International Journal of Biological Macromolecules 220 (2022) 866–877

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Active-intelligent and biodegradable sodium alginate films loaded with

Clitoria ternatea anthocyanin-rich extract to preserve and monitor
food freshness
Luan Gustavo Santos, Gisele Fernanda Alves-Silva, Vilásia Guimarães Martins *
Laboratory of Food Technology, School of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, 96203-900, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of this study was to develop and characterize sodium alginate films loaded with 10–40 % Clitoria ternatea
Smart packaging extract (CTE) and apply to monitoring the quality of milk, pork and shrimp. Films loaded with CTE showed high
Active packaging light barrier capacity and improved tensile strength by 3.8 times over control films. The incorporation of CTE in
Freshness indicator
alginate films improved the thermal stability of the materials due to intermolecular interactions and crosslinking
Anthocyanins
Biodegradable packaging
of polymeric networks. The addition of 40 % of CTE generated films with antibacterial action against E. coli. The
alginate films showed biodegradable characteristics in soil and beach sand in 15 days. The food simulant test
revealed that the loaded films show good compatibility with aqueous and acidic foods due to the release of
higher levels of polyphenols and anthocyanins. The films showed great colorimetric potential due to their ability
to change color at different pH (pink-green), ammonia gas (blue-green) and sterilization process (blue-yellow).
When the film loaded with 40 % CTE (F40) was applied to monitor the freshness of milk and meat products
(shrimp and pork), its blue color changed to purple and green, respectively. Therefore, the F40 has great po­
tential to be used as a biodegradable indicator of freshness.

1. Introduction used as food packaging [3,7–9]. The incorporation of bioactive com­

Synthetic petroleum-based polymers are widely used in the pack­
aging industries due its great capacity to form materials with good
mechanical performances and barriers, protecting the packaged food
from external conditions (e.g., moisture, light, oxygen and mechanical
stress) [1]. These synthetic packaging are not biodegradable materials,
being associated with several environmental impacts due to the low
recycling rate [2,3]. In this way, materials developed with natural
polymers (e.g., polysaccharides and proteins) have non-toxic, low cost,
biocompatible and biodegradable characteristics, being an environ­
mentally friendly alternative for the production of food packaging and
replacement of synthetic materials [4,5].
Sodium alginate is a linear-chain polysaccharide obtained mainly
from brown algae and has great ability to form flexible films, hydro­
philic and resistant to oil and oxygen permeability, being able to protect
and maintain food quality and safety [3,6]. However, alginate films have
poor mechanical properties compared to synthetic films, and can be
loaded with bioactive compounds (e.g., vitamins, peptides, essential oil,
phenolic compounds) as a technique to improve their performance to be
pounds in biopolymer films generates materials with active and intelli­
gent characteristics, capable of prolonging and monitoring the quality of
the packaged food, respectively [2].
Active packages are materials loaded with active components that
interact with the food or the empty space inside the package, reducing
the deterioration process caused by microorganisms, enzymes or
chemical oxidation reactions [4]. Previous studies show that the incor­
poration of plant extracts in biopolymer packaging increases their
antioxidant and antimicrobial activity, making them able to extend the
shelf-life of foods [3,4,10,11].
Smart or intelligent packaging is responsible for detecting environ­
mental changes (e.g., humidity, gas and pH) of the food during storage,
informing the consumer of the quality index of the packaged product
[12]. The smart systems can be inserted as labels, printing or incorpo­
rated into the matrix of food packaging and act as O2 and CO2 detectors,
pH-sensing indicators and time-temperature sensors due to the presence
of dyes or enzymes [1,13]. In the past, intelligent packaging was
developed with the incorporation of synthetic dyes in the polymer ma­
trix, however, the release of these chemical components can

* Corresponding author.
E-mail addresses: luansantos.ea@outlook.com (L.G. Santos), giferalves@gmail.com (G.F. Alves-Silva), vilasiamartins@gmail.com (V.G. Martins).

https://doi.org/10.1016/j.ijbiomac.2022.08.120
Received 9 May 2022; Received in revised form 16 August 2022; Accepted 17 August 2022
Available online 20 August 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
L.G. Santos et al.
compromise food safety, cause diseases in consumers and environmental
problems [12]. In this way, intelligent packaging has been manufactured
from the addition of natural-dyes sensitive to pH, temperature and gases
[5,10,12,14].
Anthocyanins are classified as phenolic compounds and represent a
wide group of water-soluble natural-dyes responsible for different colors
(e.g., red, blue and purple) of flowers, leaves, skins and pulps of various
vegetables [15]. There are over 600 types of anthocyanins in nature, of
which cyanidin, petunidin, petunidin, pelargonidin, delphinidin and
malvidin are the six commonly found in vegetable and food waste
[6,16]. Recent studies show that the incorporation of anthocyanins in a
biopolymer matrix contributes to the improvement of packaging per­
formance (mechanical, barrier and physical properties) due to electro­
static interaction and hydrogen bonding [3,10,17]. Moreover,
antioxidant and metal chelating characteristics of the anthocyanins
contribute to the development of active packaging, delaying the oxida­
tion process of packaged foods [4]. The anthocyanins are capable of
changing color according to environmental conditions (e.g., tempera­
ture, ammonia content and pH), being widely used in the development
of freshness indicators [10,12,18,19]. Active-intelligent films incorpo­
rated with anthocyanins from fruits [10], seeds [4], bark [3] and flowers
[19] have been reported and show that the source of anthocyanins in­
fluences the functional and physical properties of the materials.
The blue flower of C. ternatea demonstrates great antioxidant, anti-
hemolytic and anti-hypertensive activity due to its high content of
phenolic compounds, such as kaempferol, quercetins and anthocyanins
[20], being an interesting source of bioactive compound for application
in active and intelligent material. Recently, soy protein isolate [18],
methylcellulose [12] and chitosan-poly(vinyl alcohol) [19] films were
functionalized with Clitoria Ternatea extract (CTE), showing better
packaging performance and high capacity to preserve and monitor the
quality of fish, shrimp and beverages. However, to our knowledge, so­
dium alginate-based films incorporated with CTE have not been previ­
ously characterized and applied as a freshness indicator in foods.
The novelty of this study was to obtain sodium alginate-based films
loaded with CTE with good packaging properties, biodegradable char­
acteristics, bioactive capacity, color sensitivity to pH, ammonia and to
the sterilization process, and with the function of detecting the freshness
of protein foods. To achieve this, films loaded with different concen­
trations of extract (10, 20 and 40 % v/v) were evaluated for their oxygen
barrier properties, color and opacity, surface morphology (SEM),
chemical structure (FT-IR), crystallinity (XRD) and thermal properties
(DSC). In sequence, the antioxidant and antimicrobial capacity of the
films were determined, and their color change ability was evaluated in
different pH-values, ammonia gas and sterilization process. Finally, the
films were checked for their potential application in monitoring the
freshness of milk, shrimp and pork. An illustrative scheme of the study
overview is shown in Fig. 1.
Fig. 1. Schematic illustration of obtaining and characterizing sodium alginate films loaded with Clitoria ternatea extract.
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International Journal of Biological Macromolecules 220 (2022) 866–877
2. Material and methods
2.1. Materials
The Clitoria ternatea powder was supplied by C2Alimentos (São
Paulo, Brazil) and stored at − 20 ◦ C until further analysis. Sodium algi­
nate, glycerol and ethanol were purchase from Êxodo Científica
(Sumaré, Brazil). The ABTS ((2, 2′ -Azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid)) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) reagents were
purchased from Sigma-Aldrich (São Paulo, Brazil). Pasteurized milk,
freshwater shrimp and pork were purchase from local market (Rio
Grande, Brazil). The chemical reagents used in this study were of
analytical grade.
2.2. Extraction of bioactive compounds from Clitoria ternatea
The phenolic-rich extract from C. ternatea was obtained according to
a previously study [21], with some modifications. Briefly, 1.25 g of
C. ternatea powder was homogenized with 100 mL of 60 % ethanol (v/v)
for 30 min in a jacketed reactor coupled to an ultrasonic machine
(Q800W - Ecosonics, Brazil) equipped with a titanium probe (Ø = 4 mm)
and operating at 20 kHz, 350 W and 45 ◦ C. The mixture was centrifuged
at 8709 xg for 10 min, filtered through a nylon cloth and the obtained
extract stored in amber flasks under refrigeration (4 ◦ C). The extraction
was carried out on each day of analysis and preparation of the films.
2.2.1. Extract characterization
The content of total phenolic compounds (TPC) and total monomeric
anthocyanins of CTE was determined by spectrophotometric technique.
The TPC quantification was performed using aliquots of 500 μL of the
extract, 2.0 mL of Folin-Ciocalteu (10 % v/v) and 2.5 mL of sodium
carbonate (7.5 % w/v). This mixture was incubated at 50 ◦ C for 15 min,
cooled to room temperature (25 ◦ C) and read at 760 nm in a UV-VIS
spectrophotometer [22]. A gallic acid standard curve (y = 12.585×)
was used for the determination of TPC, and the values were expressed as
milligrams of gallic acid equivalent per gram of film (mg GAE/g).
The total monomeric anthocyanins were determined by the pH-
differential method [23]. Briefly, the extract diluted was mixed with a
potassium chloride buffer (pH 1.0) or sodium acetate buffer (pH 4.5) and
read at 535 nm and 700 nm in a UV-VIS spectrophotometer (IL592,
Kasuaki, Brazil). Total anthocyanins content (TAC) was determined as
mg cyanidin-3-glucoside equivalents (CGE) per 100 g of C. ternatea
powder (mg CGE/100 g). The TAC in CTE was 136.37 ± 3.86 mg CGE/g.
2.3. Preparation of sodium alginate-based films
The films were produced by the casting technique. The filmogenic
solution was obtained by mixing sodium alginate (1.5 % w/v) and
L.G. Santos et al.
glycerol (40 % w/w of the polymer) in distilled water at 70 ◦ C for 1 h.
Subsequently, the mixture was cooled to 40 ◦ C and concentrations of 10,
20 and 40 % (v/v) of CTE were added. The concentration of extract
added to the filmogenic solution was based on a previous study [3]. A
control film was developed without the addition of the extract. After
homogenization, 15 g of the filmogenic solution was placed in petri
dishes (Ø = 9 cm) and dried at 40 ◦ C for 24 h in an air circulation oven
(Q316M - Quimis, Brazil). The dried films remained for 48 h in desic­
cators at 55 % relative humidity (RH) before being analyzed. The control
film and the films incorporated with 0, 10, 20 and 40 % (v/v) of CTEs
were named as FC, F10, F20 and F40, respectively.
2.4. Characterization of physical, mechanical and barrier properties of
films
The thickness was measured with a micrometer (IP54 – Insize, Brazil)
at ten random positions of the sample. The mechanical properties of
tensile strength (MPa) and elongation at break (%) were determined
with a texture analyzer (TA.XTplus - Stable Micro Systems, England)
according to D-882-02 standard method [24]. The films (n = 10) were
cut into strips (50 × 25 mm) to be analyzed and the test was carried out
with an initial separation of the grips of 50 mm and a crosshead speed of
1 mm/s.
Water vapor permeability (WVP) was evaluated according to E96–00
standard method [24]. Briefly, plastic flasks were filled with 15 g of the
silica gel and sealed with films, allowing a permeation area of 1.60 cm2.
The flasks were placed in a desiccator at 25 ◦ C and 75 % RH. The flasks
were weighed every 24 h for 7 days and WVP was determined as
described in Eq. (1). Where the W, L, A, t and ΔP is the mass gain of the
silica (g), film thickness (mm), permeation area (m2), time of mass gain
(day) and vapor pressure difference across of the film (kPa),
respectively.
( ) W×L
WVP g mm kPa− 1 day− 1 m− 2 =
A × t × ΔP
Moisture (%) and water solubility (%) was determined by gravimetry
[3]. The films (150 mg) were placed in an aluminum capsule, dried at
105 ◦ C in an oven (A15-E – DeLeo, Brazil) to determine the weight of the
initial dry film. The moisture of the samples was determined by the
difference between the weight of the wet film and the initial dry film. For
the determination of water solubility, the initial dry films (wi) were
submerged in 50 mL of distilled water and stirred at 150 rpm for 24 h in
an orbital shaker (TE420 - Tecnal, Brazil). The insoluble materials were
dried at 105 ◦ C for 24 h and the weight of the final dry films (wf) was
determined. The water solubility was calculated by the ratio between wi
and wf.
The wettability of the films was evaluated by contact angle analysis
[10]. The films were fixed in a goniometer and drops of distilled water
(n = 5) were placed on the surface of the material. The angle formed was
recorded by a digital microscope after 5 s of contact and the mean values
were obtained by the ImageJ software.
The absorbance spectra of the films were obtained with a UV–Vis
spectrophotometer (UV-1800, Shimadzu) at a wavelength of 200–700
nm and used to evaluate the light barrier abilities of the films. Color
parameter and opacity of the films were evaluated using a colorimeter
(CR-400 – Konica Minolta, Japan) and the Illuminant D65 standard [3].
The CIELab scale was applied to determine the L* (luminosity), a*
(green/red) and b* (yellow/blue) parameters, used to determine the
color difference (ΔE) between the samples (Eq. (2)). The opacity (%) of
the samples was determined by the ration between the black and white
standards.
[ ]1/2
ΔE = (L* − L0 )2 + (a* − a0 )2 + (b* − b0 )2
Surface and cross-sectional images of the films were obtained by
scanning electron microscopy (SEM) using a microscope (JSM-6610LV,
International Journal of Biological Macromolecules 220 (2022) 866–877
Jeol). The films were fixed on aluminum stubs, coated with gold and the
images were obtained at 10 kV.
Thermal properties of the films were evaluated by differential
scanning calorimetry (DSC) with a temperature range from − 20 to
250 ◦ C using a calorimeter (TA-60WS, Shimadzu) at a heating rate of
10 ◦ C/min. Functional groups present in the films were determined in
spectra obtained by Fourier transform infrared spectroscopy (FT-IR) in
the range of 400–4000 cm− 1 in an FT-IR equipment (IRPrestige-21,
Shimadzu). The crystallinity characteristics of the films were evaluated
by an X-ray diffractometer (D8 Advance, Bruker) with Cu radiation
(1.5418 Å, 40 mA and 40 kV) and the x-ray diffraction pattern (XDR)
obtained at 2θ from 5 to 70◦ .
2.5. Biodegradability assay of films in soil and sand
Due to the packaging being discarded in sanitary landfills or inap­
propriately ending up on the coasts, the biodegradability assessment
was carried out in soil and beach sand (moisture content of 40 %).
Separately, trays were filled with soil from the Rio Grande region (RS,
Brazil) and sand from Cassino beach, keeping them at 25 ◦ C and 75 %
RH [10]. The films were cut (2 × 2 cm), placed on plastic grids and
buried in trays containing soil or sand. The samples were taken
randomly at 3-day intervals until complete degradation, being recorded
by photographs to visualize the degradation of the sample.
2.6. Evaluation of the release of active compounds in food simulants
The release of bioactive compounds of the films was evaluated using
water, 50 % ethanol (v/v) and 3 % acetic acid (v/v) as simulant medium
of aqueous, fatty and acidic foods, respectively, according to European
Regulation 10/2011 [25]. The film (100 mg) was immersed in the
simulant (10 mL), homogenized at 150 rpm for 4 h and centrifuged at
8709 ×g for 10 min. The supernatant obtained was used to determine
the TPC (according to item 2.2.1) and antioxidant activity.
(1) The antioxidant activity was determined by ABTS and DPPH
methods. The ABTS radical was obtained after diluting the mixture of
persulfate (140 mM) and ABTS (7 mM) in ethanol until a reading of 0.70
± 0.05 at 734 nm was obtained. The DPPH radical was obtained by
mixing with ethanol at a concentration of 80 mM. The analyzes were
performed by mixing 200 μL of the extract and 2.8 mL of each radical
separately, and this mixture was incubated at 35 ◦ C for 30 min. The
antioxidant capacity by ABTS and DPPH assays was determined as
radical scavenging activity (%) after reading the mixtures in a spectro­
photometer at 734 and 517 nm, respectively [3].
2.7. Antibacterial assay of sodium alginate-based films
The antibacterial activity of sodium alginate-based films was eval­
uated against Staphylococcus aureus (ATCC-6538) and Escherichia coli
(ATCC-8739) by the disc diffusion method [3]. The films were cut into
discs (Ø = 15 mm) and each side was sterilized in UV-light for 7 min. The
bacterial suspension (0.5 Mc Farland standard) was spread on the sur­
face of plates containing brain heart infusion agar and the sterilized
films discs were placed. The plates were incubated at 37 ◦ C for 24 h and
the zones of inhibition (mm) were measured.
2.8. Application of the sterilization process and exposure to ammonia gas
of sodium alginate films
For the sterilization process, the films were cut (2 × 2 cm), sealed
inside hermetic flasks and submitted to an autoclave at 127 ◦ C for 15
min [5]. The ammonia exposure test was performed by suspending the
films in flasks containing 50 mL of aqueous ammonia solution (8 mM)
(2)
for 30 min [26]. The color change of the films exposed to sterilization
process and ammonia gas was evaluated by a colorimeter based on L*,
a*, b* and ΔE parameters of the CIELab scale (according to item 2.4).
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L.G. Santos et al.
2.9. pH-sensitivity (halochromic) of films
To determine the color changes of the films caused by pH variation,
the films were cut (2 × 2 cm) and immersed in pH 1 to 12 buffer solu­
tions for 1 min [10]. The effects of the pH on the films were evaluated by
L*, a*, b* and ΔE parameter (according to item 2.4).
2.10. Practical application of the films as freshness indicators
Sterile flasks containing 20 mL of pasteurized milk were kept at 25 ◦ C
until spoilage. To assess the ability to detect milk spoilage, a film (2 × 2
cm) was immersed in milk for 30 s at different storage times (0, 24 and
48 h) and then the color change of the films was photographed. At the
same time interval, the pH of the milk was evaluated using a pH meter
[27].
The ability of intelligent film to monitor shrimp and pork freshness
was determined as previously reported [27]. Briefly, a fresh sample (10
g) was placed in sterile petri dishes, covered with a lid containing the
film (2 × 2 cm) attached internally and stored at 25 ◦ C for 48 h. The
color change of the film was monitored at intervals of 0, 24 and 48 h and
the freshness index of the samples (shrimp and pork) were determined
by total volatile basic nitrogen (TVB-N) and pH.
2.11. Statistical analysis
The analyzes were performed in triplicate and the results presented
as mean ± standard deviation. The comparison between two means was
performed by the t-student-test (p < 0.05). Analysis of variance
(ANOVA) and Tukey's test (p < 0.05) were performed to compare the
Fig. 2. FT-IR spectrum (A), XRD patterns (B), DSC curves (C) and transmission spectrum of sodium alginate films loaded with Clitoria Ternatea extract.
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International Journal of Biological Macromolecules 220 (2022) 866–877
mean among three assays.
3. Results and discussion
3.1. Physical and thermal properties of the films
The FT-IR analysis was performed to investigate molecular in­
teractions and functional groups present in CTE and sodium alginate
films (Fig. 2A). The CTE spectrum showed a wide peak at 3288 cm− 1
(-OH group), 2978 cm− 1 (-CH asymmetric stretching), 1645 cm− 1 (C–C
stretching) and 1043 cm− 1 (C–H deformation of the aromatic ring)
[12,28]. Peaks at 3288 cm− 1 and between 1000 and 1100 cm− 1 indicate
the presence of hydrophilic compounds (e.g., polyphenols, flavonoids
and anthocyanins) in the extract, as previously shown [19].
The alginate films showed characteristic bands at 3550 cm− 1 (-OH
group), 2154 cm− 1 (C– – C alkyne group), 1651 cm− 1 (COO- asymmet­
–
rical elongation vibration), 1440 cm− 1 (COO- symmetrical elongation),
1138 cm− 1 (C-O-C elongation) and 829 cm− 1 (Na–O bond vibration)
[12,18,19,29]. The peak at 3550 cm− 1 is attributed to the hydroxyl
groups (-OH) present in the hydrophilic structure of sodium alginate and
the plasticizer glycerol [29]. Loading the extract into the alginate matrix
caused the peak to increase and shift to 3547, 3527 and 3512 cm− 1 in
F10, F20 and F40, respectively.
Moreover, the peak at 1651 cm− 1 of FC is associated with the -OH
flexions of the water molecules of the alginate [28] and, after being
loaded with 20 and 40 % of CTE, it was shifted to 1649 and 1647 cm− 1,
respectively. The shift of these peaks (3547 and 1651 cm− 1) indicate the
strong intermolecular interaction by hydrogen bonds of the functional
groups of the extract (hydroxyl, carbonyl and carboxyl) and the sodium
L.G. Santos et al. International Journal of Biological Macromolecules 220 (2022) 866–877

alginate, glycerol and water molecules [19,30]. Previously, similar Table 1

behavior was observed in smart films incorporated with phenolic-rich Physical, mechanical and barrier properties of sodium alginate films incorpo­
extract from Clitoria ternatea [18,28], cabbage [30], Carissa [19] and rated with Clitoria ternatea extract.
blackcurrant [31]. Properties FC F10 F20 F40
Furthermore, the reduction of the peak intensity and the broadening Thickness (mm) 0.033 ± 0.039 ± 0.041 ± 0.044 ±
of the band at 2154 cm− 1 may indicate a crosslinking process due to 0.004a 0.005a 0.008a 0.006a
electrostatic interaction between the charged oxygen atom of the an­ Tensile strength (MPa) 3.13 ± 9.99 ± 10.18 ± 12.03 ±
thocyanins and the hydroxyl groups of the biopolymer molecules [18], 0.26c 0.21b 0.29b 0.28a
Elongation at break (%) 31.30 ± 28.37 ± 21.15 ± 18.10 ±
causing the immobilization of biocompounds and functionalization of
1.07a 1.53a 2.62b 1.28b
the films. The interactions between biopolymers with anthocyanins Water vapor permeability 3.81 ± 4.28 ± 4.68 ± 4.78 ±
occur by intermolecular and electrostatic interaction [19,32], causing (g mm/kPa day m2) 0.16c 0.12b 0.15a 0.11a
cross-linking and modifications of the structural, thermal and mechan­ Moisture (%) 13.43 ± 17.05 ± 20.86 ± 25.79 ±
ical properties of the films. 1.50d 1.63c 0.69b 1.17a
Water solubility (%) 89.16 ± 92.29 ± 95.03 ± 94.80 ±
The XRD analysis was performed to evaluate the effects of CTE 0.33b 1.26ab 0.10a 2.12a
incorporation on the crystallinity properties of sodium alginate films. In Contact angle (θ ◦ ) 60.31 ± 52.26 ± 46.11 ± 38.28 ±
XRD patterns (Fig. 2B), it was evident that all films presented semi- 1.47a 2.10b 1.68c 1.69d
crystalline structures, with peaks visible at 14 and 30◦ , as previously L* 96.89 ± 84.84 ± 74.86 ± 63.14 ±
0.21a 1.46b 0.27c 1.45d
reported [29,33]. Sodium alginate films loaded with CTE showed wider
a* − 0.76 ± − 2.74 ± − 1.73 ± 1.48 ±
and lower intensity peaks, promoting the reduction of the crystallinity 0.01b 0.17d 0.05c 0.27a
index from 46 % of FC to 45, 41 and 39 % in F10, F20 and F40, b* 3.18 ± − 5.69 ± − 15.10 ± − 24.43 ±
respectively. This modification of the peaks is a result of the electrostatic 0.07a 0.64b 0.16c 0.92d
interaction or hydrogen bonding between alginate-CTE, resulting in the ΔE 1.51 ± 17.10 ± 28.15 ± 43.11 ±
0.11d 0.16c 0.30b 1.71a
packing of the biopolymer chains and reduction of the crystalline
Opacity (%) 9.13 ± 12.29 ± 14.53 ± 20.47 ±
structure of the material [34]. 0.27d 0.17c 1.03b 0.69a
The thermal properties of the films were evaluated by DSC analysis
FC, F10, F20 and F40 correspond to the control films (without extract) and to the
(Fig. 2C). It is observed that the endothermic peak, referring to the
films incorporated with 10, 20 and 40 % (v/v) of Clitoria ternatea extract,
melting temperature, was given at 104.25 ◦ C and that, with the increase
respectively. Means with different subscript in the same line indicate significant
of the CTE loading in the films, the peak shifted to 106.05, 108.32 and differences (p < 0.05).
118.45 ◦ C in F10, F20 and F40, respectively. This phenomenon occurs
due to the intermolecular interactions previously shown by the FT-IR
agreeing with the transmittance spectra shown above. Similar behavior
analysis, generating reinforced and compact materials with greater
was observed in the development of biopolymer films added with
thermal stability [6,10]. Similar behavior was obtained in methylcel­
anthocyanins-rich extracts from purple onion peel [3], jambolão skin
lulose films functionalized with CTE [12]. According to the authors, the
[10] and açaí seed [4].
7 % increase in the melting temperature is associated with the formation
Physical, mechanical and barrier performances of sodium alginate-
of hydrogen bonds between the hydroxyl chains of the polymer and CTE,
based films are shown in Table 1. Thickness is a property that is
requiring higher temperatures to break. On the other hand, the
related to the mechanical and barrier characteristics of the films, not
exothermic peak at 216 ◦ C represents the decomposition phase of the
being statistically different (p > 0.05) by the incorporation of CTE in this
samples. It is evident that the incorporation of CTE did not cause
study. Moreover, the tensile strength (TS) and elongation at break (EB)
changes in the decomposition temperature of the films, indicating good
of the films were altered by the addition of the extract. Sodium alginate
compatibility between alginate-CTE. Similar results were obtained in
films incorporated with higher concentrations of the extract showed a
previous studies of films based on alginate incorporated with different
3.8-fold increase in TS, while EB reduced 42 % compared to the control
phenolic extracts [8,33,35].
film. Modifications of the mechanical properties of biopolymeric films
incorporated with phenolic extracts have been reported [3,5,26]. The
3.2. Influence of CTE on the mechanical and barrier properties of alginate
CTE showed a TPC of 26.61 ± 0.48 mg GAE/g, being higher than those
films
previously reported [20,36]. Biopolymer films incorporated with
phenolic compounds show changes in mechanical properties due to the
The acceptance and choice of products is related to the visual and
intermolecular interactions discussed earlier in the FT-IR section,
color aspects of the packaging, being important characteristics to be
resulting in materials with different microstructural characteristics and
evaluated. The reduction in light transmittance as the concentration of
packaging properties.
CTE increased indicates that films loaded with CTE have a high light
The microstructure of the films analyzed by SEM (Fig. 3) showed that
barrier capability compared to FC (Fig. 2D). This phenomenon occurs
FC presented a smooth, dense and continuous surface, indicating good
due to the ability to absorb UV–Vis light of aromatic compounds (e.g.,
dispersion of alginate in the plasticizer glycerol. However, wrinkles on
anthocyanins) present in CTE [6,16], promoting the reduction of light
the surface of the films increased proportionally when CTE was incor­
transmittance and less transparency of the loaded films [30]. Similar
porated. This behavior may be related to the interaction of groups (-OH)
behavior was obtained in films of methylcellulose [12], chitosan-poly
between CTE-alginate, affecting the ordered arrangement of the alginate
(vinyl alcohol) [19] and starch [28] incorporated with CTE. The incor­
chains due to the crosslinking process of the films [10,12,18]. The cross
poration of extracts rich in anthocyanins has the advantage of reducing
section showed that the FC has a structure with several cracks along the
film transparency, preserving food from oxidation [3,4].
material. The increase in the concentration of CTE in the matrix reduced
Color properties of the alginate-based films were strongly influenced
the cracks, confirming that the biopolymer side chains were closer with
by the incorporation of CTE, showing statistical difference in all pa­
the crosslinking process. However, crosslinking of the alginate films
rameters evaluated, as shown in Table 1. The increase in the concen­
loaded with CTE may have occurred partially, as the cracks were
tration of CTE incorporated in the alginate films resulted in the
widened and the cross section of the films was not completely smooth
reduction of L* and b* values, indicating that the materials presented
and compacted [37]. Previously, it was shown that the intermolecular
lower brightness and bluish color, as seen in Fig. 2D. Thus, the films
interaction between polysaccharides-polyphenols modifies the poly­
added with CTE showed higher values of ΔE and opacity index, indi­
meric network, increasing the interfacial adhesion of the material and
cating that F10, F20 and F40 are more darker and opaquer than FC,

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Fig. 3. SEM images of the surface and cross-section of the FC (A and E), F10 (B and F), F20 (C and G) and F40 (D and H) films, respectively.

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L.G. Santos et al.
reducing the elasticity of the films [12,18,30,32], agreeing with the
elongation analysis.
The evaluation of the characteristics related to the sensitivity of the
films to water (e.g., moisture, permeability to water vapor, contact angle
and solubility) must be carried out to determine the type of product in
which the film can be applied. Due to the hydrophilic characteristics of
sodium alginate, all films showed solubility in water above 89.16 %,
indicating that the developed films have high biodegradability in water.
Similar behavior was observed in several biopolymer films incorporated
with CTE [12,18,19]. Soluble films are not suitable for application as
primary packaging in foods with high water activity (e.g. meat, vege­
table and lactic acid) [11]. However, they can be used as soluble sachets
for the preparation of individual portions of food [10], active packaging
for the release of antioxidant and antimicrobial compounds [2] or
applied as oral disintegration films for the delivery of drugs [8].
The reduction of 35 % in the contact angle and increase of 25 % in
WVP indicate that the incorporation of the extract increased the content
of hydrophilic compounds in the alginate matrix. This is due to the in­
crease in the content of hydrophilic phenolic compounds of CTE (i.e.,
anthocyanins) in the alginate matrix and increases the interaction of the
material's surface with water molecules [38]. Furthermore, studies show
that the incorporation of phenolic compounds in biopolymer films can
change the order of the polymeric chains of polysaccharides, causing
greater diffusion of water vapor and other gases [3–5,7].
In the field of food packaging development, materials with lower
WVP values indicate lower moisture transfer capacities between the
environment and the product, being an ideal condition to protect and
preserve the quality of the packaged food [2]. On the other hand,
freshness indicators signal possible changes in food quality through the
passage of volatile compounds related to spoilage (e.g., oxygen,
ethylene, ammonia, acids), requiring higher WVP values for these
intelligent materials to work [38].
Fig. 4. Biodegradability of alginate films in beach sand and soil.
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International Journal of Biological Macromolecules 220 (2022) 866–877
3.3. Biodegradability of sodium alginate films in soil and sand
The evaluation of the biodegradability of films derived from natural
polymers is important to determine the degradation time of the material
when exposed to microorganisms and enzymes present in the environ­
ment. The film is considered biodegradable when 90 % of the material is
degraded by biological action in a period of up to 6 months [10].
About 10 % of petroleum-derived materials are found in the sea and
coastlines, causing major environmental impacts due to their long
period of degradation [10]. In this way, the degradation capacity of
sodium alginate films in beach sand and soil was evaluated (Fig. 4). After
6 days buried in the sand, the films began to show signs of fragmenta­
tion, being completely degraded in 15 days. Sand is a material generated
from the erosion of rocks, consisting mainly of quartz, a highly hydro­
phobic material. Thus, the rapid degradation of the films exposed to
sand may have occurred due to the presence of water molecules on the
surface of the sand crystals, causing the solubilization and fragmenta­
tion of the alginate films.
On the other hand, the films buried in soil started to fragment after 9
days, being totally degraded in 15 days. Due to the presence of cations
(e.g., sodium, calcium, magnesium, potassium, aluminum, etc.), the
films exposed to the soil showed greater resistance to fragmentation
when compared to those buried in sand. This behavior may have
occurred due to the high crosslinking capacity of alginate films with
cations [7], promoting greater resistance to degradation factors (e.g.,
moisture, microorganisms and enzymes) available in the soil. Therefore,
sodium alginate films showed rapid biodegradability and can be
considered as an alternative to replace synthetic plastics.
3.4. Release of phenolic compounds and antioxidant activity from sodium
alginate films
The use of food simulants is an alternative to evaluate the ability to
L.G. Santos et al.
release bioactive compounds present in films. The films showed a
greater capacity to release phenolics and anthocyanins in the simulants
of aqueous and acidic products (Fig. 5A and B, respectively) after 4 h of
contact. This behavior is related to the high solubility and wettability of
alginate films in water, which facilitate the diffusion of the biocomposite
to the simulant [7]. On the other hand, the films exposed to the ethanol
simulant had a low content of bioactive compounds related to the other
simulants and this is due to the low solubility of alginate in an alcoholic
medium [3], making it difficult to release the biocompounds present in
the film.
As expected, higher concentrations of the loaded extract in the film
increased the TPC and TAC, providing greater antioxidant activity by the
ABTS and DPPH assays, as shown by Fig. 5C and D, respectively. In
relation to food simulants, water showed the highest ABTS radical
scavenging activity in films F10, F20 and F40, ranging from 23 to 70 %.
This may occur due to the interaction of bioactive compounds with
hydrophilic characteristics of the extract (e.g., anthocyanins) with the
polar solvent used as a food simulant [7]. In addition, the ABTS method
is used to evaluate the antioxidant capacity of hydrophilic compounds
[3], which are present in greater amounts in aqueous simulants, such as
water and 3 % acetic acid used in this study.
Regarding the DPPH assay, unlike the films loaded with CTE, FC did
not show antioxidant activity in any of the food simulants used. In
general, acetic acid was the solvent that presented the highest antioxi­
dant activity by DPPH method in relation to the other food simulants
used. This behavior may be related to the acid's ability to induce
relaxation of the alginate biopolymer chains [7], promoting higher
diffusion rates of nonpolar compounds present in the films, which have
greater DPPH• scavenging activity [3].
In view of the results obtained, it is possible to state that among the
alginate films developed, F40 has the highest antioxidant capacity and
that, when applied to foods with aqueous and acidic characteristics,
Fig. 5. Total phenolic content (A), Total anthocyanins content (B) and antioxidant activity by radical scavenging ABTS (C) and DPPH (D) of sodium alginate films.
ND = non-detectable.
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International Journal of Biological Macromolecules 220 (2022) 866–877
allows the reduction of the effects of oxidation and probably preserves
the physical, sensory, and nutritional characteristics of packaged foods.
3.5. Antibacterial activity in sodium alginate-based films
The antimicrobial evaluation of the films is important to analyze the
ability to reduce pathogenic microbial cells present in packaged foods
[2]. In this study, none of the films showed antimicrobial activity against
S. aureus, however, F40 was the only film that was able to generate zones
of inhibition (19.22 ± 3.42 mm) against E. coli, as shown in Fig. S1. The
antimicrobial effect of F40 may be related to its high TPC content
compared to other films. The antimicrobial mechanism of phenolic
compounds is associated with their partially hydrophilic nature, which
helps to interact with the cytoplasmic membrane and bacterial lipo­
polysaccharides, causing cell instability and bacterial cell death [16].
Several studies show the antimicrobial capacity of films incorporated
with anthocyanin-rich extract [2,12,31,39,40]. The antimicrobial ac­
tivity of anthocyanins is related to the formation of pores in the cyto­
plasmic membrane of bacteria due to the interaction of the flavylium
cation with negatively charged phospholipid groups, allowing the exit of
intracellular materials, physiological changes, and inactivation of the
microorganism [15].
Packaging with antimicrobial potential allows the production of
foods with reduced amounts of chemical preservatives, ensuring
increased shelf life, food safety and quality of the packaged product [2].
In this context, among the films developed in this work, F40 is the best
alternative for application as active antibacterial packaging for food
preservation.
L.G. Santos et al. International Journal of Biological Macromolecules 220 (2022) 866–877

3.6. Effects of the sterilization process and exposure to ammonia gas of
the films
Sterilization is a food preservation process aims to inactivate spores
and microorganisms in a product, being commonly performed in pres­
surized autoclaves. Due to the epidemiological crises related to food­
borne diseases, it is extremely important that the films applied as
packaging or coating on food products maintain their physical integrity
after being subjected to sterilization, ensuring the quality and safety of
food for commercialization [5].
The alginate films submitted to the sterilization process maintained
their physical integrity to the naked eye, showing no fractures or holes
and indicating that the films can withstand the heat and pressure of
sterilization. However, the color parameters of all films were signifi­
cantly changed (p < 0.05) after the sterilization process (Table 2). In
general, the increase in L* and the positive b* values indicate that the
films have become lighter and yellowish, and this may have occurred
due to the caramelization process of the alginate matrix, forming high
molecular weight compounds of dark coloration by exposure of the
material to high temperature (121 ◦ C).
In addition, the incorporation of the anthocyanin-rich extract
intensified the color change of the films from blue to yellow, as evi­
denced by the significant increase in the b* parameter and reduction of
ΔE after sterilization of the F10, F20 and F40 films. Similar behavior was
observed in polysaccharides-based films incorporated with anthocya­
nins and exposed to sterilization process [5,41]. Anthocyanins are nat­
ural pigments that have thermosensitive characteristics and, when
exposed to high sterilization temperatures, are degraded into yellow
compounds of chalcone [15], contributing to the change in the color of
the films. In this context, the alginate films incorporated into the
anthocyanin extract have potential as a colorimetric indicator of the
sterilization process, being able to inform the consumer if the packaged
product has been sterilized and has obtained the desired food quality
and safety parameters for its commercialization.
Regarding the color responses by the exposure of the films incorpo­
rated with the anthocyanin-rich extract to ammonia, it is possible to
observe that the reduction of a* values and the positive parameters of b*
indicate that the initial bluish color of the material was changed to green
after contact with the nitrogenous volatile base (Table 2). This behavior
occurs due to the reaction between the flavylium cation of the
anthocyanins with amine compounds, generating an alkaline environ­
ment and the formation of pseudo-base carbinol and chalcone, corre­
sponding to the yellowish-green color [13].
Freshness indicators for foods are developed from the loading of
anthocyanins in different types of biopolymer matrix [12,14,31,42,43]
and are able to verify and identify the spoilage rate of protein-based
products (i.e.. meat, poultry, fish, and milk). In this study, only the
F40 film showed a statistical difference (p < 0.05) for the ΔE parameter
after contact with ammonia, indicating that the color change can be
observed with the naked eye [31]. Therefore, F40 can present the best
conditions for the development of an ammonia-sensitive colorimetric
indicator.
3.7. Color response of films loaded with anthocyanin-rich extract at
different pH
The CTE and the films loaded with the anthocyanins-rich extract
were subjected to different pH buffers and the color changes were
evaluated (Fig. 6A and B, respectively). The appearance of a reddish-
purple color in acidic solutions (pH < 3) is due to the mixture of blue
and red pigments generated by the flavylium cation, an oxonium form of
anthocyanins [10]. With increasing pH (3 < pH < 7), the anthocyanins
show intense blue coloration due to their hemiketal form (quinoidal
anhydrous base) and, when subjected to basic media (pH > 7), the hy­
droxyl groups present favor the cleavage process of the central pyrrole
ring of the anthocyanins, forming yellow-green chalcone compounds
[13,15].
Hydroxypropyl methylcellulose/microcrystalline cellulose loaded
with extracts of C. ternatea [12] were able to be applied as a pH-
responsive indicator because they show color variation among red,
purple, blue and green when subjected to respective buffer solutions of
pH 1, 4, 7 and 12, being similar to the films developed in this study. As
expected, increasing concentrations of loaded extract in alginate films
provide greater changes in color parameters (a* and b*) and, conse­
quently, ΔE values were higher (Fig. 6C). Among the developed films,
the F40 showed enormous potential for application as a pH-responsive
indicator due to the ΔE values being >42 in all pH evaluated, indi­
cating that the color change can be observed with the naked eye [10].
Therefore, F40 was applied to check the freshness of milk, pork and
shrimp.

Table 2
Color changes after exposure to sterilization process and ammonia gas of alginate films incorporated with Clitoria ternatea extract.
Films Color parameters Photography

Before After Before After

Initial Sterilization process Ammonia exposure Initial Sterilization process Ammonia exposure
Aa A a
FC L* 96.89 ± 0.21 94.96 ± 0.32 97.46 ± 0.82
a* − 0.76 ± 0.01Ba − 0.98 ± 0.03A − 1.10 ± 0.03b
b* 3.18 ± 0.07Ba 6.17 ± 0.31A 2.97 ± 0.44a
ΔE 1.51 ± 0.11Ba 5.07 ± 0.40A 1.59 ± 0.42a
F10 L* 84.84 ± 1.46Ba 87.53 ± 1.50A 82.28 ± 1.75a
a* − 2.74 ± 0.17Ba − 1.48 ± 0.33A − 5.42 ± 0.16b
b* − 5.69 ± 0.64Bb 9.62 ± 0.82A 7.43 ± 0.71a
ΔE 17.10 ± 0.63Ba 13.15 ± 0.63A 17.57 ± 0.92a
F20 L* 74.86 ± 0.27Ba 82.92 ± 0.15A 75.52 ± 2.25a
a* − 1.73 ± 0.05Aa − 1.73 ± 0.28A − 8.84 ± 0.85b
b* − 15.10 ± 0.16Bb 13.52 ± 0.56A 9.05 ± 1.45a
ΔE 28.15 ± 0.30Aa 18.59 ± 0.40B 27.63 ± 1.63a
F40 L* 63.14 ± 1.23Ba 79.25 ± 1.41A 62.93 ± 5.05a
a* 1.48 ± 0.27Aa − 0.79 ± 0.12B − 11.36 ± 0.47b
b* − 24.43 ± 0.73Bb 17.22 ± 0.72A 6.75 ± 1.37a
ΔE 43.11 ± 1.42Aa 23.74 ± 1.54B 33.76 ± 2.57b

FC, F10, F20 and F40 correspond to the control films (without extract) and to the films incorporated with 10, 20 and 40 % (v/v) of Clitoria ternatea extract, respectively.
Different capital letters on the same line indicate statistical difference by the t test (p < 0.05) when the initial film was compared to the film after the sterilization
process. Different lowercase letters on the same line indicate statistical difference by the t-test (p < 0.05) when the initial film was compared to the film after ammonia
exposure.

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L.G. Santos et al. International Journal of Biological Macromolecules 220 (2022) 866–877

Fig. 6. Visual aspects of Clitoria ternatea extract (A), alginate films loaded with anthocyanin-rich extract (B) and color parameters of the films (C) by exposure to
different pH.

3.8. Application of sodium alginate-CTE film as freshness indicator

`During processing, transport and storage, foods can undergo

biochemical reactions, causing deterioration and loss of product fresh­
ness. Commonly, pH and TVB-N are analyzes that verify the freshness
index of different types of food [12,18,19,27]. During milk storage at
25 ◦ C, the initial pH of the product significantly reduced (p < 0.05) from
6.74 ± 0.02 to 4.82 ± 0.12 at the final storage time (48 h). The change
in the pH of milk during storage occurs due to the action of lactic acid
bacteria and the production of organic acids, indicating the loss of
freshness and product quality [19]. Based on the Brazilian standard
IN76–2018, fresh milk must have a pH between 6.8 and 6.6 and,
therefore, samples stored for 48 h are unfit for human consumption.
When F40 was used to check the freshness of the milk, it was observed
that the change in the pH (neutral to acidic) of the product resulted in a
change from the blue color of the film to purple (Fig. 7), in agreement
with the analysis of the pH sensitivity discussed earlier. This finding
Fig. 7. Food freshness monitoring by the F40 colorimetric indicator.
indicates that F40 has good potential as a freshness indicator of
pasteurized milk. A similar result was obtained previously, where the
freshness of milk and orange juice were monitored by smart indicators (− NH3) which increase the TVB-N content and change the pH to alkaline
based on chitosan incorporated with anthocyanins from the CTE [19]. [27,28]. Initially, the shrimp and pork samples (Fig. 8 A and B,
The evaluation of the performance of F40 as an indicator of freshness respectively) showed TVB-N values of 3.47 and 3.70 mg/100 g and after
in shrimp and pork showed that the initial blue film had its color 48 h of storage, the values increased to 55.11 and 56.73 mg/100 g,
changed to green after 48 h (Fig. 7). The deterioration of protein-rich respectively, indicating food spoilage. Fresh meat products have TVB-N
products promotes the formation of volatile organic amine compounds values lower than 15 mg/100 g [32] and thus, after 24 h of storage,
shrimp and pork are not acceptable for human consumption.

875
L.G. Santos et al.
Fig. 8. Changes in TVB-N and pH values during storage at 25 ◦ C of shrimp (A) and pork (B).
The interaction of volatile amine compounds with the anthocyanins
present in F40 promotes the formation of chalcones that change the
color of the film to green, as previously shown by the ammonia gas
sensitivity test. Similar behavior was obtained in indicators of freshness
of meat products based on starch [28], methylcellulose [12]and agar
[44] incorporated with CTE. Therefore, F40 can be applied as a colori­
metric freshness indicator due to its high color change capability.
4. Conclusion
In this study, different concentrations of CTE were loaded into so­
dium alginate films to obtain active and intelligent biodegradable
packaging. The high water-solubility of the alginate films aided in the
degradation of the material when exposed to soil and beach sand. The
addition of the extract in the alginate matrix modified and improved the
mechanical, barrier and optical performances of the films. In addition,
the films loaded with 40 % (v/v) of the extract had a higher content of
phenolic compounds, generating greater antioxidant activity and anti­
bacterial capacity against E. coli. F40 showed immense potential to act
as a colorimetric indicator, showing higher ΔE when exposed to the
sterilization process, ammonia gas and different pH values. When used
to assess the freshness of milk and meat products (shrimp and fish), F40
changed its initial blue color to purple and green, showing its enormous
potential to be used as a biodegradable indicator of food freshness.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2022.08.120.
CRediT authorship contribution statement
Luan Gustavo Santos: Conceptualization, Data curation, Method­
ology and Writing Original draft preparation. Gisele Fernanda Alves-
Silva: Conceptualization, Methodology and Writing Reviewing. Vilásia
Guimarães Martins: Supervision and Writing Reviewing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This study was financed in part by the Coordenação de
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International Journal of Biological Macromolecules 220 (2022) 866–877
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance
Code 001 and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq). This research was developed within the scope of
the Capes-PrInt Program (Process # 88887.310848/2018-00). We are
grateful to the Centro de Microscopia Eletrônica do Sul (CEME-SUL) for
the SEM and XRD analyses.
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