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Journal of Polymers and the Environment
https://doi.org/10.1007/s10924-021-02168-5
ORIGINAL PAPER
Intelligent Films from Chitosan and Biohybrids Based on Anthocyanins
and Laponite®: Physicochemical Properties and Food Packaging
Applications
Cristiane Capello1
· Gabriel Coelho Leandro1
· Talita Ribeiro Gagliardi2
· Germán Ayala Valencia1
Accepted: 5 May 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Colorimetric films containing anthocyanins can be used as intelligent food packaging materials. The present research aims
to develop, characterize, and apply chitosan films containing a biohybrid based on anthocyanins from eggplant (Solanum
melongena L.) peel and laponite®. Anthocyanins were adsorbed on the laponite surface to produce biohybrids (BH) and
then used to manufacture edible films by casting method. The BH increased the thickness and reduced the solubility in water
of chitosan films. Furthermore, the residual mass and opacity of films increased with the presence of BH. Chitosan films
containing BH showed change color properties from grey (initial film color) to red or yellow colors when exposed to buffer
solutions with acid and basic pH, respectively. Finally, chitosan films containing BH were used to monitor meat freshness
at different temperatures (− 20, 4, and 20 °C) by means of film color alterations which was correlated with total volatile
basic nitrogen produced in meats during the storage. Based on physicochemical and colorimetric results, the BH based on
anthocyanins and laponite® is an interesting material to manufacture intelligent films for the food packaging sector.
Keywords Biopolymers · Food pigment · PH freshness indicator · Synthetic layered silicate
Introduction
Intelligent materials are a new generation of advanced sys-
tems having the property of change color in response to
modifications in the surrounding environment [1]. These
materials can be used to communicate the status related to
food safety to end-users by detecting alterations in pH, tem-
perature, and freshness in the packed food products during
transportation and storage [2, 3].
Anthocyanins are natural pigments found in flower, fruits,
vegetables, and grain cereals, which change color with alter-
ations in the pH [4, 5]. In this way, anthocyanins show red/
pink, purple/blue, green, and yellow colors at pH 1–3, 4–8,
9–11, and 12–13, respectively [2]. Based on the change color
response, several intelligent films containing anthocyanins
have been used to monitor the freshness in chicken, fish,
meat, pork, and pork [2, 6–8].
Most intelligent films have been manufactured by the
casting method, in this approach, the intelligent films are
obtained after drying the film-forming solution based on
a polymeric solution blended with an acidified anthocya-
nin extract [2, 6, 9–11]. However, in casting method, high
amounts of acidified anthocyanin extract are used, impacting
negatively on the physicochemical properties of the poly-
meric matrix [6].
Eggplant (Solanum melongena L.) is a fruit native of
Southeast Asia, characterized by having peel with a dark
purple color. Eggplant peels have a high anthocyanin
content, unfortunately, these peels are considered agri-
food wastes with no commercial value [5, 12]. Recently,
laponite® (Lap), a nanoclay, has been used to recover antho-
cyanins from agri-food wastes, as well as to improve the
stability of anthocyanins when exposed to high tempera-
tures [13, 14]. In this way, Capello et al. [12] and Lean-
dro et al. [13] adsorbed anthocyanins from eggplant and
jambolan fruit peels, respectively, on an synthetic layered
* Germán Ayala Valencia
g.ayala.valencia@ufsc.br
1
Department of Chemical and Food Engineering, Federal
University of Santa Catarina, Campus João David Ferreira
Lima, Rua Roberto Sampaio Gonzaga, s/n, PO Box 476,
Florianópolis, Santa Catarina CEP: 88040‑970, Brazil
2
Department of Cell Biology, Embryology and Genetics,
Federal University of Santa Catarina, Florianópolis, SC,
Brazil

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silicate (laponite®, Lap). The obtained powders were called
as biohybrids and showed change color properties like that
observed in anthocyanin extracts from the same fruit peels.
Furthermore, the anthocyanins showed high light stability
and improved antioxidant properties when adsorbed on Lap
surface.
BH based on anthocyanins and Lap could be an alter-
native to manufacture intelligent films without impact the
physicochemical properties of biopolymer matrix since that
low amounts of biohybrids could be used to manufacture the
colorimetric indicator films by casting method. To date, no
study has explored the application of BH based on antho-
cyanins and Lap as raw material to manufacture colorimetric
indicator films. Therefore, the objective of this research was
to develop, characterize, and apply a colorimetric indica-
tor film based on chitosan and anthocyanins from eggplant
(Solanum melongena L.) peel previously adsorbed on Lap
surface. In the present research chitosan was chosen as the
macromolecule due to its non-toxicity and good film-form-
ing properties [15, 16].
Materials and Methods
Materials
Chitosan (deacetylated grade 85% and 340,000 g/mol) was
purchased from Polymar industry (Fortaleza, Ceará, Bra-
zil). Laponite® (S-482, BYK, Germany) was used as adsor-
bent. Commercial eggplant (Solanum melongena L.) was
purchased from the local market in Florianópolis, SC, Bra-
zil. Eggplant peel was removed from eggplant fruits using
a sharp knife with thickness around 1 mm as proposed by
Capello et al. [12]. Distilled water, hydrochloric acid (37
wt%, Neon, Brazil), and glacial acetic acid (Sigma Aldrich,
Brazil) were used as solvents.
Production and Characterization of the Biohybrids
The biohybrid was produced using the methodology pro-
posed by Capello et al. [12]. Firstly, an anthocyanin extract
was obtained by mixing 1 g of eggplant peel with 60 mL of
an acidified water solution (100:1 v/v, ­
H2O: HCl 37 wt%,
pH = 1). The resulting solution was stirred under continu-
ous agitation (100 rpm) in a shaker (TE-424, Tecnal, São
Paulo, Brazil) in the absence of light at 35 °C for 80 min.
The obtained anthocyanin extract was cooled to 20 ºC, fil-
tered and then the total anthocyanin content was measured
using a UV–vis spectrophotometer (U-2900, Hitachi) and
buffer solutions with pH 1 (0.025 M potassium chloride
buffer) and 4.5 (0.4 M sodium acetate buffer) [2, 12, 13].
In sequence, Lap was added to the anthocyanin extract in a
proportion of 1 g of Lap per mg of anthocyanin, the resulting
dispersion was mixed by 15 min in absence of light. After
that, a decanted material based on anthocyanins and Lap
was observed, this material was separated, dried, and called
as biohybrid (BH).
Acid treated Lap called as M-Lap was obtained after
precipitation of Lap in acidified water solution (100:1 v/v,
­H2O:HCl, pH = 1) at 20 ºC. M-Lap was produced aiming
to understand the effect of the modified Lap in the chitosan
films [13].
Visual color characterization of M-Lap and BH were
analyzed after immersion in pH buffers (pH = 1 to 13) as
proposed by Capello et al. [12].
Preparation of the Films
Films were produced using a chitosan solution. Chitosan
solution was prepared by dissolving 1 g of chitosan in 100
mL of an acetic acid solution (1%), under constant stirring
(300 rpm) at room temperature for 2 h [2, 9]. In sequence,
the BH was added to the chitosan solution in a mas ratio of
1 g of biohybrid per gram of chitosan. The same mass ratio
was used to manufacture chitosan films containing M-Lap.
Furthermore, chitosan films without BH or M-Lap also were
produced. Chitosan films (F1) and chitosan films contain-
ing M-Lap (F2) were considered as controls and they were
compared with chitosan films containing BH (F3).
All films were produced by casting method, being that the
film-forming solution was poured into Petri dishes (8 cm-
diameter) to obtain a constant weight of dry matter equal to
8 mg/cm2
[17]. Solutions were dried in an oven with forced
air circulation at 20 °C, for 24 h. At last, it was obtained
chitosan films and chitosan films containing M-Lap or BH.
Characterization of the Films
Before characterization, films were conditioned into desic-
cators containing saturated solutions of NaBr (RH = 58%)
at 25 °C for at least seven days. All characterizations were
performed at least in triplicate for each film.
Thickness and Morphology
The average thickness of films was determined using a digi-
tal micrometer (0.001 mm; Mitutoyo), averaging ten differ-
ent positions in each film [18].
The air surface and the internal microstructure after
immersion in liquid nitrogen were analyzed using Scanning
Electron Microscopy (SEM, JSM-6390LV, JEOL, Japan) at
accelerating voltage of 5 kV. Before analysis, films were
fixed on aluminum stubs by carbon tape and then coated

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with a thin gold layer. Three micrographs were taken at ran-
dom sample spots with 100 × magnification [19].
Moisture Content (MC) and Solubility
in Water (SW)
The MC (g of water/100 g of wet material) of the films was
determined by oven drying at 105 °C for 24 h [20].
The SW (%) was calculated for each formulation, 3 sam-
ples with 2.5 cm-diameter and with known initial dry weight
were immersed in 50 mL of distilled water and placed in a
shaker (Tecnal TE/421, Brazil) under continuous stirring
(100 rpm), at 25 °C for 24 h. The material was removed
from distilled water and dried in an oven at 105 °C for 24
h, and finally, SW was calculated as dry weight difference
using Eq. (1):
where ­msi and ­
msf are the initial and final dry masses (g) of
each sample, respectively [21].
Water Contact Angle (WCA) and Water Vapor
Permeability (WVP)
The WCA (º) of the films was measured according to the
ASTM D7334 standard, using an optical tensiometer (Ramé-
Hart 250) [22]. The films were attached to the equipment
and one drop of distilled water, of approximately 4 μL, was
released over the air surface of the films using an automatic
precision syringe. The angle formed between the surface of
the film and the tangent to the drop was calculated using the
DROPimage Advanced software. The water contact angle
measurements were made by analyzing the shape of each
sessile drop after it had been placed over the samples for 5
s. A total of three measurements were taken per film.
The WVP (g⋅mm/m2
⋅h⋅kPa) of the films was determined
gravimetrically [17]. Aluminum capsules (31 ­
cm2
) contain-
ing silica gel (≈ 0% RH) were sealed with the films. The
capsules were placed in a desiccator containing distilled
water (100% RH) and weighed during 96 h. WVP was cal-
culated using the Eq. (2):
where w is the gain of capsules weight during the test, x is
the film thickness (mm), A is the permeation area ­
(m2
), t is
the time (h), and ΔP is the is the partial pressure across the
film (3.169 kPa at 25 °C).
(1)
SW (%) =
[(
msi− msf
)
∕msi
]
∗ 100
(2)
WVP = (w ∗ x)∕(A ∗ t ∗ ΔP)
Chemical Bonds and Crystallinity
Chemical bonds of films were studied using a FTIR spec-
trophotometer (FTIR, Cary 660 Agilent, USA) equipped
with Universal Attenuated Total Reflectance (ATR). FTIR
spectra were performed in the infrared region between 4000
and 400 ­
cm−1
with 20 scans, resolution of 4 ­
cm−1
and using
KBr pellets [12].
Crystalline structure of films was studied using an X-ray
Diffractometer (Rigaku MiniFlex600 DRX, Japan), operat-
ing at 40 kV and 15 mA (CuKα 1 λ = 1.5406 Å radiation).
The XRD diffractograms were recorded at 25 °C between
2θ = 2° and 70° at 20°/min [23]. The interplanar distance d
(nm) was determined from the diffraction angle at the maxi-
mum intensity of the peaks/halos found in the X-ray diffrac-
tograms and using Bragg’s law using the Eq. (3):
where λ is the wavelength (nm), and n is the reflection order
(n = 1, dimensionless).
Thermal Properties
The thermal transitions of films were analyzed by differen-
tial scanning calorimetry (Jade-DSC, Perkin Elmer, USA).
Samples (around 5 mg) were placed in a hermetically sealed
aluminum pan and then heated from -30 °C to 200 °C at a
rate of 10 °C/min, in an inert atmosphere (45 mL/min dry
­N2). An empty pan was used as a reference [17].
The thermal degradation of films was analyzed by Simul-
taneous thermogravimetric and differential thermal analyses
(TGA/DTA STA 449 F3 Jupiter, Netzsch, Germany). Sam-
ples (around 10–12 mg) were heated from 25 °C to 700 °C at
10 °C/min, in an inert atmosphere (60 mL/min dry ­
N2) [12].
Optical Properties
Color of films was analyzed after immersion in pH buffers
(pH = 1 to 13). Images from films were taken for each pH
buffer using a high-resolution digital camera (Nikon AF-
SDXNikkor 18-55mm1:3.5–5.6G VR II, 0.28 m/0.92 ft. ø
52). The images were analyzed using the software ImageJ
v 1.39 (National InstituteHealth, Bethesda, MD, USA)
equipped with the Color Space Converter plugin [2]. Color
measurements were based on CIELab coordinates namely,
L* (represents lightness index), a* (represents the tons from
red to green color), and b* (represents the tons from yellow
to blue color). The color difference (ΔE*) was calculated
by Eq. (4),
(3)
n ∗ 𝜆 = 2 ∗ d ∗ Sin(𝜃)

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where: ΔL* = L*standard – L*film; Δa* = a*standard – a*film; and
Δb* = b*standard – b*film.
Opacity (
Op
) of the films was calculated as the absorb-
ance value at 600 nm divided by film thickness. The Op
value was expressed as ­
A600/mm [24].
Application of the Films Containing
the Biohybrid for monitoring Meat
Freshness
Colorimetric indicator films were tested for freshness moni-
toring of meat. Pieces of fresh round steak were positioned
inside Petri dishes and sealed using a Petri dish cover with
attached colorimetric indicator film (1.5 × 1.5 cm) on below
side. The packed meats were placed in a freezer and a
refrigerator at -20 °C and 4 °C, respectively, as well as in an
artificial-climate incubator (20 °C, 60% RH) [2], being that
the film change color was monitored visually. Additionally,
the total volatile basic nitrogen (TVB-N) values in pieces
of fresh and stored meats were determined as proposed by
Qin et al. [25].
Statistical Analyses
Analysis of variance (ANOVA) and Tukey test of multi-
ple comparisons were accomplished with a significance
level of 5% using the Software Origin (OriginPro, ver-
sion 8.5, OriginLab, US). The results were expressed as
means ± standard deviation.
Results and Discussions
Characterization of the Biohybrid
The biohybrid (BH) displayed a purple color after drying
typical of materials containing anthocyanins (Fig. 1a). The
total anthocyanin content in the BH remained around 14
mg of anthocyanin per gram of Lap, this value was similar
to those informed by Capello et al. [12] (15 mg of antho-
cyanin per gram of Lap) and Leandro et al. [13] (16 mg of
anthocyanin per gram of Lap), who adsorbed anthocyanins
from eggplant and jambolan fruit peels on the Lap surface,
respectively. In contrast, the modified Lap (M-Lap) had
a white color similar to that observed in unmodified Lap
(Fig. 1b) [13].
The color response of the BH as function of pH is shown
in Fig. 1c. The biohybrid displayed red and pink colors at
pH values between 1 and 3 due to the presence of flavylium
(4)
ΔE ∗ =
[
(ΔL ∗)2
+ (Δa ∗)2
+ (Δb ∗)2
]1∕2 cation in anthocyanins. At pH values between 4 and 11, the
BH showed purple and blue colors, in different tonalities,
being associated to the formation of quinoidal bases. Finally,
the BH exhibited yellow tonalities at pH ≥ 12 as a conse-
quence of the anthocyanins oxidation to chalcone form [2,
12, 13].
The M-Lap did not show change color response when
exposed to the buffer solutions with different pH values
(results not shown). This result was due to the absence of
anthocyanins in the modified Lap.
Thickness and Morphology
The incorporation of M-Lap or BH increased the film thick-
ness when compared with chitosan film, suggesting that
those nanomaterials (M-Lap or BH) altered the density of
the samples (Table 1). Alteration in film thickness could be
correlated with the high molecular weight of Lap (2286.9
g/mol) [26, 27]. In contrast, Valencia et al. [18] did not
observe film thickness modifications with the addition of
Lap until a mass ratio of 6% (based on the weight of biopoly-
mer). Discrepancies between the results can be credited to
the chemical modification of Lap during the acid precipita-
tion used to produce M-Lap and BH.
Chitosan films showed surface area and cross-section
images typical of a uniform matrix with compact structure
and no pore formation (Fig. 1d and 1g), suggesting that
chitosan was efficiently dispersed in the solvent during the
production of films. In contrast, the incorporation of M-Lap
or BH altered the surface morphology and cross-section of
the films, in general, these materials had irregular surfaces
due probably to the poor M-Lap and BH dispersion into
de film-forming solution (Fig. 1e, 1f, 1h, and 1i). Irregular
morphologies in chitosan films containing M-Lap or BH
suggest which these materials have phase separation. Previ-
ously, Leandro et al. [13] observed that BH based on antho-
cyanins and Lap are not water soluble.
Moisture Content (MC) and Solubility
in Water (SW)
The presence of M-Lap and BH did not affect the MC in
the films (p > 0.05). In the current research, the MC of the
films remained around 22 ± 1% and they were typical of chi-
tosan-based materials [2]. In contrast, SW values decreased
in films containing M-Lap and BH (Table 1), this reduc-
tion can be credited to the poor water solubility of the acid
treated Lap (M-Lap and BH) [12, 13].

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Fig. 1 (a) Biohybrid powder; (b) Modified laponite powder; (c)
Change color of biohybrid powder as a function of pH. Micrographs
of: (d) Chitosan films (F1); (e) Chitosan films containing modified
laponite® (F2); (f) Chitosan films containing biohybrid (F3). Cross-
section micrographs of: F1 (g); F2 (h); F3 (i). Red arrows indicate the
presence of aggregates in each film. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the Web
version of this article.)
Table 1 Thickness, solubility in water (SW), water contact angle (WCA), and water vapor permeability (WVP) of chitosan film (F1), chitosan
film containing modified laponite® (F2), and chitosan film containing biohybrid (F3)
All values were expressed as mean ± standard error (n = 3)
Means in the same column followed by different capital letter are significantly different (p < 0.05)
Film Thickness (mm) SW (%) WCA (°) WVP (g⋅mm/m2
⋅h⋅kPa)
F1 0.03 ± 0.01A
66.10 ± 3.76A
111.83 ± 11.25A
0.48 ± 0.04A
F2 0.16 ± 0.05B
41.90 ± 6.11B
111.74 ± 7.84A
0.52 ± 0.07A
F3 0.17 ± 0.03B
45.36 ± 1.25B
105.84 ± 4.90A
0.49 ± 0.07A

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Water Contact Angle (WCA) and Water Vapor
Permeability (WVP)
The presence of M-Lap and BH did not change (p > 0.05) the
WCA and WVP values in the films. Hence, WCA and WVP
values remained around 108 ± 3 º and 0.50 g⋅mm/m2
⋅h⋅kPa
in the films, respectively (Table 1). Based on WCA results is
possible to suggest that the manufactured films have hydro-
phobic surfaces and they could be used as packaging mate-
rial in food products with high water content [6]. However,
WVP values were typical of permeable materials, being that
the vapor permeability across the films was only controlled
by the hygroscopic nature of chitosan [17, 18]. In the cur-
rent research, WCA and WVP in F3 are comparable with
those informed in gelatin films (WVP: 0.37 g⋅mm/m2
⋅h⋅kPa)
[21], collagen films (0.4 g⋅mm/m2
⋅h⋅kPa and WCA: 89 º),
chitosan/polyvinyl alcohol films (WCA: 116 º) [6], and
hydroxypropyl methylcellulose films (WVP: 0.50 – 0.60
g⋅mm/m2
⋅h⋅kPa) [19, 28], all considered biopolymer films
with potential applications as food packaging.
Chemical Bonds and Crystallinity
The films had chemical bonds typical of chitosan structure
(Fig. 2a). In this way, FTIR spectra of films showed sev-
eral bands at 3320, 2920, 1640, 1411, 1150, 1070, and 1022
­cm−1
associated to the vibration of hydroxyl groups, C–H
stretches of hydrogen atoms, C–N stretching of amide I of
chitosan, absorption of the C–O stretching, anti-symmetric
stretching of C–O–C bridge of saccharide structure, skel-
etal vibration involving the C–O stretching of saccharide of
structure, and C–O stretching of C6 of chitosan, respectively
[2, 6].
The same characteristics peaks were observed in the
FTIR spectrum of chitosan films containing M-Lap or BH
(Fig. 2a). However, the presence of M-Lap or BH reduced
the intensity of chitosan bands, suggesting that these mate-
rials probably worse interacted with the polymer chains of
the matrix [6]. Based on FTIR spectrum, it is possible to
suggests that chitosan chains interact with M-Lap and BH
by means of hydrogen bonds [2].
Chitosan films exhibited a typical XRD spectra of par-
tially crystalline materials (Fig. 2b), with two defined
peaks at 2θ = 8.8 º (10.0 Å) and 11.5 º (7.7 Å) associated
with the intermolecular lateral packing distance between
the molecular chitosan chains (crystalline phase) and a
broad band centered at 2θ = 22.1 º (4.0 Å) related to the
diffuse scattering of chitosan chains (amorphous phase)
[29]. The incorporation of M-Lap or BH promoted peak
displacements in the XRD spectra of chitosan films from
2θ = 11.5 º to 2θ = 11.9 º (7.4 Å) and from 2θ = 22.1 º to
2θ = 23.5 º (3.8 Å) and 24.0 º (3.7 Å) in chitosan films
containing M-Lap and BH (Fig. 2b). These alterations in
chitosan films with the presence of M-Lap and BH can be
associated with an increase in the molecular spacing of
the chitosan chains in the films as previously discussed in
thickness results [30].
Thermal Properties
All films exhibited DSC thermograms typical of partially
crystalline materials during the first heating cycle (Fig. 3a),
being observed an endothermic peak with melting tempera-
ture ­(Tm) and enthalpy (∆Hm) oscillating between 45 and 58
ºC and between 304 and 326 J/g, respectively, both without
statistical difference (p > 0.05). In the second heating cycle,
no thermal transition was observed in the films, suggesting
which these materials were completely melted during the
first scan (results not shown). Based on DSC thermograms
it is possible to suggest that the addition of M-Lap or BH did
not alter the crystalline and amorphous phases of chitosan
[18].
Thermogravimetric curves of chitosan films exhibited
three main decomposition stages as the water evaporation
from 25 ºC to 110 ºC, with a maximum decomposition rate
at 70 ± 5 ºC. In sequence, the second region of mass loss
was observed between 110 ºC and 410 ºC, with a maximum
decomposition rate at 281 ºC, associated to the chitosan
chain decomposition. Finally, the third region at tempera-
tures higher than 410 ± 5 ºC were associated to the biochar
or unburnt carbon formation (Fig. 3b). Chitosan films con-
taining M-Lap or BH showed the same three decomposition
stages, however, the maximum decomposition rate at the
second stage was lower to that observed in chitosan films
(Fig. 3c and d). This result suggests that the presence of
M-Lap or BH could reduce the chitosan chain interactions,
decreasing the thermal stability of the films. Similar behav-
ior was observed by Capello et al. [6] in blends of chitosan
and polyvinyl alcohol added of anthocyanin extracts.
Another important result was observed at the las of the
third stage. Chitosan films had a residual mass of 38 ± 1%,
whereas the films added of M-Lap and BH had a residual
mass about 53 ± 2% (p < 0.05) (Fig. 3b-3d). The increase
of residual mass in chitosan films containing M-Lap and
BH can be correlated with the inorganic nature of Lap
[31]. These films could be used as soil nutrient after their
application as food packaging [32]. Higher residual mass in
chitosan films containing M-Lap or BH could explain the
reduction of solubility in water (SW) values in the same
materials (see Sect. “Moisture Content (MC) and Solubility
in Water (SW)”).

7. Journal of Polymers and the Environment
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Optical Properties
The initial color of films was affected by the presence of
M-Lap or BH. Chitosan films displayed grey color with low
luminosity (Fig. 4a and Table 2). Films containing M-Lap
or BH showed grey color in other tonalities (Fig. 4a and
Table 2). The most remarkable color modification was
observed in chitosan films containing BH, this film color
was similar to that observed in anthocyanins expose pH = 4
and 5 (Fig. 1c). In the current research, the film-forming
Fig. 2 (a) Fourier-Transform
Infrared spectra and (b) X-ray
diffractograms of chitosan films
(F1), chitosan films contain-
ing modified laponite® (F2),
and chitosan films containing
biohybrid (F3). (For interpreta-
tion of the references to color in
this figure legend, the reader is
referred to the Web version of
this article.)

8. Journal of Polymers and the Environment
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Fig. 3 (a) Examples of differential scanning calorimetry thermograms
of chitosan films (F1), chitosan films containing modified laponite®
(F2), and chitosan films containing biohybrid (F3). Thermogravimet-
ric and differential thermogravimetric curves of: (b) chitosan films
(F1); (c) Chitosan films containing modified laponite (F2); (d) Chi-
tosan films containing biohybrid (F3). (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the Web
version of this article.)
Fig. 4 Images of films based on: (a) Chitosan films (F1), chitosan
films containing modified laponite® (F2), and chitosan films contain-
ing biohybrid (F3); (b) Visual aspect color of the F3 film after contact
with buffer solutions at different pH values (ranging between 1 and
13). (For interpretation of the references to color in this figure legend,
the reader is referred to the Web version of this article.)

9. Journal of Polymers and the Environment
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solutions based on chitosan had a pH = 4.5, confirming the
color of anthocyanins in the films.
Color difference among the films was evaluated by means
of ΔE* values since that color difference is perceptible to the
human eye when ΔE* > 3 [33]. In this way, films contain-
ing M-Lap or BH had visual perceptible differences when
compared with chitosan films (Table 2).
Films containing M-Lap or BH were more opaque when
compared with chitosan films (Table 2). Increasing in Op
values is desired since these materials can be used as barrier
to light to reduce the degradation of nutrients and pigments,
as well as off-flavours formation in foods when exposed to
visible and ultraviolet light [6].
Change color response of chitosan films containing BH
was evaluated after contact with different buffer solutions
with pH values varying between 1 and 13 (Fig. 4b). The
films showed change color response as a function of pH,
being attributed to the structure modification in anthocya-
nins as previously explained in Sect. “Characterization of the
Biohybrid”. Based on ΔE∗
values it is possible to conclude
that chitosan films containing BH have visual color modifi-
cation perceptible to the human eye in the pH rage studied
with exception of pH = 5, (pH value close to the pH of the
film-forming solution) (Table 3). In the current research, the
change color response of chitosan films containing BH was
similar to those observed in chitosan/polyvinyl alcohol films
containing anthocyanins from different sources such as jam-
bolana fruit, jabuticaba fruit, and purple sweet potato [2, 6].
Application of the Films Containing
the Biohybrid for monitoring Meat
Freshness
Chitosan films containing BH were applied to show the meat
freshness at − 20 °C, 4 °C, and 20 °C. Chitosan films con-
taining BH did not change color visually during the storage
time (72 h) of meat at − 20 °C (Fig. 5). In contrast, at storage
temperatures of 4 and 20 °C, the color of chitosan films con-
taining BH changed during the storage time. In general, most
evident color alterations were observed after 24 h and 72 h
in meats stored at 20 and 4 ºC, respectively (Fig. 5). This
color alteration in the films was attributed to the anthocya-
nin oxidation to chalcone form after reaction with ammonia
and amines compounds produced during the microbiological
degradation of the meats [6, 34, 35].
TVB-N results were correlated with film change color. In
this way, meats stored at -20 ºC had a constant TVB-N value
(10.10 mg/100 g) typical of fresh or frozen meats [36]. In
contrast, the TVB-N values in meats stored at 4 ºC/72 h and
20 ºC/24 h increased until 20.56 mg/100 g and 29.32 mg/100
g, respectively. Based on the literature, TVB-N values higher
than 20 mg/100 g are typical of spoiled meats [36]. In the
current research, chitosan films containing BH successfully
detected the meat deterioration by means of color alteration
in the films from purple to yellow color (Fig. 5).
Table 2 Color parameters (L*,
a*, b*, ΔE*, and opacity, Op) of
chitosan film (F1), chitosan film
containing modified laponite®
(F2), and chitosan film
containing biohybrid (F3)
All values were expressed as mean ± standard error (n = 3)
Means in the same column followed by different capital letter are significantly different (p < 0.05)
The ΔE* values of films were calculated using Eq. (4) and considering the color parameters of F1 as a
standard
Film Color parameters Op ­(A600/mm)
L* a* b* ΔE*
F1 2.50 ± 0.37A
− 0.83 ± 0.14A
2.94 ± 0.16A
0.0C
5.13 ± 0.18B
F2 0.39 ± 0.08B
− 1.21 ± 0.08A
7.95 ± 0.47B
5.8 ± 0.45B
10.94 ± 3.83A
F3 26.40 ± 0.16C
− 1.71 ± 0.08B
0.57 ± 0.14C
27.79 ± 0.20A
11.79 ± 0.36A
Table 3 Color parameters (L*, a*, b*, and ΔE*) of chitosan films
containing biohybrid (F3) after contact with buffer solutions at differ-
ent pH (1 – 13)
All values were expressed as mean ± standard error (n = 3)
The ΔE* values of films were calculated using Eq. (4) and consider-
ing the color parameters of chitosan films containing the biohybrid
before contact with the buffer solutions as a standard
pH Color parameters
L* a* b* ΔE*
1 33.38 13.32 2.1 16.64
2 33.81 3.27 6.9 10.95
3 28.20 1.45 5.72 6.31
4 30.34 -0.24 4.44 5.72
5 25.69 -0.38 1.71 1.89
6 30.78 -0.83 2.79 5.00
7 37.00 -0.98 3.36 11.00
8 33.79 -1.02 2.72 7.73
10 34.19 -0.65 1.18 7.90
11 35.25 -0.82 2.92 9.21
12 45.08 -0.30 2.27 18.81
13 26.40 -3.76 16.52 16.08

10. Journal of Polymers and the Environment
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Conclusions
In the current research were manufactured intelligent edible
chitosan films containing a biohybrid (BH) based on antho-
cyanins and laponite®. The surface micrographs showed that
the BH was not dispersed into chitosan films. The presence
of the BH increased the film thickness at least 500% and
reduced the solubility in water by almost 35% when com-
pared with chitosan films. Furthermore, crystallinity, and
optical properties of chitosan films were altered with the
presence of BH. These physicochemical modifications were
attributed to the increase in molecular spacing of chitosan
chains in the presence of BH. Chitosan films containing BH
had grey color due to the pH (4.5) of the film-forming solu-
tion based on chitosan used to manufacture the films. These
materials change color to red and yellow after contact with
buffer solutions with acid and basic pH, oscillating between
1 and 13, respectively. Change color results in chitosan films
containing BH suggests that the adsorbed anthocyanins in
the BH have chemical activity. Chitosan films containing BH
could be applied to monitor freshness in round steak stored
at different temperatures (− 20 °C, 4 °C, and 20 °C).
Fig. 5 Change color of the chitosan films containing biohybrid (F3) used to monitored meat freshness. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)

11. Journal of Polymers and the Environment
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Acknowledgements The authors gratefully acknowledge to CAPES
(Coordination for the Improvement of Higher Education Personnel),
for the fellowships of the first and third author; and to CNPq (National
Council for Scientific and Technological Development) for the research
grant (405432/2018-6). Also, the authors gratefully acknowledge the
Central Laboratory of Electronic Microscopy (LCME), Control of
Polymerization Laboratory (LCP) and Central Chemical Analysis of
Chemical Engineering and Food Engineering for the analyses.
Declarations
Conflict of interest The authors declare no conflict of interest.
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