Food Control 126 (2021) 108046

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

CMC and CNF-based intelligent pH-responsive color indicator films

integrated with shikonin to monitor fish freshness
Parya Ezati, Ruchir Priyadarshi, Yeong-Ju Bang, Jong-Whan Rhim *
BioNanocomposite Research Center, Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea

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

Keywords: Intelligent pH-responsive color indicator films based on carboxymethyl cellulose (CMC) and cellulose nanofibers
Carboxymethylcellulose (CNF) were prepared using shikonin extracted from Lithospermum erythrorhizon roots. The addition of shikonin
Cellulose nanofiber did not affect the thermal stability and water vapor barrier properties of the film; however, the surface hydro­
Shikonin
phobicity increased significantly. The shikonin-added films showed high antioxidant activity, especially in
pH-responsive
Color indicator film
aqueous solutions. The color indicator films showed a noticeable color response function for various pH values,
showed reddish-pink at pH below 7, and turned reddish-purple, purple, and light blue as the pH increased from 7
to 12. When the indicator film was used to monitor the fish freshness, it showed a reddish-pink for fresh fish (pH
= 5.7) and turned blue-violet after 36 h, indicating that it was spoiled (pH = 6.9). The shikonin-added color
indicator films with a distinct discoloration function between a weak acid and weak alkali are likely to be applied
to monitor seafood quality.

1. Introduction
As the market continues to increase packaged fresh food, especially
for raw meat, seafood, and dairy products, finding ways to monitor food
quality in the supply chain has become paramount. Since changes in the
pH of food are the first sign of food spoilage, a lot of research has been
done over the past few years on developing pH indicators to monitor the
freshness of food (Mohammadalinejhad, Almasi, & Moradi, 2020). In
fact, pH is used as one of the most crucial factors in determining food
quality because changes in food pH are direct evidence of food quality
changes. Most spoilage microorganisms increase pH by producing tri­
methylamine (TMA) in seafood. In addition, the spoilage process is
caused by a group of aerobic microorganisms rather than a single
microorganism, which increases the likelihood of microbial contami­
nation, which increases the potential for TMA production in seafood
during storage. Therefore, there is a strong and positive correlation
between pH changes in meat products and total volatile basic nitrogen
(TVBN), total viable count (TVC), and aerobic microorganisms (Dong
et al., 2020; Huang et al., 2019). In particular, there have been many
reports of the use of natural pigments in the manufacture of
pH-responsive color indicators for monitoring the freshness of packaged
foods in recent years (Aghaei, Ghorani, Emadzadeh, Kadkhodaee, &
Tucker, 2020; Alizadeh-Sani, Mohammadian, Rhim, & Jafari, 2020a;
Alizadeh-Sani, Tavassoli, et al., 2020b; Roy & Rhim, 2020b). However,
most of the research focuses on anthocyanins, and other natural pig­
ments have not been studied much compared to anthocyanins. One of
such highly applicable but underutilized pigments is shikonin, a naph­
thoquinone compound present as a secondary metabolite in the roots of
Lithospermum erythrorhizon. Shikonin is a characteristic red,
alcohol-soluble pigment that has traditionally been used in herbal
medicine as a powerful pharmaceutical substance (Assimopoulou &
Papageorgiou, 2005; Chen, Yu, Hsu, Tsai, & Tsai, 2018). Shikonin has
various medicinal properties such as antioxidant, antibacterial,
anti-inflammatory, and wound healing (Sasaki, Yoshizaki, & Abe,
2000). Structurally, shikonin is composed of a naphthazarin group and a
chiral six-carbon side chain. The naphthazarin moieties are highly
chemically reactive with acids and bases, resulting in red at low pH and
blue at high pH (Han, Weng, & Bi, 2008).
The pigment must be immobilized on a suitable solid substrate or
matrix to make a pH-responsive color indicator film. Therefore, the
proper selection of the immobilization surface is of utmost importance in
determining the effectiveness and performance of the indicator (Pour­
javaher, Almasi, Meshkini, Pirsa, & Parandi, 2017). The surface material
must be permeable to gases and water vapor to interact with trapped
pigments and cause color changes. Since most biopolymer-based films
are more gas permeable than synthetic polymers, various biopolymer

* Corresponding author.
E-mail address: jwrhim@khu.ac.kr (J.-W. Rhim).

https://doi.org/10.1016/j.foodcont.2021.108046
Received 1 December 2020; Received in revised form 15 February 2021; Accepted 27 February 2021
Available online 3 March 2021
0956-7135/© 2021 Elsevier Ltd. All rights reserved.
P. Ezati et al.
films have been widely used for this purpose over the past few years.
Cellulose is the most abundant natural biopolymer and has attracted
researchers because it is renewable, biodegradable, non-toxic, and
biocompatible (Oun & Rhim, 2017). Cellulose and its derivatives have
been widely used in the manufacture of food packaging films due to their
excellent film-forming properties and their ability as carriers for active
ingredients such as antioxidants, antibacterial agents, and pH indicating
pigments (Kono, 2014). Carboxymethyl cellulose (CMC) and cellulose
nanofibers (CNF) are the two most widely used cellulose derivatives
used in food packaging applications. CMC is a water-soluble cellulose
derivative with anionic carboxylic groups that can exist either as the free
acid or its sodium salt or mixtures (Priyadarshi, Kumar, & Rhim, 2020).
CNF consists of nano-sized cellulose fibrils with a high aspect ratio that
is a few nanometers wide and micrometers long (Ezati, Rhim, Moradi,
Tajik, & Molaei, 2020). Unlike cellulose nanocrystals (CNC), CNF has
more amorphous regions in its molecular structure, resulting in higher
mechanical strength and flexibility. Therefore, it can manufacture
stand-alone films or reinforce other biopolymer films (Vilela et al.,
2017). We have recently used cellulose paper as solid support for
immobilizing shikonin for intelligent packaging applications (Ezati,
Bang, & Rhim, 2021). However, the paper-based indicator did not
possess adequate water vapor permeability, water contact angle, trans­
parency and mechanical properties. The food industry is increasingly
interested in developing packaging films with adequate integrity and
mechanical strength to meet food preservation, facilitate distribution
and marketing, and prevent breakage or leakage during packaged food
storage. Therefore, these properties are important parameters that must
be considered for the application of packaging films. The CMC and
CNF-based films have better film properties than the paper-based film.
Therefore, we used cellulose-based biopolymers (CMC and CNF) as
support materials in the current study.
The main objective of this study was to develop smart pH-responsive
color-indicating films based on CMC and CNF incorporated with shi­
konin for food packaging applications.
2. Material and methods
2.1. Materials
CNF (TEMPO-CNF, width: 10–20 nm; length: 3–350 μm) and CMC
(MW: 90,000 g/mol) were obtained from Moorim P&P Co., Ltd. (Ulsan,
Korea) and Junsei Chemical Co., Ltd. (Tokyo, Japan), respectively. ABTS
(2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (2,2-
diphenyl-1-picrylhydrazyl) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Lithospermum erythrorhizon root for shikonin extrac­
tion was obtained from a local market in Jecheon, Korea. Buffer solu­
tions were procured from Thermo Fisher Scientific (Chelmsford, MA,
USA). Ammonia, glycerol, ethanol (C2H6O, 99.9%), and acetic acid
glacial (CH3COOH, 99.7%) were procured from Daejung Chemicals &
Metals Co. Ltd. (Siheung, Gyeonggi-do, Korea). Fish fillet (mackerel)
was purchased at a local seafood market for the packaging test.
2.2. Extraction of shikonin
Shikonin was extracted from the roots of Lithospermum erythrorhizon
following the method of Chen et al. (2018). For this, 500 mL of 95%
ethanol was added to 100 g of the ground plant root powder, stirred
continuously at 25 ◦ C for 24 h. The solution was filtered to remove
insoluble substances, then freeze-dried using a freeze dryer, and the
obtained powder was stored in a dark place at 4 ◦ C before testing.
2.3. Fabrication of pH indicator films
pH-indicator films were fabricated using a solution casting method
(Priyadarshi, Kim, & Rhim, 2021). For this, 4 g of CMC or CNF was
dissolved in 145 mL distilled water containing 1.2 g glycerol as a
2
Food Control 126 (2021) 108046
plasticizer with vigorous stirring at 90 ◦ C for 30 min. Also, shikonin
solutions were prepared by dissolving 40 mg of the pigment (1 wt% of
polymer) in 5 mL ethanol. The shikonin solution was then added to the
CMC or CNF solution, stirred for 10 min to allow them to mix thor­
oughly. The film-forming solution was then cast onto a leveled Teflon
film (Cole-Parmer Instrument Co., Chicago, IL, USA) coated glass plate
(24 cm × 30 cm) and dried at room temperature (23 ± 2 ◦ C) for two
days. The dried films were peeled off from the glass plate and condi­
tioned at 25 ◦ C and 50% RH for 48 h. Also, the control CMC and CNF
films were prepared using the same procedure without adding shikonin
for comparison.
2.4. Characterization and properties of the film
2.4.1. FT-IR spectroscopy and optical properties
Infrared spectra of cellulose-based films were recorded using a
Fourier Transform Infrared (ATR-FTIR) spectrophotometer (TENSOR 37
spectrophotometer with OPUS 6.0 software, Billerica, MA, USA) in
attenuated total reflection (ATR) mode at 4 cm− 1 resolution within the
range of 4000-400 cm− 1. The light absorption spectra of the CMC and
CNF-based films were recorded using a UV–vis spectrophotometer
(Mecasys Optizen POP series UV/Vis, Seoul, Korea) in the range of
200–800 nm.
2.4.2. Moisture content, water solubility, and water absorption of the film
The moisture content (MC) of the film was determined using a
gravimetric method. The film sample (2.5 cm × 2.5 cm) was dried at
105 ◦ C for 24 h, the weight of the film sample before and after drying
was measured, and then the MC of the film was calculated as the weight
loss percentage.
For the determination of water solubility (WS), a dried film sample
(2.5 cm × 2.5 cm) was immersed in 50 ml distilled water in a beaker for
2 h with mild stirring. The film sample was removed from the water and
dried at 105 ◦ C for 24 h. The WS was determined as the percentage
change in weight between the initial and final dry matter of the film.
The water absorption (WA) capacity of the film was determined by
immersing a film sample (2.5 cm × 2.5 cm) in distilled water at 25 ◦ C for
1 h. The film was collected, and surface water was removed using
blotting paper. By measuring the weight of the film before and after
water immersion, the WA of the film was calculated as a percentage of
the weight increase.
2.4.3. Water vapor permeability (WVP) and water contact angle (WCA)
The WVP was measured using a gravimetric procedure according to
the ASTM E96-95 standard methods. Film samples (75 mm × 75 mm)
were mounted on top of a cup (depth: 25 mm; diameter: 68 mm) con­
taining 18 mL of distilled water and sealed in such a way that the
evaporation of water from the cup takes place only through the film. The
assembled cup was weighed and stored in a humidity chamber (FX1077,
Jeio Tech Co. Ltd., Ansan, South Korea) controlled at 25 ◦ C and 50% RH.
The WVP cup was weighed every hour for 8 h, and the weight loss of the
cup versus time was plotted to determine the water vapor transmission
rate (WVTR, g/m2.s) from the slope of the plot. The WVP (g.m/m2.Pa.s)
of the film was calculated as follows:
ΔW × L
WVP = (1)
t × A × ΔP
where ΔW is the weight change of the WVP cup (g), L is the mean film
thickness (m), t is the elapsed time (s), A is the effective permeation area
of the film (m2), and ΔP is the partial water vapor pressure difference
across the film (Pa). ΔW/(t × A) in Eqn (1) is the WVTR of the film.
The WCA of the film samples was measured using a WCA analyzer
(Phoenix 150 Water Contact Angle Analyzer, Surface Electro Optics Co.
Ltd., Gunpo, Korea). The film sample (30 mm × 100 mm) was placed on
a horizontal Teflon coated steel stage on the analyzer. A drop of distilled
P. Ezati et al.
Fig. 1. Light transmittance spectra of the CMC and CNF-based films.
water (~10 μL) dropped on the film surface using a micro-syringe, and
the WCA was measured immediately.
2.4.4. Thermal stability and tensile strength
The thermal stability of the film was evaluated using a thermogra­
vimetric analyzer (Hi-Res TGA 2950, TA Instrument, New Castle, DE,
USA) (Roy, Shankar, & Rhim, 2019). The film sample (~5 mg) was taken
in an aluminum pan and heated from 30 to 600 ◦ C at a heating rate of
10 ◦ C/min under a nitrogen flow of 50 cm3/min.
The ASTM A882-88 standard method was used using an Instron
universal testing machine (Model 5565, Instron Engineering Corpora­
tion, Canton, MA, USA) to measure mechanical properties. Each film
sample was cut into specimens of dimensions of 25 mm × 150 mm using
a precision double blade cutter (Metrotec LB.02/A, Metrotec, SA, San
Sebastian, Spain). The machine was operated in tensile mode with an
initial grip separation of 50 mm and a 50 mm/min cross-head speed.
2.5. Antioxidant activity
The antioxidant activity of the films was estimated using 2,2-
diphenyl-1-picrylhydrazyl radical (DPPH•) and 2,2′ -azino-bis(3-ethyl­
benzothiazoline-6-sulfonic acid) (ABTS•+) free radical scavenging
methods. For the DPPH radical scavenging test, a film sample (50 mg)
was added to 10 mL of a DPPH solution prepared by dissolving 4 mg of
DPPH in 100 mL of methanol and reacted at room temperature in the
dark for 30 min and measured the light absorbance of the reaction so­
lution using a UV–vis spectrophotometer at 517 nm (Ezati & Rhim,
2020b). For the ABTS radical scavenging test, a 7 mM ABTS solution was
prepared, and potassium persulfate was added so that the final con­
centration was 2.45 mM. The mixture was kept in the dark for 16 h and
then diluted with distilled water to obtain an absorbance of about 1.00
at 734 nm. About 50 mg of a film sample was added to 10 mL of ABTS
solution, incubated at room temperature for 30 min, and measured the
absorbance at 734 nm. Simultaneously, a control test was performed
without a film sample following the same procedure. In both cases, the
radical scavenging activity was calculated as follows:
Ac − As
Free radical scavenging activity(%) = × 100
Ac
where Ac was the absorbance of the control DPPH or ABTS solution and
As was the absorbance of DPPH or ABTS solution of the test film.
Food Control 126 (2021) 108046
2.6. Color response efficiency
Various buffer solutions were used to determine the color change of
the indicator film at different pH (Pourjavaher et al., 2017). The indi­
cator film (3 cm × 3 cm) was soaked into buffer solutions for 5 min at
room temperature, then a photographic image of the indicator film was
captured after removing the solution. The Hunter color values of the
indicator film were measured using a Chroma meter (Konica Minolta,
CR-400, Tokyo, Japan) with a standard white plate (L = 97.75, a =
− 0.49, and b = 1.96) as a background. The total color difference (ΔE)
was calculated as follows:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = (ΔL)2 + (Δa)2 + (Δb)2 (3)
where ΔL, Δa, and Δb are the difference between each color value of the
standard color plate and film sample, respectively.
The indicator film was hung over a sealed container with a solution
of acetic acid (99.7%) or ammonia (0.8M) for 5 min and recorded color
images to evaluate the sensitivity of the indicator film to acid and base
gases (Tirtashi et al., 2019).
2.7. Packaging test
For the packaging test, 50 g of fresh fish samples were placed in a PP
tray (17.5 × 12.2 × 3.5 cm3) and covered with a polyethylene film with
attaching an indicator film (3 cm × 3 cm) below the PE film so as not to
direct contact with the fish sample, and stored at room temperature (21
± 2 C) for 36 h. The photographic images and color parameters (L, a,
◦
and b) of the indicator film and the pH change of the sample were
measured at predetermined time intervals (12, 24, and 36 h) during
storage. The pH of the sample was measured using a digital pH meter
(Thermo Scientific, Orion Star, A211-pH meter, Indonesia) after ho­
mogenizing a 10 g sample in 90 mL of distilled water in the stomacher
bag for 3 min. The packaging tests were performed in triplicate.
2.8. Statistical analysis
The film properties measurements were performed in triplicate with
individually prepared film samples and presented as mean ± standard
deviation. One-way analysis of variance was performed by SPSS soft­
ware, version 13.0. A significant difference between treatment groups
was separated by the Student’s t-test and Duncan’s multiple range test at
p = 0.05. Correlation between pH and ΔE parameter was evaluated by
Pearson’s correlation method.
3. Results and discussion
3.1. Properties of composite films
3.1.1. Optical properties
All the CMC and CNF-based films were flexible and freestanding. The
neat CMC and CNF films were very transparent without any color. In
contrast, the CMC and CNF films with the addition of shikonin had a
bright red color and had relatively low transparency. The light trans­
mission spectra of the CMC and CNF-based films are shown in Fig. 1.
Both CMC and CNF films exhibited a light transmittance of 80% or more
in the 300–700 nm range, indicating that they are transparent, as also
seen by visual observation. At wavelengths below 300 nm, the light
(2) transmittance of the CNF film was lower than that of the CMC film. It
showed a shoulder around 250 nm, which can be due to the aldehyde
group generated during the TEMPO-mediated oxidation of the CNF
preparation (Fukuzumi et al., 2009). On the other hand, the addition of
shikonin significantly reduced the light transmittance of both films in all
wavelength ranges. This may be due to blocking light through reflection
and scattering by polyphenolic compounds in the pigment (Jingrong Liu
et al., 2019; Roy & Rhim, 2020a). Nevertheless, the visible light
3
P. Ezati et al. Food Control 126 (2021) 108046

Table 1
Light transmittance, moisture content, water solubility, water absorption, water vapor permeability, and water contact angle of cellulose-based films.
9
Films T280 (%) T660 (%) MC (%) WS (%) WA (%) WVP ( × 10− g m/m2.Pa.s) WCA (deg.) TS (MPa)
d b a a a
CMC 62.7 ± 1.3 85.7 ± 0.4 15.6 ± 1.3 ND ND 1.4 ± 0.2 41.9 ± 2.5 45.1 ± 1.4a
CMC/shikonin 31.4 ± 0.1b 79.4 ± 1.5a 13.5 ± 0.9a ND ND 1.4 ± 0.0a 61.6 ± 1.2c 46.2 ± 2.0a
CNF 54.2 ± 1.1c 87.7 ± 0.3b 14.0 ± 1.0a 34.4 ± 3.0b 355.2 ± 23.6b 1.5 ± 0.1a 59.3 ± 3.1b 78.9 ± 2.6b
CNF/shikonin 22.8 ± 0.7a 81.5 ± 0.9a 12.9 ± 1.1a 26.0 ± 2.9a 287.6 ± 26.8a 1.4 ± 0.2a 73.4 ± 2.6d 79.4 ± 3.3b

The values are presented as a mean ± standard deviation. The values in the same column, followed by the same letter, are not significantly different (p > 0.05). ND: Not
determined due to the dissolution of the film.

UV-barrier property of the shikonin-added CMC and CNF-based films

was due to the UV light absorption function of shikonin (Dong et al.,
2020). A similar UV protective effect of shikonin has been observed in
silk textile fibers (Dhandapani & Sarkar, 2007) and cellulose-based film
(Dong et al., 2020).

3.1.2. FT-IR analysis

FT-IR analysis was performed to test the effect of shikonin addition
on the chemical structure of the CMC and CNF films, and the results are
as shown in Fig. 2. Regardless of the addition of shikonin, the CMC and
CNF films exhibited characteristic absorption bands for each polymer.
For the CMC film, a broad absorption band around 3270 cm− 1 was due
to the O-H stretching vibrations, while the band at 2884 cm− 1 was
attributed to the C-H stretching vibrations of the CH2 groups of CMC (Su,
Huang, Yuan, Wang, & Li, 2010; Zaïdi et al., 2011). The absorption

Fig. 2. FT-IR spectra of the CMC and CNF-based films.

transmittance of the shikonin-added films was still over 60%, making it

useful for food packaging applications. The shikonin-added films
showed a minimum light transmittance at about 525 nm, which can be
attributed to the absorption of green light by shikonin (Ozgen, Miloglu,
& Bulut, 2011).
The UV and visible light transmittance values of the CMC and CNF-
based films are shown in Table 1. The T280 of the CMC film decreased
from 62% down to 31% by adding shikonin, indicating that the UV-
barrier property of the CMC film increased significantly as much as
two times. On the other hand, the T660 decreased slightly from 85% to
79% by adding shikonin, indicating that the transparency of the film was
not reduced significantly. Similarly, the addition of shikonin to the CNF
film reduced the T280 significantly from 54% to 22%, with a slight
decrease in the transparency from 87% of the T660 to 81%. The increased Fig. 4. Antioxidant activity of the CMC and CNF-based films.

Fig. 3. (a) TGA and (b) DTG thermograms of the CMC and CNF-based films.

4
P. Ezati et al. Food Control 126 (2021) 108046

Fig. 5. Color response efficiency of the CMC/shikonin and CNF/shikonin films. (a) Color change in response to different pH buffer solutions, (b) variation in color
parameters, (c) response of pH indicator films towards ammonia and acetic acid vapors. (For interpretation of the references to color in this figure legend, the reader
is referred to the Web version of this article.)

peaks at 1587 cm− 1 and 1413 cm− 1 corresponded to the carboxylic
groups’ asymmetric and symmetric vibrations. The C-O stretching in the
polysaccharide structure was indicated by the bands at 1103 cm− 1 and
1023 cm− 1 (Chai & Isa, 2013; Taleb, El-Mohdy, & El-Rehim, 2009). For
the CNF films, the absorption bands at 3339 cm− 1 and 2899 cm− 1 were
due to the O-H stretching and C-H stretching, respectively (Oun & Rhim,
2017). Besides, the absorption band at 1428 cm− 1 corresponding to
O-C-H and H-C-H deformation was observed. Also, the bands at 1316
cm− 1 and 898 cm− 1 were related to the C-H vibrations (Cheng et al.,
2014). There was no significant change in the positions of the absorption
bands of the shikonin-added films, indicating that no covalent bond was
formed between shikonin and the polymer. However, a slight shift in the
bands corresponding to the -OH stretching towards the lower wave­
number was observed in both CMC and CNF, indicating hydrogen

5
P. Ezati et al.
Table 2
The response of the CMC and CNF-based films to the changing pH of the packed fish samples.
CMC/shikonin
Time (h) pH L a b ΔE
0 5.7 ± 0.07a 73.7 ± 2.4c 14.9 ± 0.8c 4.5 ± 0.3b
12 6.1 ± 0.04b 65.3 ± 0.7b 10 ± 0.07b 3.1 ± 0.1a,b
24 6.8 ± 0.02c 64.2 ± 0.5b 9.7 ± 0.1b 2.6 ± 0.5a
36 6.9 ± 0.02c 61.2 ± 0.2a 7.3 ± 0.1a 2.6 ± 0.4a
bonding between the polymers and the pigment (Ezati, Bang, & Rhim,
2021a).
3.1.3. Moisture content (MC), water solubility (WS), water absorption
(WA), water vapor permeability (WVP), and water contact angle (WCA)
The MC, WS, WA, WVP, and WCA of the CMC and CNF-based films
are shown in Table 1. The MC of the neat CMC was 15.6%, and the MC of
CNF film was slightly lower than that of CMC film. However, the addi­
tion of shikonin reduced their MC significantly, which was mainly due to
the hydrophobic property of shikonin (Ezati et al., 2021a).
The WS of CMC and CMC/shikonin films could not be measured as
these films were immediately dissolved in water, indicating low water-
resistance of the CMC-based films (Deng, Jung, & Zhao, 2017). On the
other hand, CNF-based films were more water-resistant than CMC-based
films which indicated that the CNF film had more water holding ca­
pacity and good swellability (Roy & Rhim, 2021). The WS of CNF and
CNF/shikonin films were 34.4% and 26.0%, respectively. The signifi­
cantly increased water-resistance of the CNF/shikonin film (i.e., reduced
WS) was also due to shikonin’s hydrophobicity.
The WA of CMC-based films was also not measurable due to its low
water resistance. On the other hand, the WA of the CNF film was
355.2%, and the addition of shikonin to the CNF film reduced WA to
287.6%, which was also due to the hydrophobic properties of shikonin
pigments (Tatsumi et al., 2016).
The WVP of the neat CMC and CNF films were 1.4 × 10− 9 and 1.5 ×
10 g m/m2.Pa.s, respectively, in good agreement with the previously
− 9
reported values (Oun & Rhim, 2015). With the incorporation of shiko­
nin, no significant effect was observed on the water vapor permeability
of CMC films, while a slight decrease was observed in CNF films. The
addition of shikonin did not affect the vapor barrier properties of the
film, probably due to the lack of chemical interaction between the
polymer matrix and shikonin, as observed in the FT-IR results. There was
no change in the free hydrophilic hydroxyl groups of the cellulose
molecules and consequently did not affect the WVP of the film. Similar
results were observed in previous studies in which curcumin was added
to the CMC film. No significant change in the WVP was observed in the
CMC films by adding curcumin due to the lack of chemical interaction
between the CMC and the curcumin (Roy & Rhim, 2020c).
The WCA of the neat CMC and CNF films were 41.9◦ and 59.3◦ ,
respectively. The significantly higher WCA of the CNF film than the CMC
film may be due to the increased crystallinity of the CNF through the
TEMPO-oxidation process and the surface roughness of the CNF film
(Yamamoto et al., 2015). The addition of shikonin significantly
increased the surface hydrophobicity of the CMC and CNF films to 61.1◦
and 73.4◦ , respectively. The increased WCA of the shikonin-added CMC
and CNF films was also attributed to the hydrophobic nature of shikonin.
In general, a film with its surface WCA greater than 65◦ is considered to
be hydrophobic (Vogler, 1998). It is interesting to note that the CNF film
became hydrophobic by the addition of shikonin.
3.1.4. Thermal stability and tensile properties
The thermal stability of the CMC and CNF-based films was deter­
mined using the thermogravimetric analysis, and the results are shown
in Fig. 3. The CMC-based films showed a two-step decomposition, while
the CNF-based films showed a three-step decomposition. Initial weight
6
Food Control 126 (2021) 108046
CNF/shikonin
L a b ΔE
27.5 ± 2.6a 64.6 ± 1.1c 18.1 ± 1.1c 8.6 ± 0.7c 33.9 ± 1.01c
27.8 ± 0.7a 57.1 ± 0.9b 11.7 ± 0.8b 2.8 ± 0.7b 37.8 ± 0.8b
29.9 ± 0.5b 56.3 ± 0.5b 8.5 ± 0.1a 0.1 ± 0.1a 37.06 ± 0.4b
32.3 ± 0.3c 53.3 ± 0.2a 9.1 ± 0.8a − 1.1 ± 0.1a 40.2 ± 0.5a
R = 0.90 R = 0.85
loss was observed in the range of about 60–150 ◦ C in all films due to the
evaporation of water molecules adsorbed to the surface due to the
breakdown of hydrogen bonds (Oun & Rhim, 2017; Wang & Rhim,
2017). The CMC-based films showed a second and substantial weight
loss around 240–310 ◦ C with a maximum at 280 ◦ C, attributed to the
evaporation of glycerol and the polymeric decomposition cellulose
structure (Zong et al., 2017). In contrast, the CNF-based films showed
separate glycerol evaporation and polymer degradation; the former
occurred at about 200–300 ◦ C, the latter at around 320–380 ◦ C with the
corresponding maximum decomposition temperatures of 270 ◦ C and
350 ◦ C, respectively. However, the addition of shikonin did not show
any remarkable changes in the thermal stability of the CMC and CNF
films.
The tensile properties of the CMC and CNF-based films are shown in
Table 1. The tensile strength of the CMC and CNF films were 45 MPa and
79 MPa, respectively. The higher tensile strength of the CNF film is due
to the fibrillated nanostructure of the polymer matrix. Unlike the
random arrangement of CMC polymer chains in CMC films, the cellulose
nanofibers in CNF films can form highly oriented structures to have
higher mechanical properties (Sehaqui et al., 2012). A slight increase in
tensile properties was observed with the addition of shikonin, but the
increase was not statistically significant (p > 0.05), which may be due to
the absence of covalent bonding between the polymer chains and shi­
konin, as suggested by the FTIR data.
3.1.5. Antioxidant activity
The antioxidant activity of the CMC and CNF-based films was
determined by the ABTS and DPPH methods, and the results are shown
in Fig. 4. The ABTS and DPPH radical scavenging activities of the neat
CMC film were 2.3% and 1.3%, respectively, and the neat CNF film was
8.6% and 7.0%, respectively. The higher antioxidant activity of CNF
films than CMC films could be attributed to the higher surface area of
CNF, as this increased the exposed -OH groups capable of interacting
with free radicals while acting as electron donors. The addition of shi­
konin significantly increased the antioxidant activity of CMC and CNF
films. Still, the degree of increase was dependent on the polymer matrix
and the antioxidant activity measurement method. By adding shikonin,
the DPPH and ABTS free radical scavenging activity increased to 13.7%
and 93.5%, respectively. Hydrophilic CMC film easily absorbs the ABTS
aqueous solution, enhancing the expansion of the polymer chain and the
release of shikonin, enhancing the interaction with ABTS, increasing the
antioxidant activity. On the other hand, methanolic DPPH solution is not
easily absorbed by the hydrophilic CMC polymer, resulting in relatively
low interaction between shikonin and DPPH solution, resulting in lower
scavenging activity (Ezati et al., 2020). A similar trend was observed in
CNF films with the addition of shikonin, and 57% and 33.3% scavenging
activities were observed for ABTS and DPPH methods, respectively.
Because CNF is a fiber-based film, individual fibers are separated from
the solution, making it easier to release shikonin in aqueous ABTS and
methanolic DPPH solutions, resulting in high antioxidant activity in
both methods. In addition, hydrophilic CNF films easily swell and swell
in aqueous solutions, leading to higher antioxidant activity in aqueous
ABTS than in methanolic DPPH solutions (Ezati et al., 2020). The anti­
oxidant activity of shikonin is attributed to many phenolic groups pre­
sent in the pigment, which react with the free radicals and stabilize them
P. Ezati et al. Food Control 126 (2021) 108046

Fig. 6. The apparent color change of (a) CMC/shikonin and (b) CNF/shikonin indicator films indicating the quality change of packaged fish at 25 ◦ C. (For inter­
pretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

(Ezati & Rhim, 2020a; Z.; Wang et al., 2010). Besides, the antioxidant
activity of functional films depends on several factors such as the rate of
release of active substances, polymer microstructure, and interaction
between active components and free radicals (Liu et al., 2017).
3.1.6. Color response efficiency
The color response properties of the shikonin-added CMC and CNF
color indicator films are shown in Fig. 5. Visual color changes of CMC/
Sikonin and CNF/Sikonin films were observed in various buffers with pH
varying in the 2–12 range. Both color indicator films showed a reddish-
pink color in the acid to neutral pH range (i.e., pH = 2–7). As the pH
passed through neutral and increased towards the alkali, both films’
color began to show a purple shade. At pH 9, the film was reddish-
purple, turned completely purple at pH 10, and then turned light blue
at pH 12. The color values of the films at different pH are shown in Fig. 5
(b). The L-value (brightness) of the color indicator film was highest at pH

7
P. Ezati et al.
7 and lowest at pH 12, indicating that the indicator film was bright at
neutral pH and dark at alkaline pH. The a-value (redness/greenness) of
the film increased with increasing pH up to 4 and then significantly
decreased with increasing pH up to 12. On the other hand, the b-value
(yellowness/blueness) of the film did not change significantly until pH
= 7 but then decreased with increasing pH, resulting in blue color at pH
12. The total color difference (ΔE) increased significantly as the pH
increased toward alkali, allowing people to check the color difference in
the film visually. Moreover, the films were very stable and showed no
color change even after long-term storage (Ezati, Bang, & Rhim, 2021b).
The results for the change in color parameters as a function of the pH of
the film were consistent with the pH-responsive visual color change of
the film (Fig. 5).
The color stability of the pH indicator is directly affected by the
structural properties of the solid support and the quality and purity of
the color material. The color stability of the shikonin-adsorbed color
indicator paper was evaluated as a function of time using different pH
buffers. The tristimulus color values for the color indicators at the start
of storage and after 4 months are shown in Table 1. This indicator with
high color stability is considered suitable for the long-term storage of
packaged food.
The color response sensitivity to ammonia and acetic acid vapors was
tested to verify the indicator film’s color response to gases with different
pH values. As shown in Fig. 5(c), the initial reddish-pink color of both
indicator films turned bluish-purple in response to ammonia vapors. In
contrast, they turned a darker reddish-pink color in response to acetic
acid vapor. Since spoilage in fish and seafood products releases volatile
amines, the color indicator film, which shows a noticeable color change
in response to ammonia vapor, can be applied as a gas sensor to monitor
pH changes in packaged seafood.
3.2. Packaging test
Raw and processed food products are perishable and tend to spoil
over time due to biochemical or microbial actions (Gram et al., 2002).
Food degradation by microbial action is more common in foods with
high water activity, such as meat and seafood-based products (Gram &
Dalgaard, 2002). These products tend to release volatile amines as a
result of microbial growth (Comi, 2017). The volatile amines accumu­
late inside the package resulting in a pH change inside the package,
which can be detected by the color change of the pH-responsive color
indicator film.
The pH changes of the fish sample and color parameters of the in­
dicator films during the storage period are shown in Table 2. Fish
products have a pH threshold of 7, and fishes with higher pH values
cannot be consumed (Ezati, Tajik, Moradi, & Molaei, 2019). The initial
pH value of fresh fish was 5.7, which increased to 6.9 after 36 h at room
temperature, and the color of the indicator changed accordingly. Over
time, fish samples’ quality deteriorated, making the fish unacceptable to
consumers (Ezati, Tajik, & Moradi, 2019). While the L, a, and b-values of
both CMC/shikonin and CNF/shikonin indicators were decreased, the
ΔE increased up to 32.3 and 40.2 after 36 h of storage. As shown in
Fig. 6, both indicators changed color during the storage, responding to
the fish spoilage. The indicators were reddish-pink when the initial fish
was fresh and turned blue-purple when the fish completely decayed after
36 h. It is noteworthy that the CNF/shikonin indicator showed a more
pronounced color change compared to the CMC/shikonin indicator. This
is because CNF polymer has a higher specific surface area and a high
capacity for color change in response to pH (Ezati et al., 2020). Since the
pH change mainly causes the color change of the film, the color change
of the film depending on the pH change was investigated using the
Hunter color system (Table 2). Pearson’s regression method was used to
evaluate the correlation between the ΔE of the indicator films and the pH
of the fish samples. There was a strong, high, and positive correlation
between the ΔE of the indicator films and the pH of the fish sample with
a high correlation coefficient (R) of 0.90 and 0.85 for the CMC and CNF
8
Food Control 126 (2021) 108046
films, respectively. The CMC/shikonin and CNF/shikonin indicators can
be used as freshness indicators for fish quality monitoring, as Dong et al.
(2020) and Huang et al. (2019) suggested.
4. Conclusions
CMC and CNF-based pH-responsive color indicator films were pre­
pared by adding shikonin extracted from the roots of Lithospermum
erythrorhizon for monitoring the freshness of fish. The addition of shi­
konin increased the surface hydrophobicity of the film without changing
other film properties. The shikonin-added color indicator film showed
distinctive color changes depending on the pH from 2 to 12, showing
reddish-pink below pH 7, reddish-purple at pH 9, turned purple and
bright blue at pH 9–12. The color indicator films were used to monitor
the freshness of packaged fish samples over 36 h. The films were reddish-
pink when the fish was fresh (pH = 5.7), which changed to bluish-violet
after 36 h of storage, indicating spoilage of fish (pH = 6.9). The CMC/
shikonin and CNF/shikonin color indicator films can be used in smart
packaging applications to monitor fish and seafood freshness in real-
time during storage and distribution.
CRediT authorship contribution statement
Parya Ezati: Conceptualization, Investigation, Methodology, Data
analysis. Ruchir Priyadarshi: Data analysis, Writing – original draft,
review and editing. Yeong-Ju Bang: Investigation, Methodology. Jong-
Whan Rhim: Writing – review & editing, Supervision, Project admin­
istration, Funding acquisition.
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.
Acknowledgment
This research was supported by the Korea Research Fellowship
program funded by the Ministry of Science, ICT and Future Planning
through the National Research Foundation of Korea
(2019H1D3A1A01070715), and the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MSIT) (No.
2019R1A2C2084221).
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