Food Hydrocolloids 84 (2018) 238–246

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

Development and evaluation of a novel antioxidant and pH indicator film T

based on chitosan and food waste sources of antioxidants
Mia Kurek∗, Ivona Elez Garofulić, Marina Tranfić Bakić, Mario Ščetar, Verica Dragović Uzelac,
Kata Galić
Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, Croatia

A R T I C LE I N FO A B S T R A C T

Keywords: Chitosan based smart films are developed using blueberry and blackberry pomace extracts as active agents at
Chitosan film different concentrations (1, 2 and 4% w/v). The whole concept of film production can be considered as eco-
Blueberry pomace friendly contributing to the reduction of generally wasted material, fruit pomace. Blueberry and blackberry
Blackberry pomace pomace showed excellent antioxidant potential that was not diminished after the film production. Chitosan
Antioxidant activity
matrix was not significantly changed to influence permeability to oxygen and mechanical properties, while
Visual pH indicator
water vapour permeability slightly decreased. Only the film stiffness increased with the addition of extract.
Visible and significant colour changes of dry pH indicator films occurred with changing pH. The film colour was
visibly transformed from pale yellow for control film, to blue-green and purple (with negative and positive a*
values, respectively) with the addition of blueberry and blackberry pomace extracts, respectively. With changing
pH from 2 to 10, films with blueberry changed from rose to blue green and with blackberry from red to dark
violet. The most significant change was observed in the pH range from 4 to 7 that is important for determination
of pH change due to the food spoilage in real foodstuff. Blackberry pomace extract had 4× more polyphenols
than blueberry one. As expected, when extracts were added to chitosan films an increase in polyphenol content
was also determined and antioxidant activity significantly increased. Films with blackberry pomace extract
showed the highest antioxidant capacity probably due to the fact that already pure blackberry pomace extract
was better antioxidant than the blueberry one. This result pointed high antioxidant activity of all produced films.

1. Introduction association of chitosan with a hydrophobic and moisture resistant

Recently there is strong interest in so called advanced smart
packaging where a total packaging concept synergistically benefits from
active (antimicrobial, antioxidant, etc.) and intelligent part (sensing
and sharing information about product freshness, safety, storage temp.,
etc.) (Vanderroost, Ragaert, Devlieghere, & De Meulenaer, 2014; Yam,
Takhistov, & Miltz, 2005). Globally, there is a strong trend towards eco-
friendly concept, from packaging materials (natural active compounds
and biodegradable polymers) to methods used for their obtaining (de
Moraes Crizel et al., 2018; Medina-Jaramillo, Ochoa-Yepes, Bernal, &
Famá, 2017). Chitosan stands out for its excellent film forming prop-
erties with unique physico-chemical characteristics regarding good
mechanical performance and great barrier capacity (Kurek, Guinault,
Voilley, Galić, & Debeaufort, 2014; Martins, Cerqueira, & Vicente,
2012). However, its high sensitivity to moisture presents limitations for
its use as food packaging material, so it is frequently modified to satisfy
different technological demands. To overcome these drawbacks
polymer from renewable sources, modification by crosslinking agents
or reinforcing with fillers is proposed (Bhuvaneshwari, Sruthi,
Sivasubramanian, Kalyani, & Sugunaba, 2011; Huang, Ma, Zhang, &
Quan, 2017; Suyatma, Copinet, Tighzert, & Coma, 2004).
Active antioxidant packaging concept became very popular in order
to avoid/stop oxidative reactions that can significantly change quality
attributes of packed product such as discoloration and rancidity and
consequently lead to accumulation of food waste. Following pre-
servative free concept, synthetic antioxidants (e.g. butylated hydro-
xyanisole, butylated hydroxytoluene) can be replaced with new natural
compounds, generally derived from plants that can control oxidative
reactions in foods and at the same time they are presumed to be safe
and well accepted by consumers. In addition to the antioxidant activity
phenolic or conjugated substances, such as anthocyanins can change
their structural forms when there is a variation in pH (Shahid &
Mohammad, 2013) making them interesting as active and intelligent
agents. They are natural, nontoxic, water soluble pigments that belong

∗
Corresponding author.
E-mail address: mkurek@pbf.hr (M. Kurek).

https://doi.org/10.1016/j.foodhyd.2018.05.050
Received 30 March 2018; Received in revised form 28 May 2018; Accepted 28 May 2018
Available online 29 May 2018
0268-005X/ © 2018 Elsevier Ltd. All rights reserved.
M. Kurek et al.
to flavonoids and are responsible for red, purple and blue hues of plant
fruits, flowers and leaves (Chandrasekhar, Madhusudhan, &
Raghavarao, 2012; Zhang, Lu, & Chen, 2014). In order to sense the food
quality and product freshness during the supply chain, up to date an-
thocyanins have been used to produce colorimetric indicators based on
pH variations that can detect the presence of volatile amines progres-
sively formed as the food is spoiled (Heising, van Boekel, & Dekker,
2015; Kuswandi & Nurfawaidi, 2017). Some literature examples include
red cabbage (Chen & Gu, 2013; Pereira, de Arruda, & Stefani, 2015),
grape skin (Ma & Wang, 2016), purple sweet potato (Choi, Lee, Lacroix,
& Han, 2017) or orchid tree extracts (Zhang et al., 2014), among others
incorporated in agar potato starch (Choi et al., 2017), gellan gum (Wei,
Cheng, Ho, Tsai, & Mi, 2017) or chitosan (Yoshida, Maciel, Mendonça,
& Franco, 2014).
Despite excellent potential of natural antioxidant extracts, the main
drawback is their temperature, light and storage instability. Nowadays,
antioxidants from berry extracts are mainly obtained by conventional
solvent extraction with a major drawback i.e. use of toxic solvents (Qin
et al., 2015). Therefore, there is a growing interest in innovative green
extraction concepts, non-toxic to human health, such as high pressure,
ultrasound, pulsed electric fields, microwave-assisted extraction (MAE),
and supercritical fluids, among others. These methods allow obtaining
extracts rich in valuable active compounds (Roselló-Soto et al., 2015).
The benefits of MAE are rapidity, high extraction yield, low degradation
of anthocyanins and efficient separation (Sun et al., 2016; Zheng et al.,
2013). According to some authors, blueberry juice extraction residue
(pomace) contains approximately 52% antioxidants (on a dry basis)
with high content of polyphenols (Ancillotti et al., 2017; Avram, 2017,
p. 2375; Lee & Wrolstad, 2004; Reque et al., 2014). Furthermore,
blackberry pomace is rich in ascorbic acids, phenolic compounds, an-
thocyanins, flavonols, chlorogenic acids and procyanidins (Mi, Howard,
Prior, & Clark, 2004; Vulić et al., 2011). Using MAE, there is also an
advantage in avoiding degradation or isomeration of flavanols and
anthocyanins from blackberry and blueberry pomace (Li et al., 2016).
In addition, Sun et al. (2016) found that MAE method has higher an-
thocyanin yield from blueberry pomace with better colour quality,
shorter extraction time and higher hydroxyl radical scavenging activity
(Zhang, Tchabo, & Ma, 2017). Under the optimal conditions, an average
validation value of anthocyanin extraction rate from blueberries is
73.73% (Zheng et al., 2013).
Only diminished literature data is available dealing with in-
corporation of blueberry and blackberry pomace extracts in chitosan
films. It is noteworthy that studies aimed at using waste from the food
agroindustry have increased in importance. Some articles describe po-
mace as a polymer filler (blueberry in cassava and corn starch (Luchese,
Garrido, Spada, Tessaro, & de la Caba, 2018; Luchese, Sperotto, Spada,
& Tessaro, 2017)), use of already commercialised powdered antho-
cyanins, or fruit residues (blueberry in gelatine films (de Moraes Crizel,
Haas Costa, de Oliveira Rios, & Hickmann Flôres, 2016) or beet residues
in gelatine (Iahnke, Costa, De Oliveira Rios, & Flôres, 2016)). Active
and intelligent films were prepared from plantain starch and pre-gela-
tinized plantain flour with and without the addition of blackberry pulp
as a natural filler using the casting methodology (Gutiérrez, 2017).
More studies including applications on real food items should be per-
formed. Blueberry and blackberry pomaces are frequently discarded
even though they are proven to contain large amounts of highly valu-
able compounds. The incorporation of their extracts in chitosan films,
especially those from blackberry one, has not been investigated and
published so far. Moreover, in the present work, assessing films on real
foodstuff for further proof of concept gives value in the valorisation of
agricultural by-products (both polymer and fruit pomace extract), thus
closing the product lifecycle. In addition, solvent free extraction tech-
nique is combined in order to keep natural concept of final bio based
films. At least, films can be classified as eatable coatings and improve
aroma compounds to maintain the characteristic flavour of the food
products (Ščetar & Galić, 2018).
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Food Hydrocolloids 84 (2018) 238–246
Therefore, a novel biobased films with blueberry and blackberry
food waste extracts, as sources of wasted antioxidants and anthocyanins
used as pH indicators, incorporated in chitosan based matrix were de-
veloped. In this paper, extracts were prepared using microwave assisted
extraction as green technology. Influence of extract type and con-
centration incorporated in chitosan films on its functional (active and
intelligent) and physicochemical properties was investigated. Colour,
solubility, water vapour and oxygen barrier, and mechanical properties
were investigated in order to check the possibility of its use as smart
films and coatings for different foodstuff. Active concept was checked
by the antioxidant activity (total phenolic compounds and ferric redu-
cing antioxidant power (FRAP)) and the intelligent aspect was con-
firmed by measuring colour change after exposure to different pH.
2. Materials and methods
2.1. Materials and reagents
Commercial grade chitosan (CS) (France Chitine, France, powder
652, having a molecular mass of 165 kDa, degree of deacetylation
of > 85%) was used as the film forming matrix. Blackberry (Rubus
fruticosus agg) and blueberry (Vaccinium corymbosum L.) pomace were
used for the extraction of antioxidants and used as active agents. Acetic
acid (glacial 100%, Merck, Darmstadt, Germany), pure ethanol (96%,
Gram-mol, Croatia), deionised water and glycerol (Fluka Chemical,
98% purity, Germany) were used as reagents in the preparation of the
film forming solutions (FFS) and fruit pomace extracts. Commercially
available pH buffers (KEFO, Croatia; pH2 – citrate, HCl; pH4 – citrate,
HCl;, pH5 – citric acid (1-hydrate), NaOH; pH6 – citric acid (1-hydrate),
NaOH;, pH7 - phosphate, pH8 - H3BO3, NaOH, HCl; pH10 – H3BO3, KCl,
NaOH; pH12 – sodium phosphate, NaOH) were used for characterisa-
tion of colour changes under different pH. No further purification of
chemicals has been done and freshly prepared solutions were always
used.
2.2. Extracts preparation
Fresh fruits were pressed and their pomace was grained using home
stick blender. This was used for preparation of extracts using micro-
wave-assisted extraction (MAE). For MAE a single-mode focused mi-
crowave reactor (Milestone, Start S Microwave Labstation for Synthesis,
Bergamo, Italy) operating at 2450 MHz with adjustable microwave
power output was used. General extraction parameters were: time re-
quired to achieve extraction temperature 2 min, stirring 75%, ventila-
tion after extraction 2 min. Extraction was carried out at constant
temperature of 50 °C using ethanol as a solvent and microwave power
level of 600 W for 10 min. Afterwards, the extracts were cooled, filtered
through Whatman no. 40 filter paper (Whatman International Ltd.),
transferred in 500-mL volumetric flasks and solvents were evaporated
until all the alcohol was removed. Final extracts were stored at −18 °C
until used.
2.3. Film preparation
A chitosan solution was prepared by dissolving the chitosan powder
in a 1% (v/v) aqueous acetic acid, to obtain 2% (w/v) film forming
solutions. To achieve a complete dispersion of chitosan, the solution
was stirred for 2 h at room temperature. Glycerol (20% w/w polymer
dry matter) was added to the chitosan solution under stirring. To obtain
antioxidant and pH indicating films, blueberry (B) or blackberry (K)
pomace extracts were added to chitosan solution (CS) (1, 2 and 4% w/
v) and homogenised for 10 min. Films were coded regarding to extract
content, i.e. 1% blueberry CSB1, 2% blueberry, CSB2, 4% blueberry,
CSB4 etc. An exact amount of the film-forming solution was then
poured into a glass Petri dish. In order to obtain films, solvents were
removed by drying in a ventilated climatic chamber (Memmert
M. Kurek et al.
HPP110, Memmert Germany) at 30 °C and 50% RH (for 24 h). After
drying, the films were peeled off the surface and stored in a ventilated
climatic chamber (Memmert HPP110, Memmert Germany) at 50% RH
and 25 °C before each measurement.
2.4. Physical-chemical characterisation of films
2.4.1. Film thickness
The film thickness was measured with a digital gauge (accuracy of
0.001 mm) (Digimet, HP, Helios Preisser, Germany). The average value
of five thickness measurements at different position per type of film was
used in all calculations.
2.4.2. Gas permeability measurements
The gas permeance (cm3 m−2 day−1 bar−1) determination was
performed using manometric method, on a permeability testing appli-
ance, Brugger, Type GDP-C (Brugger Feinmechanik GmbH, Munich,
Germany). The increase in pressure during the test period was assessed
and displayed by an external computer. The data were recorded and the
permeance was calculated by a GDP-C Software (with temperature
compensation connection). The sample temperature (25 °C) was ad-
justed using a waterbath (HAAKE F3 with Waterbath K). The perme-
ability of oxygen (PO2) was calculated as the arithmetic product of the
permeance and of the film thickness and expressed in (cm3 m−1
day−1 Pa−1).
2.4.3. Water vapour permeability measurement (WVP)
The WVP of films was determined gravimetrically using a modified
ASTM E96-80 (1980) standard method, adapted to edible materials by
Debeaufort, Martin-Polo, and Voilley (1993), using the relative hu-
midity differential of 30–100% RH and the temperature of 25 ± 1 °C.
Prior to the WVP measurements, all the film samples were equilibrated
at 25 ± 1 °C and RH 70% for 72 h. The film samples were then placed
between two Teflon rings on the top of the glass cell containing distilled
water (RH∼100%) and the cells were stored at RH∼30% in a venti-
lated chamber (Memmert HPP110, Memmert Germany) maintained at
25 ± 1 °C. WVP (g m−1 s−1 Pa−1) was calculated, from the change in
the cell weight versus time at the steady state, using the following
equation:
Δm
WVP = ⋅x
Δt⋅A⋅Δp (1)
where Δm/Δt is the weight of moisture loss per unit of time (g s−1), A is
the film area exposed to the moisture transfer (9.08 × 10−4 m2), x is
the film thickness (m), and Δp is the water vapour pressure difference
between the two sides of the film (Pa). Three replicates for each film
type were done.
2.4.4. Mechanical properties
A universal traction testing machine (Stable Micro Systems Texture
Analyser TA.HD.plus) was used to determine the tensile strength (TS, in
MPa), Young's modulus (YM, in MPa) and percentage of elongation at
breakpoint (E, %) according to ASTM standard method D882 (ASTM,
1992). The film samples were cut as rectangular samples of 1 cm wide
and 5 cm long by a special cutter (Thwing–Albert JDC Precision Sample
Cutter; Rycobel, Deerlijk, Belgium) able to prepare test samples of film
to an exact width and parallel throughout the entire length. Before
testing, all the samples were equilibrated for 7 days at 50% of relative
humidity at 25 °C. Equilibrated film specimens were mounted in the
extension grips of the testing machine and stretched uniaxially at a rate
of 50 mm min−1 until breaking. TS, YM and E were computer recorded
from stress–strain curves. For each film five replicates were tested.
2.4.5. Water solubility
The film solubility in water was determined according to the
method reported by others (Cuq, Gontard, Cuq, & Guilbert, 1996). It
240
Food Hydrocolloids 84 (2018) 238–246
was defined by the content of dry matter solubilised after 24 h im-
mersion in distilled water. Three discs of each film (2 cm diameter)
were cut and dried to constant weight in an oven at 105 °C in order to
determine the initial dry matter content (Wi). Film discs were immersed
in 30 mL of distilled water at 25 °C. After 24 h of immersion, film pieces
were taken out and dried to constant weight in an oven at 105 °C in
order to determine the final (Wf) weight of dry matter which was not
solubilised in water. The film solubility (FS, %) was calculated using the
following equation:
Wi − Wf
FS (%) = ⋅100
Wi (2)
The average value of solubility was calculated as the mean of four
measurements for each film.
2.4.6. Film colour and opacity
The colour of the film was determined using a colorimeter (Konica
Minolta Spectrophotometer CM-3500d). Hunter L*, a*, and b* values
were averaged from three readings across for each sample, and then the
total colour difference (ΔE) was calculated according to Ghorpade, Li,
Gennadios, and Hanna (1995).
The film opacity was determined by measuring the film absorbance
at 600 nm with a spectrophotometer (UV160U, Shimadzu Corporation,
Japan). Results were reported as absorbance divided by film thickness
based on three replications. Three film specimens were used for each
replicate.
2.5. Film evaluation as a pH indicator
To verify the colour response of films, the method described by Choi
et al. (2017) was used with slight modifications. Films were exposed to
solutions at pH values 2, 4, 5, 6, 7, 10 and 12 and the colour changes
were measured using a colorimeter (Konica Minolta Spectrophotometer
CM-3500d). The colour parameters of films not exposed to pH solutions
were also measured and considered as a reference.
2.6. Antioxidant film properties
2.6.1. HPLC
Separation of phenolics was performed by HPLC analysis, using the
HPLC Agilent 1260 quaternary LC Infinity system (Agilent
Technologies, Santa Clara, CA, USA) equipped with diode array de-
tector (DAD), an automatic injector and ChemStation software on a
Nucleosil 100-5C18, 5 μm (250 × 4.6 mm i.d.) column (Macherey-
Nagel). The solvent composition and used gradient conditions were
described previously by Elez Garofulić et al. (2015). Identification of
phenols was carried out by comparing retention times and spectral data
with those of the authentic standards (anthocyanins were identified at
520 nm, flavonol glycosides at 360 nm and phenolic acids at 280 nm).
The quantifications of anthocyanins, flavonol glycosides and phe-
nolic acids were performed by the external standard method using the
calibration curves of the standards. Quantifications of tentative iden-
tified compounds were done as follows: delphinidin derivative was
determined according to delphinidin-3-glucoside, cyaniding derivative
according to cyanidin-3-glucoside, petunidin derivative according to
petunidin-3-glucoside, pelargonidin derivative according to pelargo-
nidin-3-glucoside, peonidin derivative according to peonidin-3-gluco-
side and malvidin derivatives according to malvidin-3-glucoside cali-
bration curve. Quercetin glycosides were calculated according to
quercetin-3-glucoside calibration curve and kaempherol glycosides ac-
cording to the kaempherol-3-rutinoside. Hydroxycinnamic acid deri-
vative and neochlorogenic acid were expressed according to the
chlorogenic acid calibration curve. Obtained concentrations were ex-
pressed as mg per g of pomace, as mean value of three replications.
M. Kurek et al.
2.6.2. Total phenol contents (TPC)
Total phenolic content (TPC) of blueberry and blackberry pomace
films was determined using Folin-Ciocalteu method previously reported
by Shortle et al. (2014) with slight modifications. 50 mg of each film
sample were dissolved in 10 mL of acetic acid (1% v/v) and then,
100 μL of that solution were mixed with 200 μL of Folin–Ciocalteu re-
agent and 2 mL of distilled water. Afterwards, 1 mL of 20% Na2CO3 was
added. This mixture was incubated at 50 °C for 25 min. The absorbance
was measured at 765 nm by spectrophotometer (model UV-1600PC;
VWR International, Leuven, Belgium). The blank contained 100 μL of
the solvent used for extraction instead of the extract. Extraction solvent
was used instead of fruit extract for blanks, and the analytical curve was
prepared with gallic acid. The total phenolic content was expressed as
mg of gallic acid equivalent (GAE)/g of prepared film, or mg of gallic
acid equivalent (GAE)/g of pomace. All measurements were performed
in triplicate.
2.6.3. Antioxidant activity of films
The antioxidant capacity of films as well as blueberry and black-
berry pomace was measured using a modified ferric reducing anti-
oxidant power (FRAP) method (Shortle et al., 2014). The FRAP reagent
was prepared immediately prior to the determination by mixing 0.3 M
acetate buffer (pH = 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ) solution
and 20 mM FeCl3·6H2O solution in the ratio of 10:1:1 (by volume),
respectively. All reagents including standards were incubated at 37 °C
prior to analyses. A volume of 300 μL of film or pomace extract was
mixed with 2.25 mL of FRAP reagent and incubated at 37 °C for 10 min.
The absorbance was measured at 593 nm. The blank contained 300 μL
of the solvent used for extraction instead of the extract. FRAP values
were calculated according to a standard curve of ascorbic acid and the
results were expressed in mg of ascorbic acid equivalents (AAE) per g of
prepared films, or per g of pomace. All results were multiplied by 2,
since ascorbic acid can accept two electrons while the reduction from
Fe3+ to Fe2+ ions requires one (Fegredo, Wong, Wiseman, & Preedy,
2009). All measurements were performed in triplicate.
2.7. Statistical analysis
The statistical analysis of the data was performed through variance
analysis (ANOVA) using Xlstat-Pro (win) 7.5.3. (Addinsoft, New York).
The data were ranked and statistical differences were evaluated on the
ranks with a one-way analysis of variance (ANOVA) and Tukey's mul-
tiple comparison tests. In all cases, a value of p < 0.05 was considered
to be significant.
3. Results and discussion
3.1. Oxygen and water vapour permeability
Experimental results on oxygen and water vapour barrier properties
of neat chitosan films and those with different concentrations of blue-
berry and blackberry pomace extracts are given in Table 1. Oxygen
permeability of biobased films, potentially aimed to be used as edible
coatings, is important in order to preserve packed food item. Low
barrier to oxygen presents a key factor for oxidation which causes nu-
tritional and sensorial changes of food product. So, the efficiency of
chitosan films strongly depends on its gas barrier and water vapour
barrier properties.
In general, chitosan films present good oxygen barrier. The oxygen
permeability coefficient of CS films ((8.47 ± 0.76) x 10−10 cm3 m−1
day−1 Pa−1), was similar (6.13 × 10−10 cm3 m−1 day−1 Pa−1 and
7.11 × 10−10 cm3 m−1 day−1 Pa−1) to those reported by Kurek et al.
(2014) and Muzzarelli and Rocchetti (1974), respectively. It was sig-
nificantly lower than 5.27 × 10−7 cm3 m−1 d−1 Pa−1 found by
Sathivel, Liu, Huang, and Prinyawiwatkul (2007) and from 0.08 to
31.07 × 10−3 cm3 m−1 d−1 atm−1 reported by Caner, Vergano, and
241
Food Hydrocolloids 84 (2018) 238–246
Table 1
Oxygen transmission rate and water vapour permeability of chitosan based
films.
Film type PO2 × 10−10 Thickness (μm) WVP × 10−10
(cm3 m−1 day−1 Pa−1) (g m−2 s−1 Pa−1)
CS 8.47 ± 0.76b 53.1 ± 10.24a,b 2.39 ± 0.07a
CSB1 49.44 ± 10.43a 49.5 ± 0.14a,b 1.99 ± 0.04b.c
CSB2 6.82 ± 1.56b 48.3 ± 0.71b 1.89 ± 0.11c
CSB4 9.15 ± 1.89b 60.73 ± 9.81a,b 1.95 ± 0.17b.c
CSK1 12.39 ± 5.56b 54.67 ± 0.63a,b 2.24 ± 0.06a.b
CSK2 6.28 ± 0.76b 48.2 ± 0.14b 1.96 ± 0.17b.c
CSK4 8.76 ± 0.64b 63.85 ± 0.78a 1.97 ± 0.09b.c
Different superscripts (a–c) within a column indicate significant differences
among samples (p < 0.05).
Wiles (2006). Differences with literature data might be due to different
polymer chains structure with lower volume of present voids. It is im-
portant to note that these values are significantly lower than those of
commercial polymer films, including LDPE (1.94–4.3 × 10−6 cm3 m−1
day−1 Pa−1) (Robertson, 2013), HDPE (3.9–17.5 × 10−7 cm3 m−1
day−1 Pa−1) (Hernandez, 1997), PA (1 × 10−8 cm3 m−1 day−1 Pa−1)
(Hernandez, 1997), BOPP (1.09 × 10−7 cm3 m−1 day−1 Pa−1), and
comparable to those of high barrier EVOH film (3 × 10−11 cm3 m−1
day−1 Pa−1) (Hernandez, 1997). No significant changes in film oxygen
permeability (PO2) were observed after the incorporation of both ex-
tracts. Results reported in the scientific literature are different de-
pending on the concentration and the type of the active compound
added. Likewise, some authors reported that antioxidants like ferulic
acid and ethyl ferulate as well as propolis extract led to decrease in
chitosan permeability due to the interactions between active compound
and polymer matrix (Aljawish et al., 2016; Siripatrawan &
Vitchayakitti, 2016), while others (Kurek et al., 2014) stated that PO2
increased with the addition of carvacrol due to the increased mobility of
chitosan chains after the incorporation of antioxidant. We could assume
that in this study the modification of chitosan matrix was not so im-
portant to influence the permeability to oxygen.
Results on water vapour permeability (WVP) are also given in
Table 1. This is important property that may play key function in de-
teriorative reactions in food. WVP of control chitosan was
2.39 × 10−10 g m−1 s−1 Pa−1 that was close to other studies reported
in the literature (2.92 × 10−10 g m−1 s−1 Pa−1 by Sathivel et al.
(2007), p. 4.9 × 10−10 g m−1 s−1 Pa−1 by Park (1999)). These values
are significantly higher than those of commercial polymer films, in-
cluding LDPE (9.28 × 10−13 g m m−2 s−1 Pa−1) (Robertson, 2013), PA
(7.65 × 10−12 g m m−2 s−1 Pa−1) (Hernandez, 1997), polypropylene
(6.5 × 10−13 g m m−2 s−1 Pa−1) and polyvinyl chloride
(7.1 × 10−13 g m m−2 s−1 Pa−1) (Park, 1999). Water vapour perme-
ability slightly decreased with the addition of extracts (from 6% for
CSK1 to 21% for CSB4). The lower WVP of the films incorporated with
different extracts may be due to the hydrogen and covalent interactions
between chitosan network and polyphenolic compounds which reduce
the availability of the hydrophilic groups and lead subsequently to a
decrease in the affinity of chitosan film towards water molecules. Si-
milar findings were observed by Park and Zhao (2006) and Balti et al.
(2017) who found that the incorporation of mineral or vitamin into a
biobased film matrix increased the interactions among adjacent mole-
cules leading to a decrease in diffusivity of water vapour through the
film matrix and a decrease in their hydrophilicity. Gómez-Guillén, Ihl,
Bifani, Silva, and Montero (2007) also reported a decrease in avail-
ability of hydrogen groups due to cross-linking between tuna-fish ge-
latine and antioxidant extracts from murta leaves, while others (Silva-
Pereira, Teixeira, Pereira-Júnior, & Stefani, 2015) showed that the ad-
dition of red cabbage extract to chitosan/starch blends did not sig-
nificantly change water vapour barrier.
M. Kurek et al. Food Hydrocolloids 84 (2018) 238–246

Table 2
Mechanical properties (E, Elongation at break, YM, Young modulus and TS, Tensile strength), moisture content and water solubility of chitosan based films.
Film type E (%) YM (MPa) TS (MPa) Moisture content (% H2O) Film solubility in water (%)

a c a,b a
CS 76.95 ± 15.09 35.27 ± 3.60 23.86 ± 6.01 26.31 ± 1.09 34.04 ± 2.12a,b
CSB1 50.16 ± 12.24b,c 59.18 ± 7.24a,b 21.97 ± 11.80a,b 25.33 ± 0.75a,b 33.74 ± 6.88a,b
CSB2 48.79 ± 7.59b,c 65.06 ± 7.51a,b 31.62 ± 5.56a 24.83 ± 0.94a,b 41.02 ± 8.36a
CSB4 37,24 ± 7.79b,c 51.18 ± 6.13b 19.35 ± 5.82a,b 23.53 ± 0.25b,c 23.96 ± 0.93b
CSK1 49.20 ± 17.71b,c 46.81 ± 9.62b,c 26.22 ± 10.03a,b 21.79 ± 0.22c 40.46 ± 7.54a
CSK2 26.47 ± 2.97c 69.22 ± 11.74a 18.17 ± 2.68b 23.18 ± 0.42b,c 32.71 ± 4.57a,b
CSK4 53.30 ± 6.52a,b 61.08 ± 5.60a,b 32.35 ± 2.61a 21.07 ± 0.62c 30.63 ± 4.94a,b

Different superscripts (a–c) within a column indicate significant differences among samples (p < 0.05).

Table 3
Opacity and colour changes (L*, a*, b*, ΔE) in different pH indicators of chitosan based films.
Film sample Visual film colour change L* a* b* ΔE Opacity

CS 32.14 ± 0.76a −0.29 ± 0.03d 0.82 ± 0.45a 0e 1388.58 ± 1.09g

CSB1 28.17 ± 0.29b −1.10 ± 0.05e 0.72 ± 0.57a,b 4.08 ± 0.26d 3155.56 ± 14.57f

CSB2 26.08 ± 1.17c,d −1.35 ± 0.11f 0.76 ± 0.05a 6.15 ± 1.13b,c 6523.81 ± 2.93d

CSB4 25.49 ± 0.09c,d −1.45 ± 0.02f 0.01 ± 0.01a,b 6.79 ± 0.08a,b 11151.00 ± 2.33b

CSK1 27.07 ± 0.41b,c 0.01 ± 0.03c −0.3 ± 0.68b 5.23 ± 0.24c,d 6273.39 ± 5.59e

CSK2 25.59 ± 0.21c,d 0.22 ± 0.03b −0.22 ± 0.07a,b 6.65 ± 0.19a,b 7647.99 ± 1.19c

CSK4 24.61 ± 0.23d 0.58 ± 0.02a −0.12 ± 0.05a,b 7.63 ± 0.22a 11211.69 ± 28.25a

Different superscripts (a–d) within a column indicate significant differences among samples (p < 0.05).

3.2. Mechanical film properties
In order to check if the addition of extracts influenced mechanical
behaviour of produced films, tensile strength (TS), elongation at break
(E) and Young modulus (YM) were determined. Results are given in
Table 2. Based on the requirements for packaging materials, films must
have a certain degree of resistance. Mechanical properties reflect the
ability of chitosan matrix to maintain a good integrity. TS, E and EM
could be used to describe how the mechanical properties are related to
film's chemical structure. TS value indicates the maximum tensile stress
that the film can sustain, E is the maximum change in length of a film
before breaking, and EM is a measure of the stiffness of the film.
Control chitosan film had tensile strength around 24 MPa that was
higher to those reported by Ferreira et al. (2016) probably due to dif-
ferent glycerol content. The tensile strength of chitosan based films was
not significantly affected by the addition of both extracts. Similar re-
sults were found for the addition of spirulina (Balti et al., 2017) and
grape pomace extracts (Ferreira, Nunes, Castro, Ferreira, & Coimbra,
2014) to chitosan films where at lower extract concentrations as in our
study (2.5%) tensile strength did not significantly change and it was
around 30 MPa (Balti et al., 2017).
YM is a measure that gives information about the film stiffness and
it increased with the addition of extract but no significant changes
occurred by increasing the concentration of the extracts.
The percent of elongation was lower than in pure chitosan films.
The value of E obtained by different authors are very diverse, de-
pending on the film preparation conditions (Ferreira et al., 2014). De-
crease in elongation can be ascribed to the crystalline formation of
accessible polyphenolic components in the chitosan matrix because of
nonbonding of these two substances. So, the film flexibility is reduced.
Similar results were reported by Pastor, Sánchez-González, Chiralt,
Cháfer, and González-Martínez (2013) and Siripatrawan & Vitchayakitti
(2016). These authors found that elongation decreased at high propolis
concentration. These differences may be attributed to the type of chit-
osan (solvent and molecular weight) used and the particular interac-
tions with the natural components which, in turn, are affected by re-
lative humidity, the presence of surfactants, temperature, etc.
3.3. Film solubility and water content
Results on film solubility and water content are given in Table 2.
Films with CSK4 to CSB1 extracts had slightly lower moisture content
(21.07%–25.33%, respectively) than control chitosan film (34.04%).
Among active film formulations, those with blackberry pomace extracts
(K) had lower values than films with blueberry pomace extracts (B). The
amount of water present in films provide an indication of the hydro-
phobicity of the films, hence, the hydrophilic films have higher
moisture content (Bourbon et al., 2011).
Water solubility is also an important property of edible films that
gives indication of the film's water affinity (Bourbon et al., 2011). No
significant changes occurred in active films with lower extract content
(1 and 2%) (Table 2). With increasing the extract concentration, solu-
bility decreased and it was the lowest for CSB4 films (23.96%). Re-
duction in the solubility with higher extract concentration might be due
to a decrease in the hydrophobic nature because of the loss of chitosan's
free functional groups (amino and hydroxyl). This change can indicate

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Fig. 1. Visual colour change of pH indicator films at different extract concentration and different pH.

Table 4
Colour parameters of pH indicator chitosan based films (with 2% of extracts) influenced by pH variations.
pH CSB2 CSK2

L* a* b* ΔE L* a* b* ΔE

2 24.95 ± 0.01a 0.69 ± 0.01a −1.22 ± 0.01b 7.53 ± 0.01b,c 23.20 ± 0.10a,b 0.76 ± 0.19b −0.14 ± 0.43a,b 9.05 ± 0.12a,b
4 25.68 ± 0.17a 0.58 ± 0.03a −0.35 ± 0.28a 6.53 ± 0.14c 23.23 ± 0.02a,b 1.89 ± 0.07a 0.27 ± 0.07a,b 9.18 ± 0.04a,b
5 25.08 ± 0.19a −0.26 ± 0.01b 0.03 ± 0.13a 7.10 ± 0.19c 25.09 ± 2.17a 1.58 ± 0.25a 0.28 ± 0.42a 7.36 ± 1.95b
6 23.87 ± 0.39b −1.25 ± 0.09c 0.51 ± 0.11a 8.33 ± 0.39b 22.9 ± 0.67a,b 0.15 ± 0.07c,d −0.42 ± 0.44a,b 9.34 ± 0.59a,b
7 23.83 ± 0.01b −1.35 ± 0.01c 0.64 ± 0.02a 8.25 ± 0.22b 24.50 ± 2.64a,b 0.55 ± 0.16b,c −0.39 ± 0.28a,b 7.81 ± 2.49b
10 21.53 ± 0.67c −1.77 ± 0.17d 0.5 ± 0.25a 10.71 ± 0.66a 21.25 ± 0.23a,b −0.50 ± 0.02e −0.40 ± 0.19a,b 10.96 ± 0.21a,b
12 24.77 ± 0.35a −1.47 ± 0.08c 0.57 ± 0.19a 7.46 ± 0.32b,c 20.34 ± 0.50b −0.04 ± 0.13d −0.95 ± 0.29b 11.93 ± 0.47a

Different superscripts (a–d) within a column indicate significant differences among samples (p < 0.05).

certain structural changes in the film matrix that influenced difficult
extraction of polyphenols for determination of total phenolic content,
especially at higher concentrations. Similar behaviour was noticed
when cinnamon oil was incorporated in chitosan films (López-Mata
et al., 2015) while Ferreira et al. (2014) didn't notice any significant
changes when aqueous grape pomace extract was added to chitosan
film. This indicates that behaviour depends strongly on the extract
composition and its degree of bonding to chitosan matrix.
3.4. Colour changes in films used as pH indicators
Films with different concentrations of blueberry and blackberry
pomace natural extracts were prepared in order to verify if they can
visually show the changes in the pH environment. Table 3 shows visible
colour changes of fresh and dry pH indicator film samples. The film
colour was visibly transformed from pale yellow for control (CS) film to
blue-green, CSB4 (with negative a* value) and purple, CSK4 (with

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Table 5 3.5. Colour response of pH indicator films

Composition of blueberry and blackberry pomace extracts as determined by
HPLC. Colour response of pH indicator films was evaluated by immersing
Blueberry pomace extract mg g−1 Blackberry pomace extract mg g−1 film samples in different pH buffer solutions (pH 2.0–12.0). Pictures are
given in Fig. 1. It is important to note that visually some samples seem
Anthocyanins to be damaged but that is because of mechanical failures due to
Delfinidin der 1.3604 Cyanidin der. 10.2408
handling and not because of their resistance to water (see solubility
Cyanidin der 0.5353 Malvidin der. 0.8003
Petunidin der 0.3587 Pelargonidin der. 2.8569 measurement). Colour changes are related to the colour parameters L,
Peonidine der 0.0722 a*, b* and the intensity increased for all samples at all pH values with
Malvidin der 1 1.7892 increasing concentration. Visible colour changes of each sample range
Malvidin der 2 0.5377
from bright red (pH 2, pH4) > blue/green (pH 5, pH 6, pH7) > dark
Total 4.6537 13.8979
Flavanol glycosides
green (pH 10, pH12) for blueberry (B) samples and from bright red
Quercetin glycoside 1 0.1807 Quercetin glycoside 1 0.2175 (pH2, pH4) > violet (pH 5, pH 6, pH 7) > dark blue (pH10, pH 12)
Quercetin glycoside 2 0.0723 Quercetin glycoside 2 0.2862 for blackberry (K) samples. From Table 4 it is evident that significant
Kaempferol glycoside 1 1.5230 Kaempferol glycoside 1 0.5371 variations in a* and b* as a function of pH buffers indicate that film
Kaempferol glycoside 2 0.0857 Kaempferol glycoside 2 0.4538
colour strongly depends on the pH environment. The analysis was done
Kaempferol glycoside 3 0.2017
Kaempferol glycoside 4 0.2148 for all films; however, only the results for the films with 2% extract are
Total 2.2782 1.4946 given in Table 4. Films with higher concentrations of extract had more
Phenolic acids pronounced changes and those with lower concentrations of extract
Chlorogenic acid 4.0673 p-coumaric acid 0.1377
showed smaller changes. This was consistent for all extract concentra-
Hydroxycinnamic acid der. 0.0604 Neochlorogenic acid 0.3982
Chlorogenic acid 0.1987
tions (data not given). It is observed that a* has higher values at
Caffeic acid 0.4790 pH < 7 while it is significantly lower at pH 10 (−1.77) and 12
Ellagic acid 0.0649 (−1.47). This means that the green colour has greater intensity at
Total 4.1277 1.3419 higher pH values. Similar observations were given by other authors for
chitosan/starch/anthocyanin films and chitosan/pectin films (Maciel,
Yoshida, & Franco, 2015; Pereira et al., 2015; Yoshida et al., 2014),
Table 6
who noticed that with anthocyanins films changed colour to blue green
Total polyphenol content and scavenging activity (AAE) of chitosan based films.
in the range of pH 8–10.
SAMPLES Antioxidant activity Total phenolic content In CSK films colour change was more pronounced due to b* changes
(mg AAE gfilm−1) (mg gfilm−1) as those films had blueish intensity rather than greenish as it was for
Blueberry extract 15.87 ± 0.12b 6.00 ± 0.04b,c CSB films (Table 4). In another study, Luchese et al. (2017) used starch
Blackberry extract 83.78 ± 1.07a 24.85 ± 0.11a as a matrix for incorporation of blueberry anthocyanins extract. Visual
CS 1.49 ± 0.01g Nm appearance of films was similar that of CSK films in this study. Differ-
CSB1 5.84 ± 0.03f 4.17 ± 0.19f ences could be due to the different source of colorants. Actually, in this
CSB2 7.73 ± 0.02e 5.74 ± 0.21c
study different compounds contribute to major colour changes since the
CSB4 10.16 ± 0.09d 5.63 ± 0.25c,d
CSK1 6.25 ± 0.12e,f 5.02 ± 0.17e whole fruit pomace extract was used in contrast to other studies where
CSK2 10.03 ± 0.21d 5.25 ± 0.06d,e commercial (synthetic) anthocyanes with higher degree of purity were
CSK4 13.38 ± 0.18c 6.21 ± 0.14b used.
Theoretically, colour variations are related to different structural
Nm, not measured. Different superscripts (a–g) within a column indicate sig-
changes of anthocyanins that are strongly pH dependent (He, Li, Lv, &
nificant differences among samples (p < 0.05).
He, 2015; Khoo, Azlan, Tang, & Lim, 2017). In strong acid environment
(pH < 2) predominant form of anthocyanes is the flavylium cation that
positive a* values) with the addition of blueberry (B) and blackberry
contributes to red colours. Quinonoidal blue forms appear when pH
(K) extracts, respectively. Overall colour differences between CSB and
changes from 2 to 4. Further loss of protons in the flavylium cation
CSK samples probably occurred because the majority of anthocyanins in
contributes to its hydration in the range of pH 5 resulting in colourless
blueberry and blackberry are different (Table 5). Green taint is prob-
carbinol pseudobase and a chalcone form (open form) (Castañeda-
ably due to the high chlorogenic acid content in CSB, while purpleness
Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-
in CSK is accorded mostly to cyanidin that is violet at neutral pH. The
Vidal, 2009). Mixture of flavylium cation, quinonoidal base, carbinol
colour intensity increased with increasing the extract concentration
base, and chalcone in their equilibrium exists at pH values between 4.0
(from 1 to 4%). All the samples with added extracts were significantly
and 6.0 (He et al., 2015). As pH shifts to base alkaline conditions
different from the control sample with ΔE ranging from 4.08 for CSB1
(pH > 8) anhydro bases become predominant forms (Hui & Sherkat,
film to 7.63 for CSK4 film. This result confirms that there are visually
2005).
perceptible differences to the human eye between the samples, since the
These results demonstrate that blueberry and blackberry pomace
overall colour difference was higher than 3.0 (Lee & Wrolstad, 2004;
extracts could be used as visual intelligent packaging indicator as sensor
Luchese et al., 2017). By increasing the concentration, a* parameter in
for easy and rapid detection of food quality degradation.
CSB films becomes more negative. b* parameter for blueberry added
films is positive with decreasing trend by increasing concentration,
3.6. Antioxidant film properties
while for blackberry it is negative indicating that yellow intensity in-
fluences the final film colour. Blackberry added films were opaquer
In this study the antioxidant extracts were made by microwave as-
than blueberry films, while transparency decreases with increasing the
sisted extraction in order to improve the extraction process and to
extract concentration in both extract samples (Table 3). This result is
minimise degradation of active compounds. Indeed, it is known that
similar to others where authors reported decreasing trend in opacity
microwave-assisted extractions provide significant advantages in terms
values after incorporation of active agents like pomegranate rind ex-
of extraction efficiency and time savings.
tract (Qin et al., 2015), cinnamon oil (López-Mata et al., 2015), thinned
Composition of prepared antioxidant extracts measured by HPLC is
young apple polyphenols (Sun et al., 2017) etc.
given in Table 5. Major components in blueberry pomace extract pre-
sent anthocyanins (42%) and phenolic acids (37%) while in blackberry

244
M. Kurek et al.
majority present anthocyanins (83%). Even though similar compounds
are present, the chemical composition can be different in other extracts
mostly due to genotypic variation or climatic conditions in which the
plant has been grown up (Stevenson & Scalzo, 2012).
Table 6 shows the total polyphenol contents (TPC) and the
scavenging activity of pure extracts and chitosan based films. Blueberry
pomace extract showed an average polyphenol content of
6.08 ± 0.04 mg g−1 while blackberry was significantly higher with
24.85 ± 0.11 mg g−1. The phenolic contents can be used as an im-
portant indicator of antioxidant capacity so they can be used as a kind
of screening for different products if they are meant to be used as an-
tioxidants in food packing materials (Qin et al., 2015; Viuda-Martos
et al., 2011). The influences of species, cultivar, and maturity on anti-
oxidant capacity, total anthocyanin, and total phenolic contents in
fruits is well known in the scientific literature (Wang & Lin, 2000).
As expected, when extracts were added to chitosan films an increase
in polyphenol content was also determined. Surprisingly, only slight
differences in TPC were seen at different concentrations. This was
probably due to the fact that chitosan films with higher extract con-
centrations showed lower solubility because of the modification in the
film structure. Then, the assumption is that not all polyphenols were
sufficiently extracted. They might be cross-linked with chitosan.
Table 6 shows the antioxidant activity of chitosan based films with
and without incorporated blueberry and blackberry pomace extracts,
determined by ferric reducing antioxidant power (FRAP) essay. Results
showed that the antioxidant activity of films significantly increased
(p < 0.05) with the addition of extracts. Chitosan film itself showed
very low antioxidant activity. In the literature it was reported that
scavenging mechanism of chitosan is due to the residual free amino
(NH2) groups that can act with free radical to form stable macro-
molecule radicals, and the NH2 groups can form ammonium (NH3+)
groups by absorbing a hydrogen ion from the solution (Yen, Yang, &
Mau, 2008). The results of Tamer, Valachová, Mohyeldin, and Soltes
(2016) show that free radical scavenging activity of chitosan was in-
creased via amination process. Promotion of antioxidant activity may
be attributed to replacing hydroxyl groups with free amine groups.
4. Conclusion
In the present study smart chitosan films were successfully devel-
oped. The active character was attributed to the strong antioxidant
activity of incorporated blueberry and blackberry pomace extracts
while the intelligent part was due to the colour change because of
present anthocyanes. The whole concept of film production can be
considered as eco-friendly attributing to the reduction of food wasted
material (fruit pomace). Microwave assisted extraction was used as
green technology in order to extract valuable compounds from gen-
erally wasted material, fruit pomace. Blueberry and blackberry pomace
indeed showed excellent antioxidant potential with high amounts of
total phenols. Their activity was not diminished after the production of
biobased chitosan films. The antioxidant activity also increased with
the increase of the extract concentration. Due to the presence of an-
thocyanes in both fruit pomace extracts, films showed the intelligent
character as well. Significant visual colour change occurred when films
were exposed to different pH solutions. Hence films had good colour
variation, depending on the pH value, which indicates good visual
colour variability. This also indicates the potential of its use as in-
telligent films in real food. Oxygen permeability and mechanical
properties were not significantly changed after the extract incorpora-
tion. This is important to know if these kinds of films are going to be
used as antioxidant/pH as sensing food coatings. Further experiments
are needed in order to confirm the antioxidant activity and pH sensing
on real food products. Antioxidant activity could be useful in pre-
venting the oxidation of fatty acids in meat.
245
Food Hydrocolloids 84 (2018) 238–246
Acknowledgements
Authors wish to thanks Mira Bunčić, technical associate, Laboratory
for Physical Chemistry, PBF for technical support. Authors wish to
thanks Zoran Zorić, Ph.D, from Centre for Food Technology and
Biotechnology, Zadar, Croatia, for contribution regarding the HPLC
analysis of phenolic composition of blueberry and blackberry pomace.
Authors also wish to thanks to Tomislav Bosiljkov, Ph.D., Goran
Bosanac, Eng., and Darjan Prpić, technical associate from Laboratory
for Unit Operations, PBF for contribution regarding determination of
mechanical properties and colour parameters.
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