JFS M: Food Microbiology and Safety

Chitosan Polymer as Bioactive Coating and

Film against Aspergillus niger Contamination
ISSAM SEBTI, ADELE MAR
EBTI TIAL-GROS, ADELE CARNET-PANTIEZ, STEP
ARTIAL HANE GRELIER, AND V ER
TEPHANE ONIQ
ERONIQUE COMA
ONIQUE

ABSTRA
ABSTRACT CT
CT:: The inhibitor
inhibitoryy activity of chitosan-based edible coatings and films was assessed against the A spergillus
niger food pathogen and deterioration microorganism. Spore-counting assays showed an almost total inhibition of
A. niger growth when either film-forming solution or film were used at a low concentration of chitosan (0.1% w/v).
Epifluorescence microscopic results showed the action of chitosan on the relative proportion of RNA compared with
DNA. The water vvaporapor per meability ((WVP)
permeability WVP) of chitosan film was rrelativ
elativ ely lo
elatively w compar
low ed with the poor moistur
compared e
moisture
barr ier of some polysacchar
barrier polysaccharideide films
films.. M or
Mor eo
oreo ver
eov er,, a coating with chitosan film on an agar gel, used as a food model,
induced a 30% reduction in water loss. These results showed potential applications of chitosan-based films as
bioactive packaging with properties to limit the food dehydration phenomenon.
Keywor ds: chitosan, antimicr
eywords: obial film or coating, A spergillus niger
antimicrobial niger,, epifluor
epifluorescence
escence

Introduction 1979; Kendra and others 1989; Muzzarelli and others 1990; El Ghaouth

D uring the past several years, reliable methods have been de-
veloped to extend the shelf life of food products and to avoid
health hazards for consumers (Ouattara and others 2000). As men-
tioned by Guilbert and others (1996), the increased consumer de-
mand for high quality and long shelf life has initiated the development
of only mildly preserved products that keep their natural and fresh
appearance as long as possible. An edible coating is a thin film pre-
pared from edible material that acts as a barrier to the external ele-
ments (moisture, oil) and thus protects the product and extends its
shelf life. Interest in the development of biodegradable and edible
packaging is increasing due to environmental concerns and regula-
tions. Moreover, bioactive edible coatings or packaging materials are
one of the innovative food packaging concepts that have been intro-
duced as a response to foodstuffs especially sensitive to pathogen
microorganism growth. To control undesirable microorganisms on
foods, antimicrobial films or coatings could be used in an alternative
way. Antimicrobial agents are incorporated into the packaging, and
M: Food Microbiology & Safety
and others 1991, 1997; Fang and others 1994; Chen and others 1996;
Tsai and others 2000; Coma and others 2002, 2003). This linear binary
heteropolysaccharide is a deacetylated derivative of chitin, which is
one of the most abundant natural polymers widely distributed in living
organisms such as crustaceans, insects, and fungi. It is already a com-
mon ingredient of food in Japan and its official approval is currently
pending in Europe, where it has achieved a major breakthrough in di-
etetics as a fat trap, in reducing the human body’s rate of cholesterol
absorption by 20% to 30%, and as a fiber involved in intestinal transit
time. As mentioned by Begin and Van Calsteren (1999), the positive
charges of chitosan due to the protonated form of amino reactive func-
tional group at the C-3 position, would interfere with the negatively
charged residues of macromolecules at the cell surface, rendering the
membrane leaky. Due to its ability to form active edible or biodegrad-
able films (Arai and others 1968), chitosan coating can be expected to
limit contamination on surface foods.
The main objectives of this study were to assess the potential of

the antimicrobial activity is based on the release of the biocide mole-
cule (Torres and others 1985; Halek and Garg 1989; Siragusa and Dick-
son 1992; Weng and Hotchkiss 1992; Weng and Chen 1997; Padgett and
others 1998; Ouattara and others 2000; Coma and others 2001; Sebti
and others 2002). Another concept is based on edible coatings where
functional groups that have antimicrobial activity are immobilized on
the surface of polymer films (Paik and others 1998; Vermeiren and oth-
ers 1999) or the antimicrobial activity comes from the polymer itself,
directly used as film-forming entities, leading to antimicrobial activity
especially onto food surfaces (Coma and others 2002). Based on this
concept, chitosan, composed of ␤-1,4-linked 2-acetamido-2-deoxyglu-
copyranose and 2 amino-2-deoxy-glucopyranose, is a high-molecular-
weight cationic polysaccharide that exhibits antibacterial and antifun-
gal activity as well as film-forming properties (Allan and Hadwiger
MS 20040360 Submitted 6/2/04, Revised 7/1/04, Accepted 9/13/04. Authors
Sebti, Martial-Gros, and Carnet-Pantiez are with Laboratoire de Recher-
che en Génie Industriel Alimentaire, Univ. Claude Bernard Lyon I, IUT A-
Dépt. Génie Biologique, Bourg-en-Bresse, France. Author Grelier and Coma
are with Laboratoire de Chimie des Substances Végétales, CRCM, Univ.
Bordeaux 1, 351 cours de la Libération, 33405 Talence, France. Direct in-
quiries to author Coma (E-mail: v.coma@lcsv.u-bordeaux1.fr).
chitosan coating as an antifungal polymer especially against Aspergil-
lus niger contamination on surface foodstuffs. A. niger was selected
from several fungi because it is well known as an opportunistic human
pathogen and as a microorganism commonly encountered in food
contamination cases. Moreover, some of A. niger strains produce my-
cotoxins such as aflatoxin, which is one of the most potent carcinogens
known to man and has been linked to a wide array of human health
problems. Moreover, A. niger lipase is involved in deterioration of food
products with high lipid content, for example, nuts and oilseeds. The
antimicrobial activity of chitosan coating was 1st assessed on a model
solid medium made from suitable nutrient agar. Then, the effect of
chitosan was investigated in a liquid medium by a spore numeration
assessment and by epifluorescence analysis to determine the influ-
ence of the amino-polysaccharide on spore germination ability. Finally,
vapor water barrier properties and water sorption isotherms of chito-
san films were evaluated in view of potential industrial applications.
Materials and Methods
Materials
Compounds. Chitosan (deacetylation degree, higher than 90%;

M100 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 2, 2005 © 2005 Institute of Food Technologists
Published on Web 2/3/2005 Further reproduction without permission is prohibited
Antifungal chitosan coating and film . . .
low viscosity ranged between 14 and 100 cP) was furnished by
France Chitine (Marseille, France).
Organisms and maintenance. Aspergillus niger (LCSV collection,
university Bordeaux 1, Bordeaux, France) was stored at –20 °C in
10% glycerol. The 5 d pre-culture of A. niger was performed on Sa-
bouraud agar medium (DIFCO 62176, Fisher Labosi, Elancourt,
France) at 30 °C.
Methods
Preparation of chitosan coatings and films. The film-forming
solution of 0.1% or 1% (w/v) chitosan was obtained by dispersing
chitosan in a 0.5% (v/v) aqueous 1 M acetic acid solution (57022,
Chimie-plus, Denicé, France). The pH was adjusted to 5.4 with 1 M
or 10 M NaOH (40043, Chimie-plus, Denicé, France). The prepara-
tion was filtered through 5.3␮m membrane (Millipore, VWR inter-
national, Strasbourg, France) and degassed using a vacuum pump.
To obtain chitosan films, 1% chitosan solution (w/v) in 1 M aque-
ous acetic acid was then poured in uniform layer of 1mm thickness
onto a polypropylene plate (Grosseron, Saint-Herblain, France). The
optimum conditions for casting were 60 °C for at least 2 h in a drying
oven (45003, Bioblock Scientific, Illkirch, France) under reduced pres-
sure (0.83 atm). The dried films were peeled from the plate and sam-
ples were conditioned at 23 ± 1 °C and 50% ± 5% RH before properties
measurements. A micrometer (Mitutoyo electronic micrometer,
Erichsen, Rueil-Malmaison, France) was used to measure film thick-
ness to an accuracy of 1 ␮m. Ten measurements were made on each
test sample and showed a mean final thickness of about 30 ± 4 ␮m.
Preparation of liquid Sabouraud medium supplemented or
not with chitosan. Sabouraud medium was prepared by mixing 35
g/L glucose and 5 g/mL meat peptone (Fisher Labosi, Elancourt,
France) and 5 g/mL NaCl (24069, Chimie-plus, Denicé, France) in
0.5% (v/v) 1 M acetic acid solution. The pH was adjusted to 5.4 with
1 M NaOH. The preparation was filtered through 5.3-␮m mem-
brane (Millipore, VWR international, Strasbourg, France). Sab-
ouraud medium with chitosan was prepared as follows: 0.1% (w/v)
of chitosan was added to the previous mixture, stirred until the
chitosan was totally solubilized, and filtered as described previous-
ly. The solutions were sterilized for 20 min at 120 °C.
Aspergillus niger spores suspension. The fungal spores were re-
covered from a 5 d Sabouraud culture by pouring 9 mL of sterile phys-
iological water containing 0.1% Tween 80 (SIGMA, St. Quentin Fallavier,
France) on the agar plate surface and by scraping it gently using a
sterile rake to remove spores. The spores were then transferred into
sterile tubes. When necessary, the spore density was evaluated on a
hemocytometer (Malassez, VWR, Strasbourg, France) using an optical
microscope (Zeiss, Axiovert 25, HB050, Germany, magnification 40).
The spore charge was expressed as the number of spores per milliliter
(spores/mL) and was the mean of at least 10 numerations.
Evaluation of chitosan antifungal activity
Agar plate method. Volumes of 0.1 mL or 1 mL of the A. niger sus-
pension, with approximately 100, 500, 1000, 5000, and 10000
spores, were deposited on Sabouraud agar medium dishes and
dried in a flow hood at room temperature for 30 min (Faster BH
2004, Grosseron, St. Herblain, France). On the one hand, 0.1% or 1%
(w/v) chitosan film-forming solutions at a thickness of 1 mm were
deposited on the surface of inoculated Sabouraud agar. Plates were
then dried again in the flow hood at room temperature for 1.5 h. On
the other hand, 1% (w/v) chitosan films were deposited on inocu-
lated Sabouraud agar medium dishes. In both cases, dishes were
incubated at 30 °C. Visual observations and numerations were
performed after different incubation times for 5 to 10 d. Growth
controls were conducted in parallel to ensure that viable organisms
URLs and E-mail addresses are active links at www.ift.org
were present. Spores in control plates were counted, and the per-
centage of inhibition was calculated as follows:
In addition, contamination controls were performed from films
or coatings deposited on a non-inoculated tryptose agar and incu-
bated at 30 °C and 37 °C for 12 d (DIFCO 262200, Fisher Labosi,
Elancourt, France) to test their initial contamination level. Finally,
assays with 1% aqueous 1 M acetic acid solution adjusted to pH 5.4
was conducted to verify potential residual solvent that didn’t exhibit
antifungal activity.
Liquid medium method (spores numeration and epifluores-
cence assays): Tubes of 9 mL of Sabouraud liquid medium with or
without 0.1% (w/v) chitosan were inoculated to obtain a final concen-
tration of approximately 10000 spores/mL. These tubes were then
incubated 24 h at 30 °C. 0.1 mL of each tube was deposited on Sab-
ouraud agar medium dishes. These dishes were dried in a flow hood
at room temperature for 30 min. The plates were then incubated at
30 °C and spore density was determined daily (Mallassez cell).
In parallel to the counting assays, epifluorescence microscopy
analysis on A. niger spores was performed. A volume of 0.5 mL of A.
niger spore suspension was stained with 0.5 mL acridine orange
(0.25 g/L) in darkness for 5 min at room temperature. A few drops
were put on a slide covered with a cover slip and immersion oil
(Coma and others 2002). Spores counts were done at least on 10
random microscopic fields (magnification 40) under an epifluores-
cence microscope (Zeiss). Acridin orange has a high affinity for
nucleic acids and is used as a viable cell stain. When viewed under
ultraviolet light, stained RNA and single-stranded DNA fluoresce
orange, whereas double-stranded DNA appears green. The
amount of green and orange spores was expressed as a percentage,
calculated as follows:
Evaluation of physicochemical properties
of chitosan-based film
M: Food Microbiology & Safety
Water vvapor
apor per meability deter
permeability mination. The water vapor per-
determination.
meability ( WVP) of edible films was evaluated using a modified
AFNOR standardized procedure (NF ISO 2528, 2001). An aluminum
cup containing anhydrous CaCl2 dessicant (assay cup) or nothing
(control cup) was sealed by the tested film (50 cm2 exchange film
area) with paraffin wax at 50 °C. It was placed in an environment of
controlled humidity and temperature (50% ± 5% RH and 23 ± 1 °C).
The water vapor permeability (g.m./24h m2.Pa)was determined
from the weight increase of the cup over time at the steady state of
transfer. All tests were conducted in triplicate.
Sorption isotherms. Aluminum dishes containing about 0.20 g of
dried films were weighted to the nearest 0.0001 g. The dishes were
stored in sealed glass jars containing saturated salt solutions to give
different water activities: KOH, CaCl 2, K2CO3, CuCl2, NaCl, and
KCl, respectively, for 0.10, 0.33, 0.44, 0.68, 0.76, and 0.85 aw at
25 ± 1 °C. Weight of moisture equilibrate samples was determined
after drying all films at 103 °C for 2 h. The experiment was per-
formed in triplicate, and the experimental moisture sorption iso-
therm values were averaged and fitted by the Guggenhein-Ander-
son-DeBoer (GAB) model, usually used to describe water sorption
in hydrophilic food products. The GAB model equation (Velázqu-
ez de la Cruz adn others 2001) is:
Vol. 70, Nr. 2, 2005—JOURNAL OF FOOD SCIENCE M101
 Antifungal chitosan coating and film . . .
Total water desorption rrate
ate
ate.. Total water desorption rate (TWDR)
measurements were evaluated using a modified method devel-
oped by Desobry and Hardy (1993). TWDR of open agar (3% w/w)
and of agar coated with 1% chitosan film were conducted in a cham-
ber of controlled humidity and temperature (50% ± 5% RH and
24 ± 1 °C) for 3 d. The slope of the linear part of the desorption curve
(mass loss compared with time) gives TDWR, expressed as kgwater/
m2/s. From TDWR of open agar gel, Kc was determined using the
following equation (Figure 1a):
C1a was calculated with Mollier diagram from air characteristics of
open agar surface (100% RH and 24 °C) and Cb by surrounding air
characteristics (50% RH and 24 °C).
From TDWR of agar coated with chitosan film, C2a was then de-
termined using the following equation (Figure 1b):
(in the air)
Results and Discussion
A s preliminary experiments, different chitosan contents in film-
forming solutions and films were deposited on a non-inoculat-
ed tryptose agar medium and incubated for 12 d at 30 °C and
37 °C. As a result, no initial microbial contamination was observed.
Moreover, chitosan solvent did not show any antifungal activity
(data not shown).
Chitosan antimicrobial activity was evaluated by the agar plate
method. Two chitosan concentrations (0.1% and 1% [w/v]) were
tested for anti–A. niger activity and data are presented in Table 1.
Control plates, performed in the absence of chitosan, showed after
5 d of incubation, increasing spore amounts as inoculation rate was
increased from 100 to 10000 spores. A linear relationship was ob-
tained: SAf = 53 × SAi (R2 = 0.96), where SAi and SAf were, respec-
tively, initial (before incubation = inoculation rate) and final (after
M: Food Microbiology & Safety
incubation = spores in control plates) spores amounts. Very low
standard deviations were also observed, indicating a good repeat-
ability. In the presence of 0.1% and 1% (w/v) chitosan, A. niger was
completely inhibited, indicating that 0.1% (w/v) chitosan concen-
tration was sufficient to maintain a total fungal growth inhibition
for at least 10 d (Figure 2). Consequently, for the following assays,
only 0.1% (w/v) chitosan film-forming solution was tested.
The same conclusions were obtained when the 1% (w/v) chitosan
Figure 1—Models of surface water evaporation and of
water diffusion in 1% (w/v) chitosan film: (a) open agar gel
and (b) agar gel coated with 1% (w/v) chitosan film
M102 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 2, 2005
film was deposited on an inoculated Sabouraud agar medium (data
not shown). These results showed that the use of chitosan-based
films or coatings offers a great advantage in preventing A. niger
growth on food surfaces. A key feature of chitosan is its positive
charge on the amino group at C-2 position below its pKa (about 6.3).
This creates a polycationic structure, which can be expected to inter-
act with microbial cell surface. El Ghaouth and others (1997) men-
tioned that chitosan applied as a coating reduced lesion develop-
ment caused by Botrytis cinerea and Rhizopus stolonifer and delayed
the ripening process of strawberry, bell pepper, and cucumber fruit.
Chitosan is known to increase cellular leakage, and such alteration
could affect the ability of the undesirable microorganisms to initiate
infection. Planscencia-Jatomea and others (2003) obtained a strong
inhibition of A. niger spore germination (73%) on Czapeck agar me-
dium supplemented with 30 g/L of chitosan. Using scanning elec-
tron microscopy, these authors showed that chitosan produced
spore aggregation and morphological anomalies affecting swelling,
germ tube emergence, and polarization. For these authors, a great
synergism was obtained when low temperature was used while intro-
ducing chitosan. The high level of our inhibition percentage could be
explained also by the low oxygen availability potentially produced
by chitosan coatings or films, leading to a limitation of A. niger spore
germination. In fact, when chitosan was used as an edible film, Jeon
and others (2002) proved a significant reduction in moisture and ox-
ygen exchange. In our conditions, we used a chitosan with a low
molecular weight and a low viscosity that ranged between 20 and 100
cP. This kind of chitosan was used also by Shahidi and others (2001)
and Kamil and others (2002). These authors found that the limitation
of oxidation of lipids in different food products was chitosan concen-
tration–dependent and highest for low chitosan molecular weight
(14 cP). Chitosan films would reduce oxygen exchange associated to
antifungal activity due to the polycationic character, probably ex-
plaining total inhibition of aerobic A. niger strains.
Chitosan antimicrobial activity
evaluated by liquid medium method
Further experiments were conducted to analyze chitosan antifun-
gal activity in liquid medium, and results are presented in Table 2.
Figure 2—Illustration of anti–Aspergillus niger property of
1% (w/v) chitosan coating tested on Sabouraud agar me-
dium previously inoculated with 5000 spores and incubated
10 d at 30 °C
URLs and E-mail addresses are active links at www.ift.org
Antifungal chitosan coating and film . . .

Table 1—Effect of 0.1% and 1% (w/v) chitosan film form- Table 2—Effect of chitosan incorporation at 0.1% (w/v) in
ing solution on Aspergillus niger previously inoculated on Sabouraud liquid medium on Aspergillus niger growth for
Sabouraud agar medium at different rates from 100 to different spore inoculation rates after 24 h incubation at 30 °Ca
10000 spores and incubated 5 d at 30 °C a
Inoculation rate Control plates
Chitosan film-forming solution (spores/mL) (spores/mL) Inhibition (%)
Inoculation rate
Inhibition (%) 100 162245 ± 7b 95 ± 1b
(spores per Control plates
500 122361 ± 6b 91 ± 1b
Petri dish) (spores/mL) 0.1% (w/v) 1% (w/v) 1000 61567 ± 8b 100 ± 0c
100 8738 ± 4b 100 ± 0c 100 ± 0c 5000 25533 ± 4b 100 ± 0c
500 30700 ± 7b 100 ± 0c 100 ± 0c 10000 88167 ± 4b 100 ± 0c
1000 123583 ± 4b 100 ± 0c 100 ± 0c a Volumes of 0.1 mL were spread out on Sabouraud agar medium exempt from
5000 206917 ± 5b 100 ± 0c 100 ± 0c chitosan and were incubated 5 d at 30 °C. Values, followed by their standard
10000 551833 ± 6b 100 ± 0c 100 ± 0c deviation, are means of 3 experiments.
b Values are statistically different (P < 0.05).
a Values represent the means and standard deviations from at least 3 c Values are not statistically different (P < 0.05).
experiments.
b Values are statistically different (P < 0.05).
c Values are not statistically different (P < 0.05).

induce consequently high WVP. The moisture sorption isotherm of chi-

Spore amounts in control plates decreased as inoculation rate in-
creased. This result, verified at least 3 times, could probably be ex-
plained by a competition between microorganisms for the substrate.
As previously obtained in solid medium, 0.1% (w/v) chitosan inhibito-
ry activity was very high and close to 90% to 100%. To improve under-
standing of the effect of chitosan on the physiology of the fungal
strain, spore numeration was performed daily and was completed, in
parallel, by epifluorescence analysis on spores after staining with acri-
din orange. Results are presented in Figure 3. In chitosan-free medium,
a typical growth curve was observed. The epifluorescence analysis
showed a high percentage of green spores during the lag phase (about
20 h). The exponential phase was characterized by high spores
amount and a significant percentage of orange spores up to 50%. Spore
germination can also be observed (Figure 4a). According to McFeters
and others (1991), the outcome of the acridin orange staining reaction
can predict physiological activity, and it is possible to distinguish lag
and stationary phase on the basis of color. After staining with acridin
orange and when viewed under ultraviolet light, high amounts of RNA
and single-stranded DNA induce high acridin orange staining with
orange fluorescence, indicating microorganism viability. When chito-
san was incorporated, 100% green cell percentage and no variation in
spores numbers indicated clearly an antifungal effect of the molecule
(Figure 4b). Chitosan adsorption on spore membrane surfaces prob-
ably induced a reduction in nutriment exchange through spores
tosan films was then carried out. Chitosan film moisture sorption in-
creased rapidly at high aw, from 0.5 to 0.85, while increasing slowly or
moderately at intermediate and low aw, suggesting a swelling phenom-
enon as water activity increased (Figure 5). Sorption isotherms of these
chitosan films and the fact that chitosan films were found to be com-
pletely soluble in water may be detrimental to “in-use” chitosan coat-
ings on food products. Accordingly, further experiments were also con-
ducted to confirm moisture barrier properties of chitosan coatings in
conditions close to food applications. An agar gel, used as a high water
activity food model, was coated with chitosan film forming solution. The
TWDRagar was then determined by evaluating water loss during the
chosen storage conditions. Results are presented in Table 3. TWDRagar
obtained from our experiments (3.37.10–6 kg/m2.s) was lower than
those determined by Desobry and Hardy (1993) (about 5.10–6 kg/
m2.s) in the same conditions. This could be due to the experimental ar-

M: Food Microbiology & Safety

membranes. The green spore coloration showed with epifluorescence
analysis also indicated that chitosan probably acts on nucleic acids.
Shahidi and others (1999) mentioned that a binding of chitosan with
DNA and inhibition of mRNA synthesis occurs via chitosan penetrat-
ing the nuclei of the microorganism and interfering with the synthe-
sis of the mRNA and proteins. Figure 3—Aspergillus niger growth (spore concentration
versus time) in the presence or absence of 0.1% (w/v)
chitosan in Sabouraud broth. Inoculation rate was 10000
Evaluation of physicochemical spores/mL, and the incubation temperature was 30 °C for
properties of chitosan-based films several days. Histograms correspond to epifluorescence
microscopic analysis on spores stained with acridin orange.
Moisture barrier properties of chitosan film were evaluated at Values are means of at least 3 experiments.
24 ± 1 °C and 50% ± 5% RH by measuring the WVP. These conditions
reproduced a situation commonly encountered during food storage.
The obtained WVP was (8.94 ± 0.4) 10–7 g.m./ 24 h. m2 Pa. Because of
the hydrophilic character of the chitosan biopolymer, a significant
water vapor transfer was expected. This latter was relatively low com-
pared with poor water vapor barrier of polysaccharides in general. For
example, hydroxy propyl methyl cellulose films exhibited a 4 times
higher WVP in the same conditions (Coma and others 2001; Sebti and
others 2002). According to Fringant and others (1996), the 1st step of
WVP corresponds to the sorption of water molecules on the specific hy- Figure 4—Epifluorescence microscopic photos at J + 8 d of
Aspergillus niger spores in the absence (a) or presence (b)
drophilic groups of the amorphous phase. Afterward, water permeabil- of 0.1% (w/v) chitosan in Sabouraud broth previously inocu-
ity depends on the capacity of the polymer. Strong water vapor sorption lated with 10000 spores/mL and incubated for several days

URLs and E-mail addresses are active links at www.ift.org Vol. 70, Nr. 2, 2005—JOURNAL OF FOOD SCIENCE M103
 Antifungal chitosan coating and film . . .

Table 3—Impact of 1% (w/v) chitosan film, coating 3% agar Acknowledgments

gel, on dehydration phenomenona The authors would like to thank Romain Guillas, Fathi Beladgham,
Agar coated with 1% and Sébastien Fimbeau for their excellent technical assistance.
Open agar gel (w/v) chitosan film
Thickness (␮m) 54.1 References
TWDR (10–6 kg/m2.s) 3.4 2.3 Allan CR, Hadwiger LA. 1979. The fungicidal effect of chitosan on fungi of vary-
C1a or C2a (10–3 kg/m3) 22.0 18.5 ing cell wall composition. Exp Mycol 3:285.
Arai K, Kinumaki T, Fujita T. 1968. Toxicity of chitosan. Bull Tokai Reg Fish Res
a Values are means of 3 experiments. TWDR = total water desorption rate. Lab 56:89–92.
Begin A, Van Calsteren MR. 1999. Antimicrobial films produced from chitosan.
Int J Biol Macromol 26:63–7.
Chen MC, Yeh GHC, Chiang BH. 1996. Antimicrobial and physicochemical prop-
erties of methylcellulose and chitosan films containing a preservative. J Food
rangement because Desobry and Hardy (1993) put their samples in a Proc Pres 20:379–90.
Coma V, Deschamps A, Martial-Gros A. 2003. Bioactive packaging materials from
chamber with circulating air, which increased water evaporation at the edible chitosan polymer. J Food Sci 68(9):2788–92.
gel surface. TWDRfilm was reduced about 30% by the chitosan-based Coma V, Martial-Gros A, Garreau S, Copinet A, Salin F, Deschamps A. 2002. Edible
anti-microbial films based on chitosan matrix. J Food Sci 67:1162–9.
coating. These results showed that chitosan film or coatings would limit Coma V, Sebti I, Deschamps A, Pichavant HF. 2001. Anti-microbial edible packag-
dehydration phenomenon of food products. ing based on cellulosic ethers, fatty acids and nisin incorporation to inhibit
Listeria monocytogenes and Staphylococcus aureus. J Food Prot 64:470–5.
Desobry S, Hardy J. 1993. Modelling of the total water desorption rate from pack-
Conclusions aged moist food. Int J Food Sci Technol 28:347–59.

C hitosan films and coatings offer a great advantage in prevent-
ing A. niger surface growth, even at a very low concentration of
chitosan. In spite of a total chitosan film solubility, the study of bar-
rier properties of chitosan films and coatings tends to prove that
the use of bioactive chitosan-based packagings could limit the food
dehydration phenomenon. The improvement of food shelf life of
real food products should be confirmed on various foods, various
contamination levels, and different microbial physiological states.
Nomenclature
C1a water concentration in air at the surface (kgwater/m3)
C2a water concentration in the air on the outer film surface
(kgwater/m3)
Cb water concentration in surrounding air (kgwater/m3)
e packaging thickness (m)
Kc effective external transport coefficient (m/s)
RH relative humidity
TWDRagar agar total water desorption rate (kgwater/m2.s)
TWDRfilm total water desorption rate of the chitosan film (kgwater/
m2packaging.s)
M: Food Microbiology & Safety
El Ghaouth A, Arul J, Ponnampalam R, Boulet M. 1991. Chitosan coating effect on
storability and quality of fresh strawberries. J Food Sci 56:1618–31.
El Ghaouth A, Arul J, Wilson C, Benhamou N. 1997. Biochemical and cytochemical
aspects of the interactions of chitosan and Botrytis cinerea in bell pepper fruit.
Posthharvest Biol Technol 12:183–94.
Fang SW, Li CF, Shih DYC. 1994. Antifungal activity of chitosan and its preserva-
tive effect on low-sugar candied kumquat. J Food Prot 56:136–40.
Fringant C, Desbriéres J, Milas M, Rinaudo M, Joly C, Eseoubes M. 1996. Charac-
terization of sorbed molecules on neutral and ionic polysaccharides. Int. J.
Biol. Macromol., 18(4):281-6.
Guilbert S, Gontard N, Gorris LGM. 1996. Prolongation of the shelf-life of per-
ishable food products using biodegradable films and coatings. Lebensm Wiss
Technol 29:10–7.
Halek W, Garg A. 1989. Fungal inhibition by a fungicide coupled to an ionomeric
film. J Food Safety 9:215–22.
Jeon YJ, Kamil JYVA, Shahidi F. 2002. Chitosan as an edible film for quality pres-
ervation of Herring and Atlantic cod. J Agric Food Chem 50:5167–78.
Kamil J, Jeon YJ, Shahidi F. 2002. Antioxidative activity of chitosans of different
viscosity in cooked comminuted flesh of herring (Clupea harengus). Food Chem
79:69–77.
Kendra DK, Christian D, Hadwiger LA. 1989. Chitosan oligomers from Fusarium
solani/pea interactions, chitinase/␤-glucanase digestion of sporelings and
from fungal wall chitin actively inhibit fungal growth and enhance disease
resistance. Physiol Mol Plant P 35:215–30.
McFeters, GA, Singh A, Byun S, Callis PR, Williams S. 1991. Acridin orange stain-
ing reaction as an index of physiological activity in Escherichia coli. J Micro-
biol Meth 13:87–97.
Muzzarelli R, Tarsi R, Filippini O, Giovanetti E, Biagini G, Varaldo PR. 1990.
Antimicrobial properties of N-carboxybutyl chitosan. Antimicrob Agents
Chemother 2019–23.
Ouattara B, Simard RE, Piette G, Bégin A, Holley RA. 2000. Inhibition of surface
spoilage bacteria in processed meats by application of antimicrobial films
prepared with chitosan. Int J Food Microbiol 62:139–48.
Padgett T, Han IY, Dawson PL. 1998. Incorporation of food-grade antimicrobial
compounds into biodegradable packaging films. J Food Prot 61:1130–1335.

Paik JS, Dhanasekharan M, Kelly MJ. 1998. Antimicrobial activity of UV-irradi-

Figure 5—Water sorption isotherms of 1% (w/v) chitosam
film. Experimental moisture sorption isotherm values
(means of 3 experiments) were averaged and fitted by the
Guggenhein-Anderson-DeBoer (GAB) model.
ated nylon film for packaging applications. Packag Technol Sci 11:179–87.
Planscencia-Jatomea M, Viniegra G, Olayo R, Castillo-Ortega MM, Shirai K. 2003.
Effect of Chitosan and temperature on spore germination of Aspergillus niger.
Macromol Biosci 3:582–6.
Sebti I, Pichavant F, Coma V. 2002. Edible bioactive fatty acid-cellulosic deriva-
tives composites used to food packaging applications. J Agric Food Chem
50:4290–4.
Shahidi F, Arackchi UJK, Jeon YJ. 1999. Food applications of chitin and chitosans.
Trends Food Sci Technol 10:37–51.
Shahidi F, Kamil J, Jeon YJ, Kim SK. 2001. Antioxidant role of chitosan in a cooked
cod (Gadus moghua) model system. J Food Lipids 9:57–64.
Siragusa GA, Dickson JS. 1992. Inhibition of Listeria monocytogenes on beef
tissue by application of organic acids immobilized in a calcium alginate gel. J
Food Sci 57:293–6.
Torres JA, Motoki M, Karel M. 1985. Microbial stabilization of intermediate
moisture food surfaces. I- Control of surface preservative concentration. J Food
Prot Preserv 9:75–92.
Tsai GJ, Wu ZY, Su WH. 2000. Antibacterial activity of a chitooligosaccharide mix-
ture prepared by cellulase digestion of shrimp chitosan and its application to
milk preservation. J Food Prot 63:747–52.
Velázquez de la Cruz G, Tores JA, Marti-Polo MO. 2001. Temperature effect on the
moisture sorption isotherms for methylcellulose and ethylcellulose films. Jour-
nal of Food Engineering 48:91-4.
Vermeiren L, Devlieghere F, Van Beest M, De Kruijf N, Debevere J. 1999. Devel-
opments in the active packaging of foods. Trends Food Sci Technol 10:77–86.
Weng YM, Chen MJ. 1997. Sorbic anhydride as anti-mycotic additive in polyeth-
ylene food packaging films. Food Sci Technol 13:1–69.
Weng YM, Hotchkiss JH. 1992. Inhibition of surface moulds on cheese by polyeth-
ylene film containing the antimycotic imazalil. J Food Prot 55:367–9.

M104 JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 2, 2005 URLs and E-mail addresses are active links at www.ift.org