Food Hydrocolloids 43 (2015) 180e188

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Active chitosan/PVA films with anthocyanins from Brassica oleraceae

(Red Cabbage) as TimeeTemperature Indicators for application in
intelligent food packaging

lia Queiroz de Arruda, Ricardo Stefani*
Valdir Aniceto Pereira Jr., Iza Nata

rio do Araguaia (CUA), Rodovia BR-070,

rio de Estudos de Materiais (LEMat), Universidade Federal de Mato Grosso (UFMT), Campus Universita
Laborato
Km 5., Barra do Garças, MT, Brazil

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

Article history: Food-packaging featuring Time-Temperature Indicators are among the so-called intelligent packages.
Received 27 January 2014 They use a system that monitors a food's condition in real-time, thus indicating the overall influence of
Accepted 17 May 2014 temperature on food product quality. This work aimed to develop and characterize a TimeeTemperature
Available online 2 June 2014
Indicator (TTI) based on a PVA/Chitosan polymeric doped with anthocyanins in order to indirectly
indicate food quality changes through the detection of changes in the pH of packaged food products
Keywords:
when subjected to improper storage temperatures. The TTI was produced from chitosan, PVA and an-
TimeeTemperature Indicator
thocyanins extracted from Brassica oleracea var. capitata (Red Cabbage). TG-DSC, FT-IR, UVeVis, as well as
Chitosan
PVA
Swelling Index (Si) techniques were used to characterize the TTI. The colour variation after activation by
Anthocyanins different pH values was measured through the CIELab scale. The mechanical properties of the TTI were
Intelligent packaging established through stress/strain tests. Due to its physicochemical features, the developed TTI offers
Brassica oleraceae attractive features for application in intelligent food packaging, even though it presents a divergent
modulus of elasticity in comparison to commercial polymers applied in food packaging. Application of
the TTI presented here is supported by an activation test on pasteurized milk, with evident changes in the
colouration of the film, which is important for indicating to consumers that the food has been subjected
to changes in its chemical composition.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction actual parameters of food quality and safety before consumption.

Timeetemperature Indicators or Integrators (TTIs) are simple,
effective and easy to use devices for monitoring, recording and
cumulatively indicating the overall influence of temperature on
quality, from manufacturing to the end consumer (Giannakourou,
Koutsoumanis, Nychas, & Taoukis, 2005). These devices are user-
friendly, can be integrated as part of the food package and most
of them allow consumers to check food quality through a colour
response that matches or correlates to the quality of a food stuff at a
certain temperature (Kim, Jung, Park, & Lee, 2012). Food packages
featuring TimeeTemperature Indicators are examples of intelligent
packaging, due to their use of a system that monitors the conditions
of the food in real time, informing consumers about the conditions
of transport and storage of these products and establishing the
* Corresponding author. Universidade Federal de Mato Grosso (UFMT), Campus

rio do Araguaia (CUA), Rodovia BR-070, Km 5., Barra do Garças, 78600-
Universita
000, MT, Brazil. Tel./fax: þ55 66 3405 5317.
E-mail addresses: rstefani@ufmt.br, ricardo.stefani@gmail.com (R. Stefani).
Thus, a modern security system that monitors and controls critical
parameters for the quality of a food product, such as storage tem-
perature, should ensure good product quality during its life cycle
with simplicity and efficiency.
Due to its simplicity, low-cost and efficiency, TTIs have been
widely applied to establish, monitor and evaluate the storage shelf-
life at a certain temperature of many chilled and frozen food
products, such as fish products and seafood (Kuswandi, Restyana,
Abdullah, Heng, & Ahmad, 2012; Mendoza et al., 2004; Pacquit
et al., 2007), vegetables (Bobelyn, Hertog, & Nicolaï, 2006), meat
(Kerry, O'Grady, & Hogan, 2006; Kim, Choi, Kim, Kim, & Lee, 2013;
Vaikousi, Biliaderis, & Koutsoumanis, 2009) and dairy products (Fu,
Taoukis, & Labuza, 1991; Lu, Zheng, Lv, & Tang, 2013; Zhang et al.,
2013). In addition, TTIs have also been applied to evaluate the ef-
ficiency of pasteurization and sterilization (Mehauden et al., 2007).
Such TTIs are an integral part of the food package, which means
that they must have physical and chemical features compatible
with the polymer used as the main raw material to develop the food
package.

http://dx.doi.org/10.1016/j.foodhyd.2014.05.014
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188
Due to their biodegradability, non-toxicity and biological prop-
erties, many natural polymers, such as chitosan, starch, clay, pectin
and other biopolymers have been used in food packaging (Avella
et al., 2005; Fabra, Busolo, Lopez-Rubio, & Lagaron, 2013; Majeed
et al., 2013; Yoshida, Maciel, Mendonça, & Franco, 2014). Chitosan is
a biopolymer that is non-toxic and has antimicrobial properties,
which are good characteristics for food packaging. Despite its ad-
vantages, chitosan has low mechanical resistance and is often
blended with other polymers such as Poly-vinyl alcohol (PVA),
which is a non-toxic and biodegradable synthetic polymer, in order
to improve its mechanical features (Bonilla, Fortunati, Atare
s,
Chiralt, & Kenny, 2014; Kittur, Kumar, & Tharanathan, 1998;
Tripathi, Mehrotra, & Dutta, 2009; Vimala, 2011). Thus, the result-
ing films from blending chitosan and PVA have modified physical
properties when compared to films prepared only from chitosan or
PVA. Moreover, chitosan blend films can be incorporated with other
substances, such as natural extracts or inorganic metal particles, in
order to improve features such as antimicrobial activity (Dutta,
Tripathi, Mehrotra, & Dutta, 2009; Vimala, 2011) or antioxidant
activity (Belscak-Cvitanovi
c et al., 2011; Lo
pez-de-Dicastillo,
Go
mez-Estaca, Catala
, Gavara, & Herna
ndez-Mun ~ oz, 2012;
Siripatrawan & Harte, 2010), or to monitor pH variations (Yoshida
et al., 2014).
In the literature, there are many reports of the use of dyes
contained in plant tissues for colourimetric determination of pH
(Terci & Rossi, 2002; Chigurupati, Saiki, Gayser, & Dash, 2002;
Mohd, Khan, & Farooqui, 2011). Colour changes in such dyes are
due to the presence of phenolic or conjugated substances, such as
anthocyanins, which are subjected to structural changes when
there is a variation in pH (Shahid & Mohammad, 2013). Thus, ex-
tracts of some plants containing anthocyanins, such as Brassica
oleraceae (Red Cabbage), can be used as natural pH indicators.
Therefore, considering that chitosan and PVA can be safely used
in food packaging and that natural extracts can be incorporated into
chitosan/PVA blends, we report here the development and char-
acterization of a TimeeTemperature Indicator sensor based on a
PVA and chitosan polymer blend incorporated with anthocyanins.
This sensor aims to establish variations in temperature indirectly
from changes in the pH of food products during a period of time
when the foodstuff is kept at different temperatures from those
recommended for storage.
2. Material and methods
2.1. Extraction of anthocyanins from Red Cabbage (Brassica
oleracea var capitata)
Anthocyanins were extracted according to Fuleki and Francis
(1968), with modifications. A sample of approximately 150.0 g
of Red Cabbage was crushed and macerated with 80 mL of
ethanol-water (7:3). The pH of the sample was adjusted to 2.0
with HCl (1 mol/L). Subsequently, the material was stored for
24 h at 5  C, protected from light. After this period, the material
was filtered and the extract was centrifuged at 2000 rpm for
10 min. The supernatant was filtered on Whatman paper # 1 and
the resulting extract was neutralized to pH 7.0 with NaOH
2.5 mol/L.
2.2. Preparation of the TTI
A PVA hydrogel was prepared by dissolving 1 g of polymer
powder (SigmaeAldrich Art. No. 363146, þ99% Hydrolysed) in
100 mL of distilled water under magnetic stirring at 70 ± 2  C until
complete dissolution had occurred. The chitosan hydrogel was
prepared by dissolving 1 g of chitosan (SigmaeAldrich Art No.
181
448877, 80% deacetylated) in 100 mL of aqueous acetic acid 1% (v/
v) under magnetic stirring for 24 h at room temperature (25  C).
The final PVA/chitosan hydrogel (TTI hydrogel) was prepared with
a casting technique at a PVA/chitosan ratio of 3:7 (v/v), with
incorporation of anthocyanin extract. The final concentration of
anthocyanin extract in the film was established as 25% of the total
volume of the mixture of hydrogels. A 1.5% solution of sodium
tripolyphosphate (Na5P3O10) 0.1% (w/v) relative to the total vol-
ume of the mixture was added to the final hydrogel in order to
promote cross-linking. The final pH of the hydrogel blend was
adjusted to 6.1 with NaOH (1.0 mol/L), with a colour ranging from
purple to blue. The hydrogel was cast (70 mL) in Petri dishes
(150 mm diameter) and then the plates were placed in an oven for
48 h at a temperature of 35  C to remove the solvent, resulting in
the final TTI films.
2.3. Determination of Swelling Index (Si)
Swelling Index was determined by the method described by
Cavalcanti, Van Der Mooter, Caramico-Soares, and Kinget (2002)
with modifications in the time interval. Initially, the TTI samples
were cut into 4.0 cm2 slices and then the samples were kept in a
desiccator with silica-gel for 7 days. After this procedure the sam-
ples were weighed and then subjected to immersion in beakers
containing 250 mL of distilled water for different time intervals
(0.5 min, 1 min, 3 min, 5 min, 7 min, 10 min, 15 min and 20 min) at
room temperature (25  C). At each time interval, samples were
removed, dried and weighed. The Swelling Index (Si%) was calcu-
lated by Equation (1):
Final weight  Initial weight
Ii% ¼  100 (1)
Initial weight
2.4. Spectroscopy and TG-DSC analysis
FT-IR spectra were obtained in a Perkin Elmer spectrophotom-
eter (model Spectrum 100) with a resolution of 4 cm1, operating in
the range of 4000e600 cm1 and with attenuated reflectance (ATR)
Fourier transform.
UVeVis spectra were obtained on a Perkin Elmer spectropho-
tometer (model Lambda 25), operating in the range of 900e190 nm.
TG- DSC was performed in a Mettler Toledo, model TG/DSC-1,
using an a-Al2O3 crucible (70 mL) with a sample mass of approxi-
mately 7 mg, heating rate 20  C min1, dry air flow of 60 mL/
min and a temperature range of 30e1000  C.
2.5. Mechanical properties of films
The tensile (stress/strain) was measured in a universal testing
machine, Model WDW e 300E Time Group Inc. using the method
described in ASTM D1708-10, which is suitable for determining
the tensile properties of plastics or the traction of films with
thicknesses ranging from 0.0025 mm to 2.5 mm. The initial grip
separation was set to 22 mm and traction speed to 12.5 mm/min,
with 150 kgf load cell. Five samples were analysed in each test
and all testing occurred at room temperature (approximately
25  C). The thickness of the individual films was measured with a
Mitutoyo® manual micrometer, with divisions of 0.01 mm, at five
random points on each sample, which were determined to have a
mean value of 0.08 ± 0.04 mm for the TTI, 0.10 ± 0.04 mm for
chitosan films and 0.07 ± 0.03 mm for PVA films. Prior to testing,
the samples were placed in an environment with 50 ± 5%
relative humidity and a temperature of 23 ± 2  C for a period of
40 h.
182 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188
2.6. Dynamic parameters e Colour (Colourimetric) analysis
The TimeeTemperature Indicator colour parameters were
determined in a MiniScan EZ Hunterlab spectrophotometer. The
values of L* (lightness), a* (red e green) and b* (yellow e blue)
parameters were recorded to evaluate colour changes due to the TTI
contacting solutions in different pH ranges (1.0e12.0). Tests were
performed in triplicate and the total colour difference was obtained
according to the Equation (2):

2
2
2 1=2
DE ¼ DL* þ Da* þ Db*
where: DL* ¼ L*L*0; Da* ¼ a*a*0; Db* ¼ b*b*0.
where L*0 , a*0 and b*0 are colour values of the standard TTI (pH
7.0).
2.7. TTI sensitivity to lactic acid and aqueous solution of monobasic
sodium phosphate
The TTI was cut into square shaped plates 4 cm2 in area. The
plates were submerged in lactic acid aqueous solution (pH 1.0, 2.0,
3.0, 4.0, 5.0 and 6.0) and aqueous sodium phosphate 1 mol/
L monobasic buffer (pH 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0).
2.8. Potential application of the TTI (Activation test in milk)
To conduct the TTI activation test with milk, the TTI was cut into
square shaped plates 4 cm2 in area and were maintained in contact
with pasteurized milk (20 mL) in glass petri plates (5.5 cm in
diameter). The plates containing milk and the TTI squares were
kept in a room with a controlled temperature of 25  C throughout
the test period (0e4 days). During the test period the plates con-
taining the milk underwent pH readings using a calibrated digital
pH metre. After colour changes occurred in the TTI due to changes
in the milk's pH, the TTI's colour was determined with an EZ
Hunterlab MiniScan spectrophotometer on the CIELab scale.
3. Results and discussion
3.1. Swelling Index (Si)
Table 1 shows the results for Swelling Index. The final value for
the swelling index of the TTI was 38.02%. Thus, the results show
that there was an equilibration period of hydration from 1 to 3 min
(46.32e47.87%).
The results also show that the final value of the swelling index
(38.02 ± 0.03%) is less than the maximum recorded during the
hydration period (47.87%). This behaviour can be explained by the
fact that the chains attached by crosslinking the polymer blend lose
their mobility after a period of hydration, hindering the access of
solvent and consequently the hydration of the film. Another
Table 1
Swelling index for the Time-Temperature Indicator.
Time (minutes) Si% of TTI
0,5 34.35% ± 0.02a
1 46.32% ± 0.02
3 47.87% ± 0.02
5 46.73% ± 0.02
7 43.47% ± 0.03
10 40.76% ± 0.02
15 38.38% ± 0.03
20 38.02% ± 0.03
a
Average of triplicate.
important point that may have contributed to this behaviour is the
crosslinking promoted by tripolyphosphate. This crosslinking may
have resulted in an interaction between the hydroxyl hydrogen and
oxygen from the pyranosidic ring of chitosan, causing a reduction in
the number of hydrogen bonds to water; that is, the hydroxyl group
of chitosan may have caused a steric effect restricting the interac-
tion of water with the polymer (Gehrke, 2000).
Furthermore, PVA is a polymer that is more hydrophilic than
chitosan, due to its large number of hydroxyl groups. Chitosan, in
turn, contributes to reducing the film's hydrophilicity due to the
(2) presence of hydroxyl and acetate groups, which can form intra- and
inter-molecular hydrogen bonds. Therefore, the final percentage of
swelling found (38.02%) for the TTI can be assigned to the per-
centage of PVA (70%) used in the production of the film, since it has
greater swelling capacity than chitosan (Costa & Mansur, 2008;
Ginani, Navarro, Nascimento, & Oliveira, 1999).
3.2. FTIR spectra
Fig. 1 shows FT-IR spectra for the chitosan, PVA and TTI films.
The spectrum for the PVA film shows absorption peaks at about
3254 cm1 (OH stretching, Table 2) and 1084 cm1 (Table 2) cor-
responding to the CeO group (Anicuta, Dobre, Stroescu, & Jipa,
2010; Rodrigues, de Camargo Forte, Azambuja, & Castagno, 2007).
In the TTI spectrum (Fig. 1), the CeH stretching band is present
at 2930 cm1, as well as a CeN stretching band at 1240 cm1, typical
of secondary amines, which is assigned to the chitosan polymer
chain. The TTI FT-IR spectrum also shows a typical CeN (amide I)
stretching band at 1642 cm1, in addition to CeO deformation of a
hydroxyl group at 1066 cm1. These bands are shifted in compar-
ison to their pure chitosan and PVA counterparts (Table 2), indi-
cating polymeric association through hydrogen bonding (Bonilla
et al., 2014).
The FT-IR spectrum for the anthocyanin extract shows a strong
absorption band with a maximum at 1015 cm1 assigned to aro-
matic ring CeH deformation, as well as bands at 1650 and
1455 cm1 corresponding to the stretching vibration of the C]C
aromatic ring. The absorption band with a maximum at 1233 cm1
is assigned to stretching of pyran rings, typical of flavonoid com-
pounds. Bands appearing between 1300 and 1380 cm1 are
assigned to CeO angular deformations of phenols. Therefore,
considering the TTI spectrum in comparison to PVA/chitosan
without anthocyanins (Fig. 1 and Table 2), changes are present in
the region between 1500 and 1600 cm1, with a clear increase in
the intensity of the bands in this region. This shows that anthocy-
anins have been incorporated into the TTI polymeric matrix, since
the modified region is typical of C]C stretching from aromatic
rings, as well as changes in the 750e1000 cm1 region, indicating
the presence of aromatic rings with ortho substitution, which
matches the FT-IR spectrum of the anthocyanin extract (Fig. 1).
3.3. Spectroscopy in the UVeVisible region e UVeVis
Since the UVeVis spectrum of the anthocyanin extract (Fig. 2)
was obtained in an acid medium (pH 2.0), the spectrum indicates
the character of acidebase equilibrium of the flavylium cation,
which has one maximum absorption band in the visible region
(535 nm) and another in the ultraviolet region (280 nm). Moreover,
the spectrum profile obtained at pH 2.0 is quite common for an-
thocyanins, in accordance with the literature (Lee, Durst, &
Wrolstad, 2005). Thus, confirming successful anthocyanins
extraction. The results for the anthocyanin extract are also in
accordance with the study by Março and Scarminio (2007), which
showed formation of the flavylium cation under strongly acidic
conditions (pH 1.0 and 3.0).
 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188 183

Fig. 1. FT-IR spectra of anthocyanins, chitosan, PVA and TTI films.

Fig. 2 shows that PVA and chitosan film without added
anthocyanin extract have no significant absorption maxima in
the visible and ultraviolet regions. In the TTI, in the UVeVis
spectrum, it is possible to observe an absorption maximum in the
UVeVis region at a wavelength of 320 nm, characteristic of an-
thocyanins in the neutral range, exhibiting a hypochromic shift
compared with the PVA/chitosan film without added anthocya-
nins (Fig. 2). Levi, Scarminio, Poppi, and Trevisan (2004)
observed the same profile for acidic/basic anthocyanin extracts.
The results confirm the incorporation of anthocyanin extract into
PVA/chitosan film.
Table 2
Characteristic wave numbers and groups of Chitosan and PVA.
3.4. TG-DSC thermal analysis
In the TG-DSC curve of the chitosan film (Fig. 3), a weight loss
was observed in four consecutive steps. The 1st step of mass loss
(8.28% (m/m)), between 30  C and 155  C with an endothermic
peak at 150  C, was due to loss of surface water from the compound.
The 2nd stage of mass loss (10.63%) occurred between 161  C and
250  C, due to the departure of intrinsic water in the film. The
decomposition process of the film started from 250  C and pro-
ceeded until 830  C in two consecutive steps, with mass losses of
51.76% and 25.87% corresponding to the 3rd and 4th stages,
respectively. Three exothermic peaks were observed at 290  C,
620  C and 700  C. The final residue of the chitosan film, 3.46%, was
due to the presence of inorganic compounds (impurities) present in
its makeup, which are stable up to 1000  C.

Material Bands (cm1) Chemical Associated groups

Chitosan 3570e3200 nOeH bonded

(3412) NeH2
Chitosan 2955e2845 nCeH2 (ass)
(2902)
Chitosan 1900e1500 Amida I: nC]O
(1632)
Chitosan 1547 dNeH Amide I
Chitosan 1340e1250 CeN (tertiary)
(1374)
Chitosan 1400 CeN (primary)
Chitosan 1260 CeN (secondary)
Chitosan 1054 e 886 nCOC (saccharide structure-b-1-4)
Chitosan 1012 CeO (cyclic)
PVA 3550e3200 n(OH)
(3254) OH…OH
PVA 2937e2870 nCeH
PVA 1650e1630 dOeH
PVA 1461e1417 d(CH) e CH2
PVA 1254 n(CeO)
PVA 1084 n(CeO)eCeOH
PVA 865 dCeC
PVA 937 dCHeCH2
Fig. 2. UVeVis spectrum anthocyanin extract at pH 2.0, pure chitosan/PVA film and TTI
PVA 1583 nC]O
film.
184 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188

Fig. 5. TG-DSC curve of TTI film.

Fig. 3. TG-DSC curve of chitosan film.

dehydroxylation of the PVA and the degradation of the chitosan,

PVA decomposed in four consecutive steps (Fig. 4). The 1st stage
comprising pyrolytic dehydration and depolymerization of the
was due to thermal dehydration, with a mass loss of 5.49% (m/m),
polysaccharide structure, promoting the formation of water, carbon
with an endotherm between 80 and 133  C. Thermal decomposi-
dioxide, methane and ammonia (Zohuriaan & Shokrolahi, 2004).
tion of the anhydrous compound (2nd, 3rd and 4th stages) occurred
The 4th stage of decomposition occurred up to 490  C, with an
between 196  C and 720  C with mass losses of 69.37%, 13.26% and
exothermic peak at 515  C and mass loss of 16.44%. The 5th stage of
11.06% (m/m), respectively, with an endothermic peak at 227  C.
decomposition occurred up to 620  C, with a mass of 12.95%. The
This peak is attributed to dehydration of its polymer chain and
6th stage of decomposition occurred up to 950  C with a mass loss
polymer melting, confirmed by the literature and qualitative
of 14.29%. The final thermal decomposition temperature (950  C) is
testing, together with the subsequent formation of volatile prod-
at least 120  C higher than the final temperature of decomposition
ucts. Another endothermic peak at 300  C is due to dehydroxylation
of the chitosan film and 230  C greater than that of the PVA film.
of the PVA (Lewandowska, 2009). The DSC for the 2nd, 3rd and 4th
The final weight of the TTI residue settled at 3.40%, which can be
stages of thermal decomposition presented four exothermic peaks
assigned to impurities (inorganic compounds) present in the chi-
between 360 and 718  C. The exothermic peaks observed in the DSC
tosan. From the DSC curves of the chitosan and PVA films it was
curve of the PVA film (Fig. 4) are due to oxidative decomposition of
possible to observe the intrinsic characteristics of these compounds
organic material in an air atmosphere without the formation of
when compared with the TTI's DSC curve, indicating physical
waste in the thermal decomposition of the PVA film.
mixing in the formation of the TTI, and that the crosslinking action
The TG-DSC curve of the TTI (Fig. 5) shows the thermal
of sodium tripolyphosphate conferred increased resistance to
decomposition occurring in six stages, with the first stage due to
thermal degradation when compared to the PVA and chitosan films
the departure of surface-adsorbed water (17.12%). The 2nd stage of
(Rabello, 2000; Sarantopoulos, Oliveira, Anjos, Alves, & Ardito,
thermal decomposition occurred between 183  C and 290  C, with
2002).
a mass loss of 5.03%, due to the removal of intrinsic water. The 3rd
The crosslinking carried out by the sodium tripolyphosphate in
stage of mass loss by the indicator occurred at 290  Ce363  C, with
the PVA/chitosan blend to form the TTI promoted the formation of
a mass loss of 30.77% and an endothermic peak at 430  C, indicating
an interpenetrating crosslinked polymer, since both the FTIR
greater thermal stability than the chitosan and PVA films sepa-
spectrum (Fig. 1) and the DSC curve of the TTI (Fig. 5) compared
rately. This step represents a thermal degradation due to
with films of pure constitution indicate this type of crosslinking,
which is effective for all polymers of the blend. This indication is
due to the fact that the polymer mixture of chitosan and PVA had
increased miscibility as well as an increased glass transition tem-
perature. Thus, the interactions between the polymer chains of the
PVA and the chitosan reduced the possibility for rotation around
the covalent linkages of polymeric chains and limited movements
of atomic clusters, thereby modifying its glass transition tempera-
ture (Mano, Reis, & Cunha, 2000; Paul & Newman, 1978), which in
the case of PVA increased from approximately 70  Ce97  C while
that of chitosan shifted from approximately 160  Ce195  C as seen
by the DSC curve in Fig. 6 in comparison with the DSC curves of
Figs. 3 and 4.

3.5. TTI sensitivity to lactic acid and aqueous solutions of

monobasic sodium phosphate

After the TTI was in contact with solutions at different pH, a

visible colour change was detectable in the samples, ranging from
Fig. 4. TG-DSC curve of PVA film. bright red to bright green from pH 1.0 to pH 12.0 (Fig. 6). The colour
 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188 185

Fig. 6. TTI colour response at different pH conditions.

change in the TTI settled at bright light red at pH 1.0, and pink Table 3 presents the mean values and standard deviations in
colouration at pH 2.0 and 3.0. When it was in contact with the lactic colour parameters L*, a*, b*, as well as full colour variation (DE) on
acid solution at pH 4.0, the TTI showed a purple colour, while at pH the CIELab scale for the TTI film according to its activation by
to 5.0 and 6.0 it showed a not very noticeable purple tone. At pH 7.0 contact with aqueous solutions of lactic acid and monobasic so-
the TTI showed a visible colour change to light blue. At pH 8.0, the dium phosphate.
TTI appeared a bright green colour. This colour intensified as the pH The L* parameter data (Table 3) showed high values according to
increased further from to 9.0, 10, 11 and 12 as shown in Fig. 6. the luminosity displayed (L* parameter). When the TTI was acti-
This spectrum of vivid colours found in the TTI (Fig. 6) is vated at pH values between 5.0 and 7.0, it did not exhibit a very
consistent with Wallach (1996), who reported that an activated strong staining, ranging from lilac to a bright green tone, which
indicator for pH changes may exhibit an expansion of its range of explains the high values of the L* (>99) parameter, which indicates
colour changes in comparison to sensors that are activated by transparency, and the fact that anthocyanin presents a more
enzymatic reactions. intense red colour when the pH is below 5.0, which can be seen in
Fig. 6.
Considering the data in Table 3, the parameter a* (green e red
axis) derived from the CIELab scale showed significant variations in
3.6. Determination of the TTI's dynamic parameters e Full colour the range which comprises the colour red, and is characterized by
variation the values found for the activated TTI in lactic acid, with a
maximum value of 19.75 ± 0.01 in pH 1.0 to pH 4.0 (2.86 ± 0.02).
The colour of the TTI film at neutral pH (7.0) was used as a set This is supported by the variation in the colour from red to pink
point to establish the parameters for total variation in the TTI's (Fig. 6), considering the pH range from 1.0 to 4.0. The red colour is
colour. However, the final pH for the TTI was set at 6.1; Table 3 assigned to the flavylium cation, which arises when anthocyanin
shows the actual values. are in a strongly acidic medium. (Terci & Rossi, 2002). In the alka-
line pH range, the TTI showed negative values for parameter a*
Table 3 starting at pH 7.0 (2.36 ± 0.01), being more evident at pH 12.0
Colour Parameters (CIELab) of non-activated and activated TTI in different pH so- (19.24 ± 0.08), with the TTI having a bright green colour.
lutions with the total colour change (DE) after activation.
Regarding parameter b* (blueeyellow axis) on the CIElab
TTI inactivated Colour parameters (Table 3) scale, negative values indicate the presence of blue colour
L*0 a*0 b*0 visually displaying a trend toward the colour purple in the TTI,
especially at pH 5.0 and 6.0, as can be seen in Fig. 8. However,
96.76 ± 0.07 1.57 ± 0.03 4.05 ± 0.07 e
TTI (DpH) L* a* b* DE several authors claim that there is an relationship between the
parameters a* and b*, as chroma (C), which represents the in-
1.0 86.88 ± 0.08 19.75 ± 0.01 9.28 ± 0.04 27.01
2.0 89.42 ± 0.02 16.96 ± 0.07 5.94 ± 0.08 22.29
tensity/brightness and hue angle (h ), representing little difference
3.0 90.01 ± 0.01 15.44 ± 0.30 5.02 ± 0.09 20.42 of colour, meaning that changes in one or the other parameter (a*
4.0 98.11 ± 0.06 2.86 ± 0.02 1.17 ± 0.04 5.45 and b*) are directly linked to the colour of the sample (Choubert &
5.0 99.49 ± 0.04 1.06 ± 0.09 2.46 ± 0.07 4.11 Baccaunaud, 2006).
6.0 99.78 ± 0.08 0.90 ± 0.09 7.51 ± 0.09 5.21
Thus, the default values for the total change in the TTI's colour
7.0 99.68 ± 0.11 2.36 ± 0.01 7.27 ± 0.08 4.41
8.0 95.19 ± 0.20 6.27 ± 0.09 7.03 ± 0.07 5.78 are valid and similar to the work of Yoshida et al. (2013), who
9.0 88.63 ± 0.06 9.28 ± 0.09 5.65 ± 0.02 11.31 developed smart chitosan/anthocyanin films to monitor pH
10.0 86.42 ± 0.07 10.66 ± 0.09 2.51 ± 0.09 13.85 changes in packaging. The total colour variation (DE) in the TTI
11.0 86.35 ± 0.04 12.13 ± 0.04 0.92 ± 0.09 15.63 developed in the present work on the CIELab scale showed a
12.0 84.30 ± 0.02 19.24 ± 0.08 2.80 ± 0.03 22.68
behaviour that justifies the visual colour change, where the highest
186 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188
Fig. 7. Tension-deformation curve of PVA film (a), chitosan film (b) and TTI (c).
values for full colour are set at the extremes of the pH range used
for the activation test (Table 3), i.e. 27.01 and 22.68 at pH 1.0 at pH
12.
3.7. Mechanical properties
Fig. 7a shows the stressestrain curve of the PVA film from the
average statistical data generated from the five specimens and the
elasticity modulus (E) that was determined by the specified linear
portion (straight-line equation). The elasticity modulus for the PVA
film was 1.29 MPa, the maximum tensile stress (smax) was 37.5 MPa,
the specific deformation (ε) was 263.5% and the break voltage (s)
was 25.6 MPa. The mechanical properties of the PVA film are in
accordance with the literature (Chiellini, Cinelli, Imam, & Mao,
2001; Costa & Mansur, 2008; Mansur, Sadahira, Souza, & Mansur,
2007). The degree of hydrolysis is a crucial influence on the me-
chanical properties of PVA (Mansur et al., 2007), as corroborated by
the divergent results on the mechanical properties of PVA found by
Costa and Mansur (2008), who obtained a specific deformation of
Fig. 8. TTI colour response in contact with pasteurized milk at 25  C and different time
intervals.
276% a tensile stress of 27.5 MPa and a maximum elasticity modulus
of 0.6 MPa. Considering the deformation tension of PVA (Fig. 8a),
PVA has a gradual transition from elastic to plastic. This means that
plastic deformation begins at the point where the stressestrain
curve ceases to be linear. At this point, called the proportional limit,
the material deforms plastically above the linear section of the
stressestrain curve, incurring a permanent deformation.
Fig. 7b shows the stressestrain curve of the chitosan film,
generated from the statistical average of the five specimens and (E)
and determined from the linear portions shown in red. The chito-
san film showed an elasticity modulus of 24.37 MPa, a maximum
tensile stress of 31.8 MPa, a specific deformation of 16.2% and a
tensile strength of 27.6 MPa.
Fig. 7c shows the stressestrain curve for the TTI. The TTI had an
elasticity modulus of 3.53 MPa, a maximum tensile stress of
9.8 MPa, a specific deformation of 26.8% and a Tensile Strength of
9.6 MPa. These mechanical characteristics of a TTI were also
observed by Bispo, Mansur, Barbosa-Stancioli, and Mansur, 2010,
where their films containing 25% chitosan and 75% PVA without a
crosslinking agent experienced a gain of 250% in the specific
deformation voltage with the addition of 1% crosslinker. Bispo et al.,
2010 proposed that films formed from polymer blends present
results with intermediate values of maximum stress compared to
pure PVA or chitosan films. Therefore, the mechanical properties of
the TTI may suggest that the PVA polymer has the most influence
on the tensile stress and maximum specific deformation. However,
the TTI exhibited a considerably smaller elastic behaviour
compared to its constituents (chitosan and PVA).
Comparing the results with the TTI with the mechanical prop-
erties of polyethylene and polyethylene terephthalate, the TTI
showed much lower specific strain, about 2.6-fold lower than that
of PET, and 3.7- and 14.8-fold less compared to polyethylene of low
(LDPE) and high density (HDPE), respectively. The elasticity
modulus of the TTI (3.53 MPa) was considerably lower when
compared to PET (2100e3100 MPa), LDPE (200e400 MPa), HDPE
 V.A. Pereira Jr. et al. / Food Hydrocolloids 43 (2015) 180e188
Table 4
Colour parameters L*, a*, b* and the total colour variation (DE) of activated TTI upon
contact with pasteurized milk kept at temperatures over cooling range.
TTI (DpH) Colour parameters of TTI enabled in milk
L* a* b*
6,7 75,95 ± 0,08 1,73 ± 0,03 4,18 ± 0,05
6,0 70,96 ± 0,10 5,26 ± 0,09 5,26 ± 0,12
5,0 69,18 ± 0,08 8,28 ± 0,02 3,55 ± 0,04
4,6 67,01 ± 0,05 19,82 ± 0,08 8,00 ± 0,09
(600e1400 MPa), PLA (350e2800 MPa), PA (1400 MPa) or PVC
(25e1600 MPa) (Boedeker Plastics, 2013; Netzsch Group, 2014;
Plastic Products, 2010). Thus, there are divergences between the
mechanical properties of the TTI and those of commercially avail-
able polymers. This limitation can be overcome, since TTIs are not
commonly used as a whole food package, but as a minor compo-
nent of it.
3.8. Potential application of the TTI (Activation test in milk)
The pH of fresh milk is slightly acidic, ranging between 6.6 and
6.8, with an average of 6.7 at 20  C or 6.6 at 25  C. When milk is at a
temperature that is above the maximum described for as ideal for
cooling (7  C), it begins to decompose, which changes the medium's
pH, reducing the stability of the milk. Many different factors, such
as microbial contamination and subsequent accumulation of lactic
acid due to microbial metabolism, can cause the pH of milk to
lower. Therefore, milk spoilage can be detected and monitored
through a colourimetric pH monitoring system such as the TTI
developed in this work.
After coming into contact with the milk, a visible colour change
was detected in the TTI samples (Fig. 8), which at pH 6.7 (unspoiled
milk) showed a dark grey colour that, as the pH decreased from 6.7
to 5.0, gradually became clearer. At pH 4.6 (spoiled milk) the TTI
changed to a dark pink colour, clearly indicating milk spoilage.
Table 4 shows the average values and standard deviations of
colour parameters L* deviations, a*, b*, and the total colour varia-
tion (DE) on the CIELab scale of triplicate samples of the TTI film
according to activation by contact with pasteurized milk that was
kept at a temperature above the cooling temperature for time in-
tervals ranging from 0 to 4 days.
The L* parameter data in Table 4 show the indicator of lightness
(L* parameter) was reduced compared to that of the standard (non-
activated TTI) values. This is due to the fact that milk has a white
opaque colour that could interfere with the TTI's transparency.
Considering the data in Table 4, it can be seen that parameter a*
(green e red axis) from the CIELab scale had values within the
range which comprises the colour red, while at pH 4.6 it showed a
maximum value for the TTI of 19.82 ± 0.08 for parameter a*.
Regarding parameter b* (blueeyellow axis) on the CIElab
(Table 4) scale, positive values indicate the presence of yellow
colour, which was not visually perceptible in the TTI sample.
Assuming full colour variation (Table 4), the greatest colour change
after activation in pasteurized milk occurred when the milk had a
pH of 4.6 (DE ¼ 36.85). At this point, the red colour was more
noticeable in the TTI (Fig. 8). The results show that the developed
TTI is very sensitive to pH changes, which is the key point for TTI
activation. This supports the use of the fabricated TTI as part of an
intelligent packaging system, due to evident and persistent changes
in the colour of the TTI films according to pH changes.
4. Conclusion
The developed TTI has good spectroscopic and physicochemical
properties for application in intelligent food packaging, although its
187
mechanical properties diverge from those of commercial purpose
polymers. The changes in colour of the film provides a cheap and
simple way to indicate that a food has undergone changes in its
chemical composition, the colour shift resulting from changes in
DE the pH of the contents that occur when food is spoiled.
21,07
26,71 Acknowledgements
29,29
36,85
The authors are thankful for the financial support of Coor-
denaça ~o de Aperfeiçoamento de Pessoal de Nível Superior and
FAPEMAT (grant no: 835372/2009). The authors are further grateful
to Dr. Adriano Buzutti for his direct contribution to the analysis of
TG-DSC, to Dr. Gilberto Fuzari for assistance with the mechanical
testing, and to Dr. Nara de Souza and Dr. Gilberto Goulart for sug-
gestions during manuscript preparation.
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