g/L HCS2 still promoted growth of C. albicans,
Many techniques are in use for testing microbial activity.
although both CS4 and HCS2 were dissolved in the acidic
Plate counts and MPN-techniques remain the most widely used
ones because of their low cost and the unequivocal results.
Microcalorimetry is a useful tool for quantitative evaluation of
the growth activity of microbial cells based on detection of C. albicans a. Control
their metabolic heat (Wadsö, 2002). One of the most prominent 2000 b. 10 mM HAc
features of the microbial growth process is the production of c. 1.4g/L HCS2
heat. Microbial activity may be quantified by the detection of c d. 1.4g/L HCS2+10 mM HAC
1500 e. 0.8g/L CS4+ 10 mM HAc
heat output accompanying all biochemical redox reaction.
When the heat is monitored by microcalorimeter, much useful
P/ µW

information, both qualitative and quantitative, may be d

1000
obtained. Calorimetry has also been particularly useful in
monitoring cellular metabolism (Lin, Liu, Liu, Qu, & Yu,
2004), and heat measurements have long been used to study 500 b
metabolism in cells and whole organism. The good reprodu-
cibility of calorimetric measurements on cellular systems has a
been demonstrated in a number of studies on heat production. e
0
Modern instruments allow heat quantities as small as micro-
0 1000 2000 3000 4000 5000
watt, e.g. evolved by bacteria, to be recorded. The method
t/min
further provides information on the bacteriostatic and
bactericidal effects of various chemicals (Yan, Liu, & Wang, Fig. 4. Power-time curves for growth of Candida albicans.
 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374 371

a. Control 60 C. albicans
450 c S. aureus
b. 12 mM HAc
c. 2.8g/L HCS1
0.3g/L CS+2.3mM HAc
d. 2.8g/L HCS1
+12 mM HAC a
40 Control
300 e. 1.5g/L CS3
+ 12 mM HAc CS0
P µ/W

CS1
b

Q /J
CS2
CS3
CS4
150 d 20 CS5
CS6

e
0 0
200 400 600 800 1000 2000 3000 4000 5000 6000 7000
t/min t/min

Fig. 5. Power-time curves for growth of Staphylococcus aureus. Fig. 7. Influence of chitosan with different Mw on the growth of C. albicans.

media. Moreover, CS5 with lower Mw also exhibited the activity of these chitosans was excluded. The molecular weight
inhibitory effect on growth of C. albicans in the acidic medium dependence of the antimicrobial activity of chitosan was more
(Fig. 7), which was contributed to poorer water-solubility of pronounced at a low concentration. CS2 and CS3 with middle
CS5 than that of HCS2. Similarly, in the presence of 10 mM Mw had higher inhibitory effect than others. Chitooligomer
HAc, CS3 at a concentration of 1.5 g/L completely prevented CS6 promoted the growth of C. albicans, but slightly inhibited
growth of both target bacteria (Figs. 5 and 6), whereas HCS1 at growth of the bacteria. It is worth noting that CS0 with the
a concentration of 2.8 g/L only slightly delayed the lag phases. highest M w exhibited much weak inhibitory effect in
These results revealed that the water-soluble chitosan HCS1 comparison with CS2.
and HCS2 in the acetic media had no significantly different
antimicrobial effect from the corresponding HAc, which were
3.3.4. Effect of chitooligomers
very different from the water-insoluble chitosan CS3 and CS4
The effect of chitooligomers on growth of the microorgan-
in the acetic media.
isms was shown in Fig. 10. The chitooligomers, especially half
N-acetylated sample HCS3 promoted growth of C. albicans.
3.3.3. Effect of Mw of chitosan On the other hand, CS6 slightly inhibited growth of S. aureus
The effect of Mw on antimicrobial activity of chitosan was and E. coli, and HCS3 possessed very weak effect. The results
shown in Figs. 7–9. The DD of chitosans CS0 to CS6 was suggested that chitooligomers at neutral media have no
similar (85–88%). Thus, the effect of DD on antimicrobial significant antimicrobial effect.

7
a. Control b. 12 mM HAc c. 2.8g/L HCS1
S. aureus
d. 2.8g/L HCS1+12 mM HAC
6
2000 e. 1.5g/L CS3 + 12 mM HAc 1.0g/L CS+8mM HAc
5 Control
CS0
E. coli d
1500 CS1
4
CS2
Q /J

a
P µ/W

CS3
3 CS4
1000
c c CS5
2 CS6

500 b
a 1

e 0
0

0 500 1000 1500 2000 0 200 400 600 800

t/min t/min

Fig. 6. Power-time curves for growth of Escherichia coli. Fig. 8. Influence of chitosan with different Mw on the growth of S. aureus.
372 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374

most. Water-soluble chitosan with DD around 50% had the

15 highest affinity for E. coli cells and was adsorbed in highest
E. coli
amounts (Strand, Vårum, & Østgaard, 2003). However, many
1.0g/L CS+8 mM HAc research results proved that chitosan oligomers had weak or no
12
Control antimicrobial activity although chitosan oligomers were well
CS0 water-soluble (Jeon et al., 2001; No et al., 2002). Moreover,
9 CS1 Park, Je, Byun, Moon, and Kim (2004) reported that 75%
CS2
Q/J

CS3
deacetylated chitosan and its degraded products showed the
CS4 highest antimicrobial activity as compared with 90 and 50%
6
CS5 deacetylated chitosan and their degraded products in medium
CS6 of pH 5.5, suggesting that the antimicrobial activity of chitosan
3 is not proportional to its DD value. In addition, the quaternary
ammonium salt of chitosan had stronger antimicrobial activity
0 against these three microorganisms in weak basic conditions
than in weak acidic conditions (Qin, Xiao, Li, Fang, Liu and
200 300 400 500 600 700 800
t/min
Chen, 2004), which coincided well with the regulation of
general quaternary ammonium disinfectants (Gu & Zhang,
Fig. 9. Influence of chitosan with different Mw on the growth of E. coli. 1990). Thus, the mode of polycation for antimicrobial activity
of chitosan in acidic media should be further discussed.
Some reported experiments confirmed that the dissolved
3.4. Discussion water-insoluble chitosan increased the permeability of cell
membrane, and ultimately disrupted bacterial cell membranes
It has been postulated that the antimicrobial action of with the release of cellular contents (Helander, Nurmiaho--
chitosan occurs as a result of several mechanisms. Chitosan Lassila, Ahvenainen, Rhoades, & Roller, 2001; Liu et al,
exhibited antibacterial activity only in an acidic medium, 2004). Our research result revealed that the dissolved water-
which was usually attributed to the poor solubility of chitosan insoluble chitosan exhibited the antimicrobial effect, whereas
above pH 6.5 and more positively charged polycation with water-soluble chitosan itself had no significant antimicrobial
stronger affinity for cells (Rabea et al., 2003). We found that effect against both bacteria and yeast. Thus, it is conceivable
water-soluble chitosan promoted the growth of C. albicans that chitosan molecules have the ability to interact with
even in acidic media whereas dissolved water-insoluble bacterial surface compounds, and is absorbed on surface of the
chitosan exhibited inhibitory effect. Actually, the theoretical cells. However, physiological pH in the cell is around neutral,
likelihood of strong interaction between microbial proteins and so water-insoluble chitosan molecules can precipitate, and
chitosan at very acidic pH values is low. This was proven by stack on the microbial cell surface, thereby forming an
Strand’s experiments. The adsorption of chitosans to E. coli impervious layer around the cell and blocking the channels,
cells increased strongly with increasing pH. Despite their low which are crucial for living cells. Such a layer can be expected
charge density, chitosans with lower DD were adsorbed in to prevent the transport of essential solutes and may also
higher amounts and reversed the cell surface charge most destabilize the cell wall beyond repair thereby causing severe
effectively. Chitosan with low molecular weight was adsorbed leakage of cell constituents and ultimately cell death (Ralston,
Tracey, & Wrench, 1964). Rhoades & Roller (2000) reported
that mild hydrolysis of chitosan enhanced its inhibitory activity
against some species of spoilage yeasts grown in complex
30 Control media, whereas highly degraded chitosan oligomers showed no
2.8g/L CS6 antimicrobial activity, which was also found by other authors
2.8g/L HCS3 (Knill, Kennedy, Mistry, Miraftab, Smart and Groocock, 2004;
48 h No et al., 2002). When the chitosan molecules are too large, the
20 chitosan layer may be not very compact. Contrarily, chitosan
12.5 h
with lower molecular weight is more difficult to precipitate and
Q /J

form the layer. The water-soluble chitosan could not form such
a layer so that water-soluble chitosan had no antimicrobial
10
activity. Interestingly, all water-soluble chitosan and chitooli-
15 h gomer as alkaline substances promoted growth of C. albicans
although the optimal pH for growth of C. albicans is 5.5.
A possible reason is that C. albicans’ cell wall contains chitin
(Maoz, & Neeman, 2000), which has the structural affinity with
0
E.coli S. aureus GlcN and GlcNAc residues. This also proved that the water-
C. albicans
soluble chitosan has low cell-toxicity. Many papers have
Fig. 10. Effect of chitooligomers on growth of the microorganisms. reported that water-soluble chitosan had better physiological
 C. Qin et al. / Carbohydrate Polymers 63 (2006) 367–374 373

activity such as antitumor activity and immuno-enhancing Khor, E., & Lim, L. Y. (2003). Implantable applications of chitin and chitosan.
effects in vivo (Jeon & Kim, 2002; Qin, Du, Xiao & Zhan, Biomaterials, 24, 2339–2349.
Knill, C. J., Kennedy, J. F., Mistry, J., Miraftab, M., Smart, G., Groocock, M. R.,
2002; Suzuki, 1996). et al. (2004). Alginate fibres modified with unhydrolysed and hydrolysed
chitosans for wound dressings. Carbohydrate Polymers, 55, 65–76.
Koide, S. S. (1998). Chitin–chitosan, properties, benefits and risks. Nutrition
4. Conclusions Research, 18, 1091–1101.
Kubota, N., Tatsumoto, N., Sano, T., & Toya, K. (2000). A simple preparation
The water-soluble half-acetylated chitosans and chitooligo- of half N-acetylated chitosan highly soluble in water and aqueous organic
mers had no significant antimicrobial activity against these solvents. Carbohydrate Research, 324, 268–274.
Kumar, M. N. V. R., Muzzarelli, R. A. A., Muzzarelli, C., Sashiwa, H., &
microorganisms. Moreover, water-soluble chitosan and chitoo-
Domb, A. J. (2004). Chitosan chemistry and pharmaceutical perspectives.
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insoluble chitosan in acidic medium exhibited inhibitory effect Kurita, K., Sannan, T., & Iwakura, Y. (1977). Evidence for formation of block
against the three tested microorganisms. The water-insoluble and random copolymers of N-acetyl-D-glucosamine and D-glucosamine by
chitosans with Mw around 5!104 in these tested chitosan samples heterogeneous and homogeneous hydrolyses. Makromolekulare Chemie,
178, 3197–3202.
exhibited the best antimicrobial action. The results suggest that
Lin, X. Y., Liu, Y., Liu, P., Qu, S. S., & Yu, Z. N. (2004). Microcalorimetric
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Liu, H., Du, Y. M., Wang, X. H., & Sun, L. P. (2004). Chitosan kills bacteria
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Maoz, M., & Neeman, I. (2000). Effect of inula viscose extract on chitin
This work was supported by the Natural Science Foundation synthesis in dermatophytes and Candida albicans. Journal of Ethnophar-
of China (20472066), and the Science and Technology macology, 71, 479–482.
Foundation of Hubei Province, China (2004AA101C59). No, H. K., Lee, S. H., Park, N. Y., & Meyers, S. P. (2003). Comparison of
physicochemical, binding, and antibacterial properties of chitosans
prepared without and with deproteinization process. Journal of Agricultural
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