Food Hydrocolloids 29 (2012) 48e56

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Food Hydrocolloids
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Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp
shell waste
M.S. Benhabiles a, R. Salah a, H. Lounici a, N. Drouiche a, c, *, M.F.A. Goosen d, N. Mameri b
a
Laboratoire des biotechnologies Environnementales et génie des procédés BIOGEP, Ecole Nationale Polytechnique, B.P. 182-16200, El Harrach, Algiers, Algeria
b
University of Technology of Compiègne, Département Génie chimique, B.P. 20.509, 60205 Compiègne cedex, France
c
Silicon Technology Development Unit, Department of Environmental Engineering, 2, Bd Frantz Fanon BP140 Alger-7-mervielles Algiers, Algeria
d
Alfaisal University, Riyadh, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: The antimicrobial activities of chito-oligosaccharides against four Gram-positive and seven Gram-
Received 11 October 2011 negative bacteria were compared to chitosan and chitin with an emphasis on the effects of biopolymer
Accepted 14 February 2012 molecular weight (Mv) and degree of deacetylation (DD). Chitin was isolated from shrimp (Parapenaeus
longirostris) shell waste by sequential chemical treatments. Chitosan and its oligomers N-acetyl chito-
Keywords: oligosaccharides and chito-oligosaccharides were prepared by deacetylation and chemical hydrolysis,
Chitin
respectively. Chitin exhibited a bacteriostatic effect on Gram-negative bacteria, Escherichia coli ATCC
Chitosan
25922, Vibrio cholerae, Shigella dysenteriae, and Bacteroides fragilis. Chitosan exhibited a bacteriostatic
Oligomers of chitin and chitosan
Valorization
effect on all bacteria tested, except Salmonella typhimurium. The oligomers exhibited a bactericidal effect
Shrimp on all bacteria tested.
Antibacterial effect Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Srinivasa Gopal, 2012). Recent studies on chitosan depolymeriza-

Much research has focused on chitosan as a source of bioactive
material during past few decades (Khanafari, Marandi, & Sanatei,
2008; Li, Dunn, Grandmaison, & Goosen, 1992; Limam, Selmi,
Sadok, & El-abed, 2011). However, the high molecular weight of
chitosan, which results in poor solubility at neutral pH and its high
solution viscosity, limits its use in the food, cosmetics, agriculture
and health industry (Xia, Liu, Zhang, & Chen, 2011). Chitosan is
natural, non toxic, copolymer of glucosamine and N-acetylglucos-
amine prepared from chitin by deacetylation, which in turn, is
a major component of the shells of crustaceans. It is found
commercially in the waste products of the marine food processing
industry (Khanafari et al., 2008; Limam et al., 2011).
Various chemical modifications have been investigated to try
and improve chitosan’s solubility and thus to increase its range of
applications (Park et al., 2010; Zhang et al., 2010). Considering this
limitation, researchers are now concentrating on conversion of
chitosan into oligosaccharides (Mohan, Ravishankar, Lalitha, &
* Corresponding author. Silicon Technology Development Unit, Department of
Environmental Engineering, 2, Bd Frantz Fanon BP140 Alger-7-mervielles Algiers,
Algeria. Tel.: þ213 21 279880x192; fax: þ213 21 433511.
E-mail address: nadjibdrouiche@yahoo.fr (N. Drouiche).
tion have drawn considerable attention, as the products obtained
are more water-soluble. Beneficial properties of chitosan and its
oligosaccharides include: antitumour (Quan et al., 2009); neuro-
protective (Pangestuti and al., 2010); antifungal and antibacterial
(Fernandes et al., 2008; Wang et al., 2007); and anti-inflammatory
(Yang, Kim, Kim, Kim, & Lee, 2010). In addition chito-oligosaccha-
rides (COS) are very promising compounds for use as natural
antioxidants in biological systems (Fernandes et al., 2010).
The antimicrobial activity of chitin, chitosan and their deriva-
tives against different groups of microorganisms, such as bacteria,
yeast, and fungi, has received considerable attention in recent years
(Khanafari et al., 2008; Li et al., 1992; Limam et al., 2011). Two main
mechanisms have been suggested as the cause of the inhibition of
microbial cells by chitosan. One means is that the polycationic
nature of chitosan interferes with bacterial metabolism by elec-
trostatic stacking at the cell surface of bacteria (Chung et al., 2004;
Je & Kim, 2006). The other is blocking of transcription of RNA from
DNA by adsorption of penetrated chitosan to DNA molecules. In this
mechanism the molecular weight of chitosan must be less than
some critical value (w5000 Da) in order to be able to permeate into
cell (Liu, Yun, Dong, Zhi, & Kang, 2001). The antimicrobial activities
of chitosan are greatly dependent on its physical characteristics,
most notably molecular weight (Mv) and degree of deacetylation
(DD). Chitosan with a higher degree of deacetylation tends to have

0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.02.013
 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56
a higher antimicrobial activity (Aharya et al., 2005). Some authors
have reported that chitosan is more effective than chito-oligosac-
charides (COS) in inhibiting growth of bacteria; for example, water-
insoluble chitosans exhibited higher antimicrobial effect against E.
coli than COS (Qin et al., 2006). Other have reported that an increase
in Mv leads to a decrease in the activity of chitosan; for instance
COS had higher antimicrobial effect against E. coli than water-
insoluble chitosans (Zheng & Zhu, 2003). Furthermore, Jeon, Park,
and Kim (2001) observed that chitosan with Mv of 224 and
1106 kDa possessed weak or no antibacterial activity against S.
typhimurium, compared to chitosan with a Mv ¼ 28 kDa.
In this study, the antimicrobial activities of chito-oligosaccha-
rides against four Gram-positive and seven Gram-negative bacteria
were compared to chitosan and chitin, prepared from shrimp shell
waste (Parapenaeus longirostris), with an emphasis on the effects of
biopolymer molecular weight (Mv) and degree of deacetylation
(DD). The long term aim of this work is to increase the application
of chitosan and chito-oligosaccharides in the food, cosmetics,
agriculture and health industry.
2. Methodology
2.1. Materials
All chemicals used in this study were analytical grade and
purchased from Sigma Chemical Co. (St. LouisMo). Shrimp shells
were obtained from a seafood restaurant. It was confirmed that all
shells were from a single species of shrimp “P. longirostris”.
2.2. Preparation of chitin and chitosan
The shrimp shells were washed under running warm tap water to
remove soluble organics, adherent proteins and other impurities. The
shells were then collected and boiled in water for 1 h to remove the
tissue, followed by drying in an oven (Prolabo, model Volca MC18,
French) at 160  C for 2 h to make them more brittle and to break down
the crystalline structure of chitin (Mukherjee, 2002). At the end, the
dried shells were ground into a fine powder using a standard grinder
(Model KU-2, PredomMesko, SkarzyskoKam., Poland).
2.2.1. Demineralization
Calcium carbonate constitutes the main inorganic component of
the shells. To remove the calcium carbonate, only dilute hydro-
chloric acid was used to prevent hydrolysis of chitin (No et al.,
1995). The hydrochloric acid concentrations ranged from 0.25 to
2 M and the reaction time was varied from 10 to 120 min. The ratio
of dried shells to acid solution used during the extraction of chitin
ranged from 1/10 to 1/30 (w/v). The experiments were carried out
at room temperature under constant stirring of 150 rpm. The
decalcified shells were collected on a 250 mm sieve, washed to
neutrality with tap water, rinsed with deionised water, and then
oven-dried at 80  C overnight. The rate of demineralization was
evaluated by determining ash contents in the solid.
2.2.2. Deproteinization
Similar experimental conditions were applied for the deminer-
alization of dried shells. The sodium hydroxide concentration was
varied from 0.5 to 5 M, the reaction time ranged from 10 to 400 min
and the temperature was varied from 20 to 100  C. At the end of this
process the material was filtrated, washed and dried, as previously
described in the demineralization process. In order to evaluate the
extent of deproteinization, the protein concentration in the
supernatant was determined according to Biuret’s method (Fine,
1935); It remains the most widely used method for protein
determination.
49
2.2.3. Decolouration
The chitin residue was mixed with acetone at a solid/solvent
ration of 1:10 (w/v) for 10 min, filtered, dried for 2 h at room
temperature, followed by bleaching with 0.315% NaOCl for 5 min at
the same solid/solvent ration (No et al., 1995). The decoloured
chitin was washed and filtered as described previously.
2.2.4. Deacetylation
The conversion of chitin to chitosan involved deacetylation
using the process suggested by Kurita et al. (2003). The parameters
employed (i.e. reaction duration, temperature and concentration of
alkaline reagent) were as follows: a suspension of 1 g of chitin in
50 mL of aqueous sodium hydroxide, as deacetylation reagent (50%
by weight) was mixed at fixed temperature (90e100  C) under
constant stirring. After 3e5 h, the solid was filtered, washed with
water and 80% (v/v) alcohol until the filtrate was neutral. Then it
was oven-dried at 80  C overnight.
2.3. Characterization of chitin and chitosan
The ash content (%) (wt/wt) was determined by the AOAC
standard method 942.05 (AOAC, 1995). The protein content was
determined, in three replicates, according to the Kjeldahl procedure
(AOAC, 1990).
The degree of deacetylation (DD) was assessed by the method of
Sabnis and Block (1997) using FTIR spectroscopy. The spectra of
chitosan samples were obtained using an I.R instrument (SHI-
MADZU-8400, Japan) with a frequency range from 4000 to
400 cm1. The DD was calculated using Baxter’s equation (Baxter,
Dillon, Taylor, & Roberts, 1992):
 
A1655 100
DD ¼ 100   (1)
A3450 1:33
where A1655 and A3450 are the absorbance at 1655 cm1 of the
amide-I band as a measure of the N-acetyl group content and
3450 cm1 of the hydroxyl band as an internal standard to correct
for film thickness or for differences in chitosan concentration
powder form. The factor “1.33” denotes the value of the ratio of
A1655/A3450 for fully N-acetylated chitosan. It was assumed that the
value of this ratio was zero for fully deacetylated chitosan and that
there was a rectilinear relationship between the N-acetyl group
content and the absorbance of the amide-I band (Domszy &
Roberts, 2003).
The viscosity-average molecular weight, Mv, was determined
with an Ubbelohde viscosimeter (Technico, ASTM D. 445) at room
temperature. The solvent used for chitin was dimethylacetamide/
LiCl and for chitosan, 0.2 M acetic acid/0.3 M sodium acetate. The
viscosity-average molecular weight (Mv) was obtained from
viscosity equation using MarkeHouwink parameters according to
the equation:
½h ¼ kðMvÞa (2)
where [h] is the intrinsic viscosity and k, a are constants. These
parameters were determined for chitin (k ¼ 0.24 cm3 g1 and
a ¼ 0.69) and chitosan (k ¼ 0.078 cm3 g1 and a ¼ 0.76) (Mirzadeh
et al., 2002).
2.4. Preparation of N-acetyl chito-oligosaccharides & chito-
oligosaccharides
N-acetyl chito-oligosaccharides (NAc-COS) were prepared by
chemical hydrolysis of chitin as follows: 1 g chitin is hydrolyzed by
50 ml 7 N HCl at 70  C during 3 h. Chito-oligosaccharides (COS)
were prepared by chemical hydrolysis of chitosan as follows: 1 g
50 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56
chitosan is hydrolyzed by 50 ml HCl 6.27 N at 56  C during 3 h and ash (20%) on a dry basis (Table 1). Our values were in agree-
(Chang, Lee, & Fu, 2000). ment with those obtained by No et al., (1995). However, it should be
noted that these results are not constant because for crustaceans
2.5. Antibacterial tests quantitative and qualitative changes in chitin may be observed due
to the physiological stage of the organism or seasonal variations,
Minimum inhibitory concentrations are important to confirm and according to the species, (Fernadez-Kim 2004; Seng 1988).
resistance of microorganisms to an antimicrobial agent and also to Thus, an appropriate extraction procedure needed to be devel-
monitor the activity of new antimicrobial agents. The minimal oped to obtain a white chitin product comparable to the commer-
inhibitory concentration (MIC) is generally regarded as the most cial chitins. Demineralization of the dried shrimp shells was
basic laboratory measurement of the activity of an antimicrobial achieved with 1.5 N HCl and 30 min reaction time at ambient
agent against an organism. Antibacterial activities of chitin, chito-
san, N-acetyl chito-oligosaccharides (NAc-COS) and chito-
oligosaccharides (COS) were examined as the inhibitory effects
against the growth of four Gram-positive bacteria: Staphylococcus
aureus ATCC 25923, S. aureus ATCC 43300, Bacillus subtilis and
Bacillus cereus and seven Gram-negative bacteria: Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella
typhimurium, Vibrio cholerae, Shigella dysenteriae, Prevotella mela-
ninogenica and Bacteroides fragilis. All bacteria were supplied by
Algeria Pasteur Institute. 0.1 g of sterile chitin, chitosan, N-acetyl
chito-oligosaccharides (NAc-COS) or chito-oligosaccharides (COS)
was added in 100 ml of cultured bacteria suspension in a flask and
incubated with shaking at 37  C. The inhibitory effect was esti-
mated periodically by measuring turbidity of the cultured medium
at 640 nm using a spectrophotometer UVeVisible mini 1240 SHI-
MADZU. The minimum inhibitory concentration (MIC) was defined
as the lowest concentration of chitin, chitosan, NAc-COS or COS
required to completely inhibit bacterial growth after incubation at
37  C for 72 h (No, Park, Lee, & Meyers, 2002).
The anaerobic bacteria (P. melaninogenica and B. fragilis) were
cultured in anaerobic atmosphere (Jeon & Kim, 2000). For deter-
mination of the minimum inhibitory concentration (MIC) of chitin,
chitosan, N-acetyl chito-oligosaccharides (NAc-COS) and chito-
oligosaccharides (COS), solutions (1% (w/w) in 1% (w/w) acid) of
each substances were added to Muller Hinton agar (supplemented
with blood for anaerobic bacteria) for final chitin, chitosan, NAc-
COS or COS concentrations of 0.1%, 0.08%, 0.05%, 0.03%, 0.01%,
0.006% and 0.003% (w/v).
Plots were made of the optical density (OD) (i.e. Absorbance at
640 nm) versus the culture time for each of the four Gram-positive
bacteria and the seven Gram-negative bacteria tested by the
shaking flask method.
Inhibitory effects against growth due to antibacterial activities
of chitin, chitosan, N-acetyl chito-oligosaccharides (NAc-COS) and
chito-oligosaccharides (COS) would be indicated by a levelling off of
the slopes of the curves.
3. Results and discussion
3.1. Extraction, demineralization and deproteinization of chitin
Chemical analyses showed that shrimp shell consisted of 24%
dry weight chitin. Shrimps can thus be considered a good source of
chitin. The shells contained a relatively high protein content (40%)
Table 1
Characteristics of chitin and chitosan produced.
Carapace brute Chitin Chitosan
% of ash (wt/wt) 20.18 0.22 0.18
% of protein (wt/wt) 40.6 3.14 1.08
Densité réelle 1.34 0.97 0.99
Densité apparente 0.46 0.21 0.19
Fig. 1. (a): Effect of concentration of HCl on extent of demineralization (Ambient
Corps gras, pigments et autres (%) 15.55 0 0
temperature solid/solvent ¼ 1/15 (g/mL) t ¼ 24 h). (b): Effect of reaction times on
Intrinsic viscosity (100 mL/g) / 15.687 0.9884
extent of demineralization, (Ambient temperature [HCl] ¼ 1.5 N Solid/Solvent ¼ 1/15
Molecular weight (Daltons) / 388.00 12.00
(g/mL)), (c): Effect of solids to solvent ratio on extent of demineralization, (Ambient
Degree of deacetylation (%) / 35 80
temperature [HCl] ¼ 1.5 N 1 g of solid t ¼ 1 h).
 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56 51
temperature (Fig. 1a and b). However, results showed that the effect determined to be 12,000 Da with an average DD of 80% (Table 1).
of the various solidesolvent ratios was not significant (Fig. 1c). In The molecular weight of chitin and chitosan depends on the sour-
fact, about 100% of the original ash content decreased at ces and the methods of preparation (Fernandez-Kim, 2004). In
solidesolvent ratio ranging from 1/10 to 1/30 (w/v). It is important a previous study of the crawfish shell waste, for example, by No and
to note that previous studies have reported that a high quality Meyers (1995) an average molecular weight (Mv) for chitin was
grade of chitosan should have less than 1% ash content (No & found ranging from 120,000 to 1,500,000 Da, and for chitosan a Mv
Meyers, 1995). of 46,000 Da. The degree of deacetylation obtained by these
The extent of deproteinization ranged from 40% to 55% (wt/wt) authors ranged from 56 to 99% with an average of 80%. The shrimp
as a result of varying the NaOH concentration, reaction time and shell chitosan produced in the current study resulted in a lower
temperature (Fig. 2aec). At 2 N NaOH the deproteinization levelled molecular weight (Mv) compared to those presented by No and
off at 40%. A similar effect was observed at reactions times greater Meyers (1995); 12,000 compared to 46,000. This difference may
than 2 h, with a maximum in deproteinization of 40%. However, the be explained by different sources (i.e. crab versus shrimp) and
extent of deproteinization levelled off at about 55% at reaction methods of preparation, as noted Fernandez-Kim (2004). The high
temperatures over 55  C. On the other hand, the solid to solvent temperature, dissolved oxygen, and shear stress encountered
ratio appeared to be the most critical parameter. Indeed, the extent during the deacetylation process can cause degradation of chitosan
of deproteinization with NaOH (2 N) over 2 h at a temperature of (Methacanon, Prasitsilp, Pothsree, & Pattaraarchachai, 2003).
45  C increased from 30% at a solidsesolvent ratio of 1/10 (w/v) to Hence, it is difficult to obtain chitosan with a specific degree of
96% with a solidsesolvent ratio of 1/20 (w/v) (Fig. 2d). This yield deacetylation without polymer chain degradation.
(i.e. 96% removal of protein) was the highest value obtained. This
result probably can be explained by the fact that protein is bound by 3.3. Antibacterial activity
covalent bonds to chitin, forming stable complex which made
difficult to obtain a yield of 100%. An almost complete removal of The minimal inhibitory concentrations (MIC) of chitin, chitosan
protein is desirable because it allows for a higher solubility of and their oligosaccharides were determined in order to assess their
chitosan after the deacetylation step. antimicrobial activity. The oligosaccharides NAc-COS and COS
showed lower MIC values (0.003%), compared to chitin and chito-
3.2. Molecular weight and degree of deacetylation of chitin and san, for all bacteria strains tested, indicating their stronger anti-
chitosan microbial activity (Table 2). The minimal inhibitory concentrations
(MIC) of chitin and chitosan depended on the bacterium being
The viscosity-average molecular weight, Mv, of chitin obtained studied. The MIC values of chitin, for example, generally ranged
in this study was determined to be 338,000 Da with a degree of from 0.006% to 0.01%; with the exceptions being P. aeruginosa,
deacetylation (DD) of 35%, whereas the Mv of chitosan was S. typhimurium and P. melaninogenica which required more than
Fig. 2. (a): Effect of concentration of NaOH on extent of deproteinization, Ambient temperature solid/solvent ¼ 1/15 (g/mL) t ¼ 24 h. (b): Effect of reaction time on extent of
deproteinization at room temperature [NaOH] ¼ 2 N solid/solvent ¼ 1/15 (g/mL). (c): Effect of temperature on extent deproteinization [NaOH] ¼ 2 N solid/solvent ¼ 1/15 (g/mL)
t ¼ 2 h. (d): Effect of solidesolvent ratio on deproteinization, [NaOH] ¼ 2 N T ¼ 45  C 1 g of solid t ¼ 2 h.
52 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56
Table 2 OD S.aureus
Determination of minimal inhibitory concentrations (MIC) of chitin, chitosan and
their oligosaccharides to assess their antimicrobial activity.
0.50 OD S.aureus + 0.1% chitin
OD S.aureus + 0.1% chitosan
Bacteria tested M.I.C. (%)
OD S.aureus + 0.1% N acetyl chito-oligosaccharides
Chitin Chitosan N-acetyl chito- chito-
Optical Density (OD) (Absorbance at 640nm)

oligosaccharides oligosaccharides ODS.aureus + 0.1% chito-oligosaccharides

0.40
Escherichia coli 0.01 0.01 0.003 0.003
Pseudomonas aeruginosa > 0.1 0.05 0.003 0.003
Staphylococcus 0.03 0.03 0.003 0.003
aureus ATCC 43300
Staphylococcus 0.03 0.03 0.003 0.003 0.30
aureus ATCC 25923
Salmonella typhimurium > 0.1 > 0.1 0.003 0.003
Bacillus subtilis 0.03 0.01 0.003 0.003
Bacillus cereus 0.03 0.01 0.003 0.003
Vibrio cholerae 0.01 0.01 0.003 0.003 0.20
Shigella dysenteriae 0.01 0.01 0.003 0.003 Time (hrs)
Enterobacter agglomerans 0.01 0.01 0.003 0.003
Prevotella melaninogenica > 0.1 0.01 0.003 0.003
Bacteroides fragilis 0.006 0.006 0.003 0.003
0.10
0.1%. Indeed, P. aeruginosa is problematic as it has intrinsic resis-
tance to several antibiotics and the capability to acquire resistance
0.00
during antibiotic therapy as reported by Beck, Cirtain, Glover,
0 1 2 3 4 5
Felsted, and Safa (1988). The MIC values of chitosan ranged from
0.006% to 0.03% except for P. aeruginosa (0.05%) and the food borne Fig. 4. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
pathogen S. typhimurium (more than 0.1%). The latter was the most oligosaccharides on the growth of Staphylococcus aureus ATCC 43300.
resistant bacteria strain studied.
The natural antibacterial characteristics of chitin, chitosan and
their oligomers, at a concentration of 0.1% (w/v), are depicted in chitosan and their oligomers against S. aureus ATCC 25923 and
Figs. 3e13 where the activity data were evaluated in terms of the S. aureus ATCC 43300, respectively, as measured by the shaking
percentage suppression of colony formation. It can be observed that flask test method. The smaller the OD of the medium, as deter-
tested samples in this study showed inhibitory effects depending mined by the lower absorbance at 640 nm, the higher the anti-
on their molecular size and the type of bacterial (Gram-positive or bacterial activity of the tested material since fewer microbial cells
Gram-negative). were produced. Chitin and chitosan exhibited similar inhibitory
growth activity against the two S. aureus strains throughout the
3.3.1. Gram-positive strains 5e6 h incubation period (Figs. 3and 4). In contrast, the amount of
Figs. 3and 4 show the plots of the optical density (OD) (i.e. bacteria increased rapidly in the control group (S. aureus). The late
Absorbance at 640 nm) versus the culture time for 0.1% (w/v) chitin, exponential phase was reached after 2 h with an OD of 0.38.
Bacteria growth was at slower rate in the experimental groups
containing 0.1% chitin and 0.1% chitosan with the late exponential
phase being reached after 4 h. The maximum OD (i.e. absorbance at
0.50 DO S, typhimurium
640 nm) in the control group was 0.4 (i.e. 1.2  108 cell/mL), but was
OD S, typhimurium + 0.1% chitin only 0.25 (i.e. 0.75  108 cell/mL) for the experiments with chitin
Optical Density (OD)( Absorbance at 640nm)

OD S, typhimurium + 0.1% chitosan and chitosan. This shows the antibacterial activity of chitin and
0.40 OD S, typhimurium + 0.1% N acetyl chito-oligosaccharides
OD S, typhimurium +0.1% chito-oligosaccharides

Optical Density (OD) (Absorbance at 640 nm)

0.50 OD B.subtilis
OD B.subtilis + 0.1% chitin
0.30
0.40 OD B.subtilis + 0.1% chitosan
OD B.subtilis + 0.1% N acetyl chito-oligosaccharides
OD B.subtilis + 0,1% chito-oligosaccharides
0.30
0.20
0.20
0.10
0.10
0.00 0.00
0 1 2 3 4 5 6 0 1 2 3 4 5
Time (hrs) Time (hrs)
Fig. 3. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito- Fig. 5. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Staphylococcus aureus ATCC 25923. oligosaccharides on the growth of Bacillus subtilis.
 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56 53
0.50 DO B.cereus 0.50 OD V.cholerae
Optical Density (OD) (Absorbance at 640 nm)
Optical Density (OD) (Absorbance

DO B.cereus + 0.1% chitin OD V.cholerae + 0.1% chitin

DO B.cereus + 0.1% chitosan OD V.choleraeogawa + 0.1% chitosan
0.40 DO B.cereus + 0.1% N acetyl chito-oligosaccharides 0.40 OD V.cholerae + 0.1% N acetyl chito-oligosaccharides
density at 640 nm)

DO B.cereus + 0.1% chito-oligosaccharides OD V.cholerae + 0.1% chito-oligosaccharides

0.30
0.30
0.20
0.20
0.10
0.10
0.00
0 1 2 3 4 5
0.00
Time (hrs)
0 1 2 3 4 5
Fig. 6. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito- Time (hrs)
oligosaccharides on the growth of Bacillus cereus.
Fig. 8. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Vibrio cholerae.
chitosan. This was consistent with the previous study by
Gerasimenko, Avdienko, Bannikova, Zueva, and Varlamov (2004)
higher at 0.264 (i.e. 0.792  108 cells/mL) and 0.22 (i.e. 0.66  108
who reported the antimicrobial activity of chitosan on S. aureus.
cells/mL) for B. subtilis and B. cereus, respectively. In terms of sup-
They further reported that chitosan with Mv of 12 kDa was more
pressing cell colony formation for B. subtilis and B. cereus, 0.1%
effective in inhibiting cell growth than the chitosan with Mv of
chitosan inhibited about 64% of the cell growth compared to the
27 kDa.
non treated control. This result indicates that Bacillus strains were
In the current study, treatment with the oligomers NAc-COS and
more susceptible to chitosan than chitin atleast at the 0.1%
COS completely inhibited the growth of S. aureus after 2 h of
concentration tested in this study.
incubation (Figs. 3and 4). Similar results were obtained by
The chitosan used in this study had a relatively low Mv of 12 KDa
Fernandes et al. (2008) who reported that 0.5% (w/v) COS with
and a corresponding low viscosity (0.9884 mL/g). No et al. (2002)
Mv < 5 kDa and DD in the range 80e85% required only 1 h to
have shown that the antibacterial activity against Bacillus sp
completely eliminate the initial population of S. aureus.
increased with decreased viscosity of chitosan. We can speculate
Treatment with 0.1% (w/v) chito-oligomers NAc-COS and COS
that this is due to the fact that the smaller chitosan chains are better
completely inhibited the growth of both B. subtilis and B. cereus
able to interact with the microbial cells.
(Figs. 5and 6). After about 3 h the optical density dropped to zero. In
the case of treatments with 0.1% chitin and chitosan, the stationary
3.3.2. Gram-negative strains
phase was reached at 4 to 4.5 h. A maximum optical density (OD) of
The antibacterial activity of 0.1% (w/v) chitin, chitosan and their
0.341 (i.e. 1.023  108 cells/mL) was obtained for control. In contrast
oligomers against pathogenic E. coli is shown in Fig. 7. In the control
the OD was only 0.123 (i.e. 0.369  108 cells/mL) for the 0.1% chi-
group the bacterial numbers increased rapidly with time. The
tosan treated samples, indicating inhibition of cell growth. While
stationary phase was reached after only 2 h of incubation. In
with 0.1% chitin treated samples the maximum OD was slightly
contrast, with 0.1% (w/v) chitin and chitosan it took twice as long
0.50 OD S.dysenteriae
0.50

Optical Density (OD) (Absorbance at 640 nm)

OD S.dysenteriae
OD S.dysenteriae + 0.1% chitin
Optical Density (OD) (Absorbance at 640nm)

OD S.dysenteriae + 0.1% chitin

OD S.dysenteriae + 0.1% chitosan
OD S.dysenteriae + 0.1% chitosan
0.40 OD S.dysenteriae + 0.1% N acetyl chito-oligosaccharides 0.40 OD S.dysenteriae + 0.1% N acetyl chito oligosaccharides
OD S.dysenteriae + 0.1% chito-oligosaccharides OD S.dysenteriae + 0.1% chito oligosaccharides
0.30 0.30
0.20 0.20
0.10 0.10
0.00 0.00
0 1 2 3 4 5 0 1 2 3 4 5
Time (hrs) Time (hrs)
Fig. 7. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito- Fig. 9. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Shigella dysenteriae. oligosaccharides on the growth of Shigella dysenteriae.
54 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56
DO P.melaninogenica
0.50
Optical Density (OD) (Absorbance at 640 nm)

OD B.fragilis
DO P.melaninogenica + 0.1% chitin
OD B.fragilis + 0.1% chitin
Optical Density (OD) (Absorbance at 640

DO P.melaninogenica + 0.1% chitosan

OD B.fragilis + 0.1% chitosan
OD B.fragilis + 0.1% N acetyl chito-oligosaccharides
0.40
OD B.fragilis + 0.1% chito-oligosaccharides
0.30
0.20
0.10
0.00
0 1 2 3
Time (hr)
Fig. 10. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Bacteroides fragilis.
with the stationary phase being reached at 4 h. The maximum OD
for the control group was 0.39 (i.e. 1.17  108 cells/mL), whereas it
was only 0.268 (i.e. 0.804  108 cells/mL) for the chitin and chitosan
treated samples. So it is clear that while chitin and chitosan were
equally inhibitory against E. coli, only 31% of the bacteria could be
killed after 4 h of incubation, compared to the untreated control.
These results were similar to those reported by others that chitosan
could inhibited the growth of E. coli (Liu et al., 2006; Tipparat &
Oraphan, 2008; Zhuang, Liu, Li, Guan, & Yao, 2004). The 0.1%
(w/v) NAc-COS and COS treated samples showed even higher
antimicrobial activity against E. coli with the optical density drop-
ping to zero after only 2 h incubation (Fig. 7). This is similar to the
results observed with S. aureus ATCC 25923 and S. aureus ATCC
43300 (Figs. 3and 4). In a related study, Li, Feng, and Yang (2010)
reported that chitosan oligomers with a molecular weight of
3 kDa exhibited higher antibacterial activity against E. coli than
chitosan with molecular weight of 50 kDa. They employed slightly
higher oligomer concentrations (i.e. 0.5% (w/v)). We can speculate
that the increase in the viscosity of a 0.5% (w/v) chito-oligomer
DO P.melaninogenica + 0.1% N acetyl chito-oligosaccharides
DO P.melaninogenica + 0.1% chito-oligosaccharides
0.5
0.4
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7
Time (hrs)
Fig. 12. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Prevotella melaninogenica.
effectiveness of the 50 KDa chitosan oligomers, as shown earlier by
4 5 6 No et al. (2002).
The antibacterial activities of 0.1% (w/v) chitin, chitosan and
their oligomers against V. cholerae, Shigella disenteriae and B. fragilis
are shown in Figs. 8e10, respectively. It appears that both chitin
and chitosan have a similar inhibitory effect against three bacterial
strains. Compared to the control group, the amount of V. cholerae
bacteria declined by 57% and 63% for chitin and chitosan treated
samples, respectively; 51% and 62% for chitin and chitosan,
respectively, against S. disenteriae; and 80% for both chitin and
chitosan against B. fragilis. However, once again 0.1% (w/v) NAc-COS
and COS showed higher antibacterial activities than native poly-
saccharides and markedly inhibited the growth of all the tested
bacteria. Almost all V. cholerae and B. fragilis were destroyed after
1.5 h and 2 h of incubation, respectively, in the presence of 0.1% (w/
v) chito-oligomers in the culture medium. Although, the required
time for killing all S. disenteriae was 1.5 h and 2.5 h for COS and NAc-
COS respectively, suggesting that COS is a more effective antimi-
crobial agent.
The antibacterial activities of chitin, chitosan and their oligo-
mers against P. aeruginosa ATCC 27853 and P. melaninogenica are
shown in Figs. 11and 12, respectively. As can be seen, the viable
population of the 0.1% (w/v) chitosan treated microorganisms was
less than that of the control throughout the incubation period
0.50 DO S, typhimurium

Optical Density (OD)( Absorbance at 640nm)

OD S, typhimurium + 0.1% chitin
solution, compared to a 0.1% (w/v) solution used in our study,
OD S, typhimurium + 0.1% chitosan
could have been partially responsible for the lower antimicrobial
0.40 OD S, typhimurium + 0.1% N acetyl chito-oligosaccharides
OD S, typhimurium +0.1% chito-oligosaccharides
0.50 OD P.aeruginosa
Optical Density (OD) (Absorbance at

OD P.aeruginosa + 0.1% chitin

0.30
0.40 OD P.aeruginosa + 0.1% chitosan
OD P.aeruginosa + 0.1% N acetyl chito-
oligosaccharides
0.30
640 nm

0.20
0.20
0.10
0.10
0.00
0.00
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6
Time (hr) Time (hrs)
Fig. 11. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito- Fig. 13. Effect of chitin, chitosan, N-acetyl chito-oligosaccharides and chito-
oligosaccharides on the growth of Pseudomonas aeruginosa ATCC 27853. oligosaccharides on the growth of Salmonella typhimurium.
 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56
(0e6 h) which indicated that chitosan could inhibit bacterial
growth at the tested concentration. However, chitin did not inhibit
the growth of both P. aeruginosa ATCC 27853 and P. melaninogenica.
In fact the curves overlapped with the controls (i.e. untreated
cultures). This is in sharp contrast to the results observed with E.
coli (Fig. 7), V. cholerae (Fig. 8), S. disenteriae (Fig. 9) and B. fragilis
(Fig. 10) where chitin and chitosan were equally effective as anti-
microbial agents. This suggests that the gram-negative strains P.
aeruginosa ATCC 27853 and P. melaninogenica are more resistant to
antimicrobial agents. Even chitosan was not as effective as shown
by the slightly higher optical densities (i.e. compare Figs. 11and 12
with Figs. 7e10). In a related study, Jeon et al. (2001) reported that
0.06% (w/v) chitosan more effectively inhibited E. coli than P. aer-
uginosa; suggesting variable sensitivities of different microorgan-
isms to antimicrobial agents.
COS exhibited higher bactericidal properties than NAc-COS
against P. aeruginosa ATCC 27853 and P. melaninogenica (Figs. 11and
12). While COS and NAc-COS completely inhibited the growth of
P. aeruginosa ATCC 27853 after exposure times of 2.5 h and 3.5 h,
respectively (Figs. 9and 10), the required time for complete inhibition
of P. melaninogenica was 4 h and 6 h for COS and NAc-COS respec-
tively. In a related study Bhatt, Kim, Nam, Oh, and Chai (2009)
reported on the antimicrobial activity of water-soluble chitosan
oligomers with different molecular weights ranging from Mv of 1 to
10 kDa and DD from 77 to 82%.
The antibacterial activity of chitin, chitosan and their oligomers
against Gram-negative, food borne pathogen S. typhimurium was
the least effective of all the microorganisms examined (Fig. 13).
There was no difference in the optical absorption at 640 nm
between the 0.1% (w/v) chitin and 0.1% (w/v) chitosan treated
groups and the untreated control. The two polysaccharides showed
no antimicrobial activities, atleast at the concentration employed in
the current study. However, under conditions of our experiment,
0.1% (w/v) NAc-COS and 0.1% (w/v) COS showed effective antibac-
terial activities against S. typhimurium, with COS being the most
effective of the two. While NAc-COS completely inhibited the
growth of S. typhimurium after 6 h of incubation, COS killed all of
the bacteria after only 3 h of incubation. In a similar study, Wu,
Zivanovic, Draughon, Conway, and Sams (2005) reported that
chitinous material extracted from mycelia of Aspergillus niger and
Mucor rouxi, in a concentration of 0.1% (w/v) reduced S. typhimu-
rium DT104 2576 cell counts by 0.5e1.5 logs during a 4 day incu-
bation in tryptic Soy broth at 25  C.
3.3.3. Mode of action of chitin, chitosan and its oligomers as
antimicrobial agents
Based on the results, chitin and chitosan can be characterized as
bacteriostatic rather than bactericidal. Hence, the mechanism of
antibacterial activity of these two later polysaccharides is that it
makes the bacteria flocculate and thus killing it presumably
through lack of nutrients and oxygen (i.e. mass transfer limitation).
However, at tested concentration of 0.1% (w/v) chitin and chitosan
there may have been insufficient polysaccharide to flocculate and
kill all the bacteria in the culture medium. The surviving cells
would have gone on reproducing. The antibacterial activities of
chitosan had exceeded the action of chitin, in particular, against
Gram-negative bacteria strains because chitosan possesses
a number of polycationic amines which can interact with the
negatively charged residues of carbohydrates, lipids and proteins
located on the cell surface of bacteria, which subsequently inhibit
the growth of bacteria (Wu, Zeng, Mo, & Ruan, 2006). In addition, if
the molecular weight of chitosan is low, its polymer chains have
greater flexibility to bind more than one cell. Thus, bridges between
bacterial cells and polymer chains of chitosan are quickly formed,
so that the bacteria are immediately inactivated (Wu et al., 2006).
55
It can be suggested that the considerable antibacterial activity
of chitin is due to the high value of the degree of deacetylation
(DD ¼ 35%) and possibly the quenching step during the extraction
of chitin from shrimp shells. The latter decreases the crystallinity
and increases the flexibility of polymer chains of chitin. In
contrast, NAc-COS and COS can be characterized as bactericidal
agents. In this case, the oligomers molecules are assumed to be
able to pass through the bacterial cell wall, composed of multi-
layers of cross-linked murien, and reach the plasma membrane.
This is supported by the E. coli work of Tsai and Su (1999) who
investigated the antibacterial activity of shrimp chitosan and by
Liu et al. (2006) who assessed the effect of Mv and concentration
of chitosan on antibacterial action. Observation by confocal laser
scanning microscopy confirmed the presence of chitosan oligo-
mers inside E. coli suggesting that oligomers molecules are able to
pass through the bacterial cell wall. In a related effort, Raafat, von
Bargen, Haas, and Sahl (2008) reported on the mode of action of
chitosan treatment of Staphylococcus simulans 22 and S. aureus
SG511 cells. Chitosan exhibited a dose-dependent growth-inhib-
itory effect. A simultaneous permeabilization of the cell
membrane to small cellular components was detected coupled to
a significant membrane depolarization. Furthermore, chitosan
treatment of S. simulans 22 cells did not give rise to cell wall lysis
with the cell membrane remaining intact. Analysis of transcrip-
tional response data of S. aureus SG511 revealed that chitosan
treatment leads to changes in gene expression related to regula-
tion of stress, autolysis and energy metabolism. Raafet et al.
(2008) went on to speculate that a possible mechanism for chi-
tosan’s activity consists of binding of the biopolymer to teichoic
acids (i.e. polysaccharides found within the cell wall of Gram-
positive bacteria), coupled with extraction of membrane lipids
that results in a sequence of events that eventually lead to
bacterial death. A good review of chitosan as an antimicrobial
agent with applications and mode of action is provided by Rabea,
Badawy, Stevens, Smagghe, and Steurbaut (2003).
4. Conclusions
Chitin exhibited a bacteriostatic effect on Gram-negative
bacteria, E. coli ATCC 25922, V. cholerae, S. disenteriae and
B. fragilis. Chitosan exhibited a bacteriostatic effect on all bacteria
tested, except S. typhimurium. The oligomers exhibited a bacteri-
cidal effect on all bacteria tested. Differences in activity were
exhibited between the type and/or molecular weight of the
chitinous material and the bacterial species. Chito-oligomers would
have advantages as new antimicrobial agents due to their higher
activity and since they are also more readily soluble in water than
the native polysaccharides. On a commercial basis N-acetyl chito-
oligosaccharides (NAc-COS) would be preferred since it is
prepared directly from chitin without the need for a deacetylation
step, while chito-oligosaccharides (COS) must be prepared from
chitosan. Both have similar antibacterial activity. Further work is
needed to better understand the mode of action of chitin, chitosan
and its oligomers as antimicrobial agents.
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