Chitosan-Aflatoxins B1, M1 Interaction: A Computational Approach

Abstract

Contamination by aflatoxins affects food; especially cereal grains are affected by aflatoxin AfB1, while AfM1 affects milk. Many efforts have been addressed to face the problem. In a recent study, we have experimentally showed that the biopolymer chitosan is capable to adsorb the AfB1; however, more sensing and capturing studies are required. In this work, the aflatoxins mentioned above and their interaction with chitosan is computationally investigated. Density Functional Theory as implemented in the GAUSSIAN 09 code is employed. The B3LYP functional together with the 6-31g(d) basis set are enough to modelate these interactions. Through a total energy calculation, it was found that the more negative charge is on the oxygen atoms of aflatoxins, being the preferred site to interact with chitosan. According to the adsorption energy, aflatoxins are physisorbed which is confirmed by the simulated IR spectra. Modification onto the HOMO-LUMO gap as well as a little shift on the simulated infrared spectra suggests sensing applications of chitosan for these aflatoxins.

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Chitosan—Aflatoxins B1, M1 interaction: A computational approach
L. A. Juárez-Moralesa, H. Hernández-Cocoletzi*a, E. Chigo-Anota1a, E. Águila-Almanza2a and M. G.
Tenorio-Arvide3b
aFacultad de Ingeniería Química, Benemérita Universidad Autónoma de Puebla Puebla, México;
bDepartamento de Investigación en Ciencias Agrícolas, Instituto de Ciencias, Benemérita Universidad
Autónoma de Puebla, Puebla, México
Abstract: Contamination by aflatoxins affects food; especially cereal grains are affected by aflatoxin AfB1,
while AfM1 affects milk. Many efforts have been addressed to face the problem. In a recent study, we have
experimentally showed that the biopolymer chitosan is capable to adsorb the AfB1; however, more sensing and
capturing studies are required. In this work, the aflatoxins mentioned above and their interaction with chitosan is
computationally investigated. Density Functional Theory as implemented in the GAUSSIAN 09 code is employed. The
B3LYP functional together with the 6-31g(d) basis set are enough to modelate these interactions. Through a total energy
calculation, it was found that the more negative charge is on the oxygen atoms of aflatoxins, being the preferred site to
interact with chitosan. According to the adsorption energy, aflatoxins are physisorbed which is confirmed by the simulated
IR spectra. Modification onto the HOMO-LUMO gap as well as a little shift on the simulated infrared spectra suggests
sensing applications of chitosan for these aflatoxins.
Keywords: Chitosan; sensing; adsorption; micotoxins; Density Functional Theory, conductivity, infrared spectra.
1. INTRODUCTION
Among the known toxins, aflatoxins belong to the more
dangerous group, their impact on humans and on animals are
very important. Depending on the exposition time they may
be carcinogenic, hepatotoxic, and teratogenic [1]. In the
world, at least 25% of the grains for food and feed are
contaminated with these substances, mainly with the
aflatoxins B1, and G1 (AfB1, AfG1, respectively) [2]; milk and
their derivatives are usually contaminated with the aflatoxin
M1 (AfM1) [3]. Aspergillus flavus and Aspergillus parasiticus
fungi produce these aflatoxins [4]. A reduction of the adverse
effects of these aflatoxins can be reached through their
isolation or their destruction. There are different approaches
to face the problem; for instance, appropriate crop stocking,
early harvest, and insect control are useful. Physical
separation of contaminated grains as well as hot and radiation
are also used. Biological degradation with enzymes and
genetically modified plants has shown efficiency [5]; the
nixtamilization process for grains is also considered a good
option [4]. Chemical methods that involve acids, aldehydes,
and gases have been traditionally used [6]. The former has led
to the fungus and/or aflatoxins more resistant, requiring larger
amounts or concentrations of chemicals, giving rise to some
damage to the grain and eventually to humans and animals,
including the environment. Aflatoxins are very dangerous
contaminants so that the European Union has established
limits for the aflatoxin contents of several foodstuffs, for these
microorganisms.
*Address correspondence to this author at the Facultad de Ingeniería
Química, Benemérita Universidad Autónoma de Puebla, C. P.: 72501,
Puebla, México; Tel/Fax: ++52-222-229-5500 ext. 7253, +52-222-229-5500
ext.7254; E-mails: heribert@ifuap.buap.mx, heribert@sirio.ifuap.buap.mx
Natural adsorbents like to be a viable solution. Volcanic
soils [7], some bentonites [8] and grape bagasse (pulp and
shell) [9] have shown to be efficient in adsorbing mainly
aflatoxin B1, reaching efficiencies as 4.73 ± 0.77 mg mg-1.
Commercial bentonites and activated carbon have been also
used [10]. Experimentally and computationally has been
shown that kaolinite, illite, and smectite clays have adsorption
affinity on the AfB1 [11].There is an increasing interest on the
usage of antifungal compounds obtained from natural
resources such as chitosan, mainly obtained from shrimp
exoskeletons. This biopolymer is non-toxic, biodegradable,
and biocompatible and it has low production cost. It has been
shown recently that synthetic chitosan is able to inhibit the
growth of both the AfB1 and the fungus that produces it [2].
Sequestering AfB1 is also possible by using natural chitosan
with efficiencies higher than that obtained with bentonites,
grape bagasse and volcanic soils [12].
There exists a variety of products consumed by humans
which are contaminated with B aflatoxins, among them are
chilli, peanut and rice [13]. Aflatoxins M1 and M2 have been
found in various diary commercial products worldwide [14].
Recently it was detected in Turkysh raw milk in high
quantities [15]. Sensing toxins is also a challenge. Dynamic
light scattering has been used to detect aflatoxin M1 in milk
[16], chemiluminescence assays have been developed to
detect aflatoxin B1 in corn samples [17]; more recently, a
reduction in the content of aflatoxin G1 in pistachio with high
efficiency was proposed [18]. Many of the available devices
for sensing use high cost precursors or they are not practical
for using. Easier techniques and low cost methodologies are
always desirable. The design of devices that meet these roles
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2 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
may begin with molecular modeling. It is useful to understand
chemical behaviors and to predict new systems, among other
applications. The interaction as well as some chemical and
physical changes may give us insights on the sensing
properties of a material. This may be the case of chitosan;
shifts on its infrared spectra and/or on the gap may suggest its
possible usage as sensor of aflatoxins among other toxic
substances. As an example, this biopolymer has been used to
develop a bioassay for the detection and monitoring of
Ochratoxin A [19].
The aim of this work is to study theoretically the
interaction of aflatoxins B1 and M1 with chitosan. Data like
preferred interaction site and adsorption energy as well as
distribution charge are reported. The
chemisorption/physisorption is demonstrated using simulated
IR spectra together with the adsorption energy. The main
distances like the interaction and the bonds are also examined.
2. SIMULATION MODELS AND METHODS
First principles total energy calculations to study the
interaction of aflatoxins B1 and M1 (C17H12O6 and C17H12O7,
respectively) with chitosan (C6H11NO4) were realized.
Chitosan is a biopolymer that exists of D-glucosamine (GlcN,
about 80%) and GlcNAc (about 20%) units obtained through
deacetylation of chitin using hot alkali [20]. The presence of
amine groups in the polimeric chain confers chitosan basic
like behaviour favoring the formation of polielectrolites; the
quelation, the antioxidant and the adsorption capacity, as well
as filmogenic effect, among others, is also provided by this
group [21], [22]; the calculations have been done using the
monomeric unit shown in Figure 1. The AfB1 is composed by
a dihydrofuran group with two oxygen atoms, one for each
hydrofuran (O1 and O2), a coumarin group with the oxygens
O3, O4, O5 and a cyclopentenone with one oxygen atom (O6).
The AfM1 is the same as the AfB1, but with an oxygen atom
(O7) in the middle of the dihydrofuran. From O1 to O7 there
exists free charge, which is responsible of the aflatoxins
reactivity. The rest of the structure is hermetically bonded, no-
free electrons exist which difficult breaking the bonds. Figure
1 contains the 2 aflatoxins.
Individually, aflatoxins and chitosan were structurally
optimized; the obtention of non-negative frequencies ensured
the stability of the systems. The GAUSSIAN 09 package [23]
with the B3LYP [24] hybrid functional and 6-31g(d) basis set
were employed. The M06 [25] and HSEh1PBE [26]
functionals and 6-311g(d) base were also tested; the negative
charge (Q= −1) and Multiplicity (=2ST+1) of 2 gave the
lowest total energy. The main results of these calculations are
shown in Table 1. The interaction was studied within the
lowest total energy atomic array. To do that, six possible sites
for AfB1 and seven for AfM1 were considered, as indicated in
Figure 1.
Total energy calculations for the chitosan-aflatoxins
systems were developed in order to find the preferred site of
interaction; to do that, the amine group was set to interact with
O1 to O7 oxygen atoms. Then, the adsorption energy [Ead =
Eaflatoxin-chitosan – (Eaflatoxin + Echitosan)] was evaluated searching
for the kind of adsorption. Simulated infrared spectra were
used to confirm the previous results; the spectra were obtained
using the GausView 5.0.8 software, directly from the
optimized systems. Finally, sensing perspectives were
analyzed within the help of the HOMO-LUMO gap (EHOMO-
LUMO = EHOMO - ELUMO), where the HOMO (highest occupied
molecular orbital) and the LUMO (lowest unoccupied
molecular orbital) are the frontier orbitals; this descriptor can
be associated to the conductivity, parameter experimentally
measured.
Parallel to simulation study, experimental work has been
developed, to know the adsorption capacity of chitosan as
AfB1-sorbent, the isotherm were prepare as follow, chitosan
sample (0.1 mg) was added to 3 mL of AfB1 solutions with
concentrations of 0.0 to 8.0 mg/L, and after 24 h of shaking
the amount of AfB1 adsorbed was determined, using a
UV/visible spectrophotometer. Data were fitted to the
Langmuir equation [12]
3. RESULTS AND DISCUSSION
3.1. Structural and Electronic Properties of Aflatoxins
The aim of this work is to study theoretically the
interaction of the AfB1 and AfM1 aflatoxins with chitosan;
results may give us insights on a possible adsorbent or sensor
for these dangerous molecules. To reach this purpose,
descriptors like polarity, reactivity, and absorption energy for
the ground state are reported. Figure 1 contains the relaxed
structure of the free AfB1, AfM1 and chitosan. The geometry
of the AfB1 coincides with that reported in the literature [27].
To our knowledge, no data exist for AfM1; however, the
former results give us confidence on this optimization. For the
two aflatoxins, the doublet (Multiplicity=2) yields the
molecules to the ground state (Table 1); for all cases non-
negative frequencies were found ensuring the stability of the
molecules. For AfB1, the cyclopentenone, the coumarin and
the inner hydrofuran are in the same plane, the outer
hydrofuran is toward the viewer with an angle of 110° formed
by the O1 and O2 and 115° in the opposite side. Something
similar happens with AfM1. For the two cases, the hydrogen
atoms of the cyclopentenone repel the methoxy group towards
the hydrofuran. The higher C-C bond is 1.56 Å, at the bond
union of the dihydrofuran; the remaining C-C bonds range
between this value and 1.33 Å. The C-O bond belonging to
the coumarin and the cyclopentenone is around 1.22 Å (the
lowest one), while this bond in the rest of the rings ranges
from 1.38 to 1.45 Å. All these parameters as well as the
formed angles are in good accordance to that obtained by
Billes et al. [27] for AfB1, information on the AfM1 has not
been reported to date. These characteristics confer the high
stability of aflatoxins, also confirmed by their cohesive
energy, 5.21, and 5.19 eV/atom for AfB1 and AfM1,
respectively. Concerning monomer of chitosan, its relaxed
configuration is shown in Figure 1a. A pyranose ring and the
amine group at the C2 compose this molecule. The C-C bonds
range from 1.51 to 1.54 Å; the C-O bonds are in the range
1.36-1.46 Å. The C-N bond has the value 1.48 Å.
The distribution charge for each aflatoxin is also shown in
Figure 1. For AfB1 this parameter ranges from -0.58 |e| to 0.58
|e|. As expected, the oxygen atoms have the higher negative
charge, the more reactive sites in the molecule, in accordance
to its high electronegativity; the O4 possess the highest charge
-0.58 |e|, almost twice of that reported by Deng et al [28]. In the
AfM1 the distribution charge extends from -0.67 |e| to 0.67 |e|,
higher values than the former. For this case, the highest charge

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 3
was found on the O7 atom. The two molecules are highly polar
justified by their dipole moment, 10.72 D for AfB1, and 10.17
D for AfM1. This feature is of high importance because it
suggests that the molecules can be solubilized in polar solvents,
essential for their isolation studies. Figure 1 also contains the
distribution charge of chitosan; the more negative value relays
on the O (-0.76 |e|) attached to the C4, which connects to the
next monomer. The N atom has a very near charge, -0.74 |e|; it
is worthwhile to mention that the amine confers chitosan
properties like filmogenic effects, adsorption capacity and
antifungal properties, among others. Its dipole moment (5.46 D)
is lower compared with that of aflatoxins but enough to be
soluble for example in acetic acid, as it is well known.
3.2. Aflatoxins—Chitosan interactions
In Figure 2 the ground state for each aflatoxin-chitosan
system is shown, corresponding to the lowest total energy
configuration; after the interaction, the aflatoxin geometry is
essentially the same, compared with the isolated aflatoxins, i. e.
chitosan does not modifies their structure. The relaxation in the
AfB1–chitosan (Figure 2a) system makes one H atom of the
amine point to the AfB1 near O6 atom at a relaxed distance of
1.67 Å, higher than that of isolated O-H bond (0.96 Å) which is
insufficient to form a bond; the other H atom is pointing far
away, being the amine group in the same plane as the
cyclopentenone. The presence of the AfB1 is reflected on a
reduction on the C-N bond of the amine from 1.48 to 1.29 Å.
Concerning the AfM1, chitosan is relaxed almost parallel to the
coumarin-cyclopentenone plane (Figure 2b), being the shortest
distance 1.81 Å, between the H atom of O7 and the O4 atom of
chitosan; amine group is not playing a significant role in the
interaction. This relaxed distance is not enough to form a bond
between the AfM1 and chitosan, as in the former case a
physisorption process is expected.
The distribution charge for the AfB1–chitosan system
ranges from -0.72 |e| to 0.72 |e|, the more negative value (-0.72
|e|) corresponds to the N atom (Figure 2a); a slight reduction on
the N charge is found, reflected in an increase on the O5 and O6
negative charge of the aflatoxin. The redistribution charge on
chitosan also increases the charge on the H atoms of amine,
giving rise to an increase on the dipole moment (from 5.46 to
15.09 D) respect the pristine chitosan; the rest of the charge is
essentially the same. The AfM1-chitosan total dipole moment is
higher (by 1.3 D) than that of pristine chitosan, associated to a
redistribution charge on both AfM1 and chitosan. As mentioned
before, these high values on the dipole moment favor the
dispersion in polar solvents, this means that when big quantities
of aflatoxins are together they will not interact themselves,
facilitating eventually the desorption from chitosan.
The presence of aflatoxins also modifies the HOMO-
LUMO gap of chitosan. This parameter for the isolated state has
the value 0.40 eV; when interacts with the AfB1 it is reduced to
0.1 eV. When the AfM1 takes place in the interaction the
HOMO-LUMO gap is also reduced to 0.1 eV. It is well known
that these changes are reflected on a shifting on the conductivity
of the systems, which can be easily measured [29]. Then, it is
proposed that chitosan could be used as a sensing material for
the AfB1 and AfM1 aflatoxins. In this sense, recently was
reported an amperometric immunosensor for simple and
sensitive determination of AfB1 [30].
3.3. Total and adsorption energy
Total and adsorption energies will give us insights on the
kind of adsorption. Among the six possible (O atoms) sites in
the AfB1, chitosan interacts with O6 through the amine group
with the lowest total energy. When the amine is set near O5 and
O4 almost the same energy is obtained, in saturation these
oxygen atoms would interact with the amine as a second
chemical option; the rest of the sites have total energies higher
than 10 meV. For the AfM1 the preferred site of chitosan was
to be the O7, consistent whit the geometry optimization and in
accordance with the shortest distance between these to
molecules. The nearest energy site is the O1 of AfM1. Actually,
the O1 and O7 sites have practically the same total energy;
chitosan will interact equally in these two sites. Like in the
AfB1, the other sites has higher total energy, consequently, are
less probable to occur the adsorption on these sites. Table 1
contains the total energy on each site for all the aflatoxins. The
zero energy corresponds to the lowest total energy taken as the
reference.
The adsorption energy was evaluated in all the sites and for
each case (See Table 2). For the lowest total energy sites the
found values are -14.73 and -14.71 eV for AfB1 and AfM1,
respectively; these are very small energies, confirming that no
bond is formed between the aflatoxins and chitosan. All these
values are in the regime of physisorption (as was proposed
previously [12]), which is consistent with the relatively large
distance between the aflatoxins and the chitosan [31]. This fact
eventually would give rise to a relatively easy desorption, i. e.
the separation of these two molecules would be possible by
using for example infrared radiation. The kind of adsorbent
determines the type of adsorption; a weak electrostatic
attraction is the responsible for the AfB1-kaolinite adsorption
(like in the present report), while moderate electron-donor-
acceptor attraction and a strong calcium-bridging linkage are
involved in the AfB1-illite and AfB1-smectatite adsorption,
respectively [11]. Also, chitosan-based foam has been proposed
for succesfullly screening of wines for ocratoxin A [19].
All the calculations were realized under neutral pH conditions,
which is usually associated with high AfB1 sorption with some
exceptions. On clays, acid pH may correlate with weathering
but weathering (mineral dissolution) is a slow process and pH
can change quickly. Thus they will correlate best in stable
conditions only [1].
3.4. Infrared analysis
Simulated infrared spectrum for chitosan and the two
aflatoxins is shown in Figure 3. For chitosan (Figure 3a) the
stretching of O-H and N-H is found at 3420 and 3255 cm-1
respectively. The signals at 2879 cm-1 corresponds to the
stretching of the C-H of the alkenes. At 1654 cm-1 the stretching
of C=O (characteristic of the amide I), at 1625 cm-1 (the amide
II), at 1570 cm-1 the flexion NH2 (deformation in the CONH
plane), at 1423 cm-1 secondary absorptions of the alkenes CH2
(stretching), at 1375 cm-1 the C-CH amide stretching are also
found. The spectra is complemented by bands at 1315 cm-1
stretching of C-N, at 1157 cm-1 the C-O-C bridge stretching, at
1079 cm-1the C-O-C symmetric stretching in the ring, and the
glycoside stretching C-O-C at 1023, 890, 713 cm-1 [32].
Concerning aflatoxins (Figure 3a), a resonance frequency at
3680 cm-1 for AfM1 was found, associated to the OH group in
the middle of the dihydrofuran; this band does not appear in the
other aflatoxin due the absence of this group. Characteristic

4 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
frequencies in the region of 3000 cm-1 associated to the CH2
group are found; very near values were reported in the literature
for AfB1 [33]. The more pronounced bands are found at 1841,
and 1832 cm-1 for AfB1, and AfM1, respectively. At 1710 cm-1
is found the CO. Around 1635, 1600 and 1525 cm-1 for the two
aflatoxins are also found. For the next, frequencies are divided
into four groups: the O-CH3 in the range 1364-1369, the CH at
1220, C-O-C at 1035 and the H at 902 cm-1. Experimental
results for the AfB1 are in good accordance with the simulated
results, which gives us confidence on the results obtained in this
work.
Also, Figure 4 contains the spectrum of AfB1, and AfM1-
chitosan systems. For the two cases the bands of aflatoxins as
well as the chitosan are contained in the spectrum, without new
bands confirming the physisorption; only a little shift (about
100 cm-1) to lower frequencies is observed. The interaction is
mainly attributed to electrostatic interaction. The shift on the
infrared spectrum can be experimentally measured, as reported
in a device designed for detecting AfB1 [30]. These results
confirm chitosan as a candidate for detecting aflatoxins AfB1
and AfM1, which can be used as a film or a membrane in a
device. Chemical structure of AfB1 and AfM1 is very similar to
that of AfG1, and then it is proposed that a chitosan-based
device can also be used for sensing this aflatoxin.
CONCLUSION
Frist principles total energy calculations to study the
interaction of chitosan with AfB1, and AfM1 afltoxins were
reported in this work. The structural optimization of the AfM1
was reported for the first time. After the interaction, the
chitosan and aflatoxins geometry is essentially the same,
compared with the pristine counteparts. The relaxed distance
between chitosan and aflatoxins is so bigger to induce a bond,
suggesting a physisorption process. In spite of amine group
plays the fundamental role in chitosan; the interaction is
developed through this group only with the AfB1, reducing the
C-N bond of the amine from 1.48 to 1.29 Å. Concerning the
AfM1, chitosan is relaxed almost parallel to the coumarin-
cyclopentenone plane, being the shortest distance 1.81 Å. The
adsorption energy is in the range of physisorption, associated
to a weak van der Waals interaction. The previous results were
confirmed by the simulated infrared spectra were no new
bands appeared. Also, aflatoxins reduce the HOMO-LUMO
gap of chitosan from 0.40 to 0.1 eV; these changes are
associated to a shifting on the conductivity suggesting sensing
applications for chitosan. This assumption was confirmed by
a little shift (about 100 cm-1) to lower frequencies observed in
the infrared spectra.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict
of interest.
ACKNOWLEDGEMENTS
This work was developed under the funds of VIEP-BUAP
and Cuerpo Académico Ingeniería en Materiales (BUAP–CA-
177). We thank the support given by the National Laboratory
Supercomputing Southeast housed at the Benemérita
Universidad Autónoma de Puebla.
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Received: March 20, 2014 Revised: April 16, 2014 Accepted: April 20, 2014

6 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
Table 1. Total energies (a.u.) for aflatoxins obtained with different functional, base, global charge and
multiplicity (M=ST+1; ST=total spin).
Aflatoxin Functional Base Total energy Charge Multiplicity
B1 B3LYP 6-31g(d) -1106.259 0 3
-1106.074 1 2
-1106.373 -1 2
-1106.236 -1 4
-1106.107 -1 6
M06 -1105.593 0 3
-1105.411 1 2
-1105.716 -1 2
6-311g(d) -1105.821 0 3
-1105.631 1 2
-1105.952 -1 2
-1105.821 -1 4
HSEh1PBE 6-311g(d) -1105.191 1 2
-1105.506 -1 2
M1 B3LYP 6-31g(d) -1181.026 0 3
-1180.829 1 2
-1181.589 -1 2

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 7
Table 2. Total relative (eV/atom) and adsorption energies (eV) in each site of interaction of aflatoxin-chitosan
systems.
Aflatoxin site Total relative
energy
Adsorption
energy
B1 1 0.154
2 0.154
3 0.120
4 0.018
5 0.013
6 0 -0.46
M1 1 0.264
2 0.264
3 0.314
4 0 -0.56
5 0 -0.56
6 0.116
7 0.258

8 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
b)
a)
1
2
3
4
5
6
O1
O2
O3
O4
O5
O6
115°
110°
O1
O2
O3
O4

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 9
Figure 1. Relaxed structures and distribution charge of chitosan a) and aflatoxins b) B1, c) M1. Gray: carbon atoms,
red: oxygen atoms, white: H atoms.
c)
O1
O2 O3
O4
O5
O6
O7

10 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
Figure 2. Ground state for each aflatoxin-chitosan system (a) AfB1, and (b) AfM1.
O4
O5
O6
AfB1
a)
O4
Chitosan
1.67 Å
O7
O4
Chitosan
AfM1
b)
1.
81
Å
O1
O3

Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 11
Figure 3. Simulated infrared spectra of a) chitosan and b) aflatoxins AfB1 and AfM1.
3500 3000 2500 2000 1500 1000 500
0
500
1000
1500
2000
2500
3000
3500
Epsilon
Frequency (cm
-1
)
Chitosan
4000 3500 3000 2500 2000 1500 1000 500
0
1000
2000
3000
4000
Epsilon
Frequency (cm
-1
)
AfB
1
AfM
1
a)
b)

12 Journal Name, 2014, Vol. 0, No. 0 Hernández-Cocoletzi et al.
Figure 4. Simulated infrared spectra of AfB1, and AfM1-chitosan systems. Spectrum of chitosan is also included as comparison.
3500 3000 2500 2000 1500 1000 500
0
2000
4000
6000
8000
10000
12000
Epsilon
Frequency (cm
-1
)
Chitosan
Chitosan-AfB
1
3500 3000 2500 2000 1500 1000 500
0
500
1000
1500
2000
2500
3000
3500
Epsilon
Frequency (cm
-1
)
Chitosan
Chitosan-AfM
1
a)
b)