Food Hydrocolloids 127 (2022) 107517

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

Two colorimetric films based on chitin whiskers and sodium alginate/

gelatin incorporated with anthocyanins for monitoring food freshness
Yuewei Zheng a, 1, Xiaomin Li a, c, 1, Yao Huang b, **, Houbin Li a, Lingyun Chen d, Xinghai Liu a, *
a
School of Printing and Packaging Engineering, Wuhan University, Wuhan, 430072, PR China
b
School of Environmental Studies, China University of Geosciences, Wuhan, 430074, PR China
c
Hubei Dan Ya Xiang Biotechnology Co., Ltd., Wuhan, 430030, PR China
d
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada, T6G 2P5

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

Keywords: Intelligent food packaging materials from natural resources has attracted increasing attention nowadays. In this
Chitin whiskers paper, two fresh colorimetric films were obtained by incorporating natural pigment anthocyanins extract (LRM)
Sodium alginate as pH-sensitive pigments into two polyelectrolyte complex films composed of chitin whiskers (CW) and sodium
Gelatin
alginate (SA)/gelatin (GE), respectively. The analysis results of FTIR, SEM and DSC show that, compared with the
Anthocyanin
Freshness detection
single polysaccharide system of SA or GE, the composite film with addition of CW has better water resistance and
thermal stability, due to the existence of hydrogen bonds and electrostatic interactions. Meanwhile, the adhesion
to LRM is also enhanced. At the same time, through Ritger–Peppas kinetic model analysis, the release process of
anthocyanins in the two film systems was analyzed. The CW/SA/LRM system membrane showed good respon­
siveness to lactic acid and was successfully used in the detection of milk freshness; while the CW/GE/LRM system
membrane showed good responsiveness to amine gases and has been successfully used in the detection of pork
freshness. The study revealed that the above colorimetric films demonstrate excellent durability and accuracy in
food freshness monitoring.

1. Introduction scientific research topic around food freshness will have good practical
application prospects as well as commercial values.
With the improvement of living standards, consumers are paying The characteristic metabolites produced during spoilage of food
more and more attention to food safety and quality. When food as a products, whether carbon dioxide or volatile salt-based nitrogen, can
commodity is processed or transported, it may come into contact with cause a change in pH within the packaging atmosphere (Balbinot-Alfaro
various physical, chemical and biological agents, which will inevitably et al., 2019; Choi & Han, 2018). Therefore, by loading pH-sensitive in­
affect the freshness and edibility of the food, and eventually food dicators into the packaging material to reflect this pH change, the
spoilage would take place(Kalpana, & Priyadarshini, & Maria Leena, & quality status of the food can be understood in real time. pH-sensitive
Moses, and Anandharamakrishnan, 2019). An important evaluation in­ indicators consist of a pH-sensitive pigment and a film-forming sub­
dicator of food safety is freshness. Nowadays, using packaging material strate to immobilize the pigment, usually in the form of a film or label
to achieve the detection of food freshness has become an attractive so­ attached to the packaging surface. There have been many studies using
lution, since it can respond to changes in food quality by sending elec­ synthetic pH indicators such as bromothymol blue, methyl red or a
trical signals or colorimetric signals, so that the consumers could mixture of indicators as color developers (Ran et al., 2021). Although
recognize the real-time freshness of food at the time of purchase(Park, & these color developers have rapid reactions and good color develop­
Nam, & Yun, & Jin, and Kwak, 2021). Even food packaging materials ment, they may migrate into food and cause food safety problems, so
can directly remove potential pollutants(Zhou et al., 2021; Linares, & many people are turning to nature to find natural color developers such
Peñaranda, & Iniesta, & Egea, and Garrido, 2022). Therefore, the as anthocyanins, curcumin, and chymosin, which are non-toxic and

* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (Y. Huang), [email protected] (X. Liu).
1
Yuewei Zheng and Xiaomin Li contributed equally to the writing of this article.

https://doi.org/10.1016/j.foodhyd.2022.107517
Received 15 October 2021; Received in revised form 9 January 2022; Accepted 17 January 2022
Available online 19 January 2022
0268-005X/© 2022 Elsevier Ltd. All rights reserved.
Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

renewable, widely available and cost-controlled. While anthocyanins, in indicators.

addition to these advantages, have excellent antioxidant properties,
protein protection and antibacterial effects (Li et al., 2019), making 2. Material and methods
them one of the best options for natural color developer (Ahmad, &
Abdullah Lim, and Navaranjan, 2020; Capello, & Leandro, & Gagliardi, 2.1. Materials
and Valencia, 2021; Capello et al., 2021b; Capello, Leandro, Gagliardi, &
Valencia, 2021a; Chen, & Zhang, & Bhandari, and Yang, 2020; Chen, Black wolfberry was purchased from Tmall Supermarket in Qinghai.
et al., 2020; Liu, & Cui, & Shang, and Zhong, 2021; Zeng et al., 2019). Chitin (M.W.:221.208 Da), sodium alginate (Analysis pure, M.
The main film-forming substrates of anthocyanin pH-sensitive in­ W.:398.31 Da) and gelatin (Chemically pure, Type B, isoelectric point at
dicators in current studies are focused on natural polymeric materials, pH 4.5~5.3) were all purchased from Shanghai Meryer Chemical
such as chitosan (Fernández-Marín, & Fernandes, & Sánchez, and Technology Co., Ltd. (China). The experimental water is ultrapure
Labidi, 2022; Ahmad, & Abdullah Lim, and Navaranjan, 2020; Li, & Wu, water. The milk and pork used were purchased from the local market in
& Wang, and Li, 2021; Zeng et al., 2019), starch (Chen, et al., 2020), Wuhan. Potassium chloride, hydrogen chloride, sodium hydroxide, so­
sodium alginate (Yenchit et al., 2020), and gelatin (Zeng et al., 2019), dium acetate trihydrate, acetic acid, ethanol, sulfuric acid, calcium
mainly due to their good film-forming properties and environmental chloride, phenolphthalein, glutaraldehyde, magnesium oxide, boric
tolerance. Sodium alginate (SA) is a natural polysaccharide compound acid, methyl red, and methylene blue were provided by Sinopharm
extracted from kelp or algae, which has good biocompatibility and Chemical Reagent Co., Ltd.
film-forming properties due to the large number of hydroxyl and
carboxyl groups in its molecular chain (Li, & Yi, & Yu, & Wang, and 2.2. Extraction of anthocyanins
Wang, 2020). Gelatin (GE), one of the most important sources of pro­
teins among biological macromolecules, is a commonly used natural The ultrasonic-assisted solvent extraction method (Wu et al., 2006)
hydrogel matrix, hydrophilic colloid, belonging to amphiphilic electro­ was used to extract anthocyanins from black wolfberry (LRM). The
lytes, chemically denatured from collagen obtained from animal skin, specific operation is as follows: the roots of the black wolfberry were cut
bone or tissue. Gelatin contains a large number of hydroxyl and amino off, and then the berries were dried in a drying box at 60 ◦ C for 24 h,
groups in its molecular structure and thus has a high hydrophilic nature crushed with a wall breaker, placed in a brown sample bottle, sealed and
(Azilawati, & Hashim, & Jamilah, and Amin, 2014). However, protected from light. 50 g of the crushed black wolfberry powder was
single-component films suffer from a brittle texture, poor mechanical added into 500 mL of acidified ethanol (ethanol solution containing 3%
properties and poor thermal stability. Therefore, the preparation of films acetic acid/hydrochloric acid) for soaking extraction under ultrasonic
by compounding two different film-forming substrates to improve their treatment for 30 min. Vacuum filtration was used to remove the insol­
physicochemical properties has been realized in several studies (Yang uble matter, and the filtrate was collected; the obtained filtrate was
et al., 2020; Ahmad, & Abdullah Lim, and Navaranjan, 2020; Capello rotary evaporated at 45 ◦ C to obtain a concentrated solution, and then
et al., 2021b; Capello, Leandro, Gagliardi, & Valencia, 2021a; Chen, & the solution was freeze-dried for 2 days to obtain LRM powder. The
Zhang, & Bhandari, and Yang, 2020; Chen, & Yan, & Huang, & Zhou, sample was stored at 4 ◦ C and protected from light.
and Hu, 2021; Liu, & Cui, & Shang, and Zhong, 2021; Sani, & Tavassoli,
& Hamishehkar, and McClements, 2021; Zeng et al., 2019). 2.3. Preparation of the colorimetric indicator films
Sodium alginate (SA) and gelatin (GE) are anionic polysaccharides
(Couvreur, 2014). The chitin whiskers(CW) are nano-scale crystals ob­ 2.3.1. Preparation of CW/SA series film
tained from the crystalline area of chitin after hydrolysis treatment Four-step preparation of chitin whiskers (Huang et al., 2015a): acid
(Zeng, & He, & Li, and Wang, 2011), which are positively charged due to hydrolysis-centrifugation-dialysis-centrifugation. The specific steps are
the protonation of amino groups (Tzoumaki, & Moschakis, and Bilia­ as follows: 10 g of chitin powder was hydrolyzed with 300 mL of 3 M
deris, 2010). Due to the existence of positive and negative charges and (mol/L) H2SO4 at 90 ◦ C, with vigorous stirring for 6 h. The resulting
hydrogen bonds, it not only can improve and enhance the water resis­ suspension was then repeatedly diluted with distilled water and
tance and mechanical properties of the composite material by the for­ centrifuged (7200 rpm) to remove excess acid, and the supernatant was
mation of polyelectrolyte composite materials (Huang et al., 2015a), but discarded. The precipitates were dialyzed against distilled water until
also anthocyanins can also be fixed in the composite film, which pre­ pH surpassed 6. In order to make the chitin whiskers more uniformly
vents the migration of anthocyanins and increases the application range dispersed, the suspensions were subjected to ultrasonic treatment at 50
of composite films(Wu et al., 2021; Liao et al., 2021; Singh, & Nwabor, & W power for 30 min. Afterwards, the chitin whisker suspension was
Syukri, and Voravuthikunchai, 2021; Zhou et al., 2021). However, further centrifuged (7200 r/min) for 10 min to remove the visible
Colorimetric films based on CW/SA and CW/GE systems have not been sediment to obtain the chitin whisker suspension (CW).
reported. For preparation of the CW/SA composite film, 0.1 M NaOH was
Therefore, in this paper, CW was introduced to improve the prop­ added dropwise to the diluted CW suspension, to keep the pH range of
erties of two natural polymers SA and GE films, and both systems were 11.0–11.5 during which the CW is negatively charged. The resultant
realized for milk/pork freshness testing. Two freshness colorimetric suspension was then subjected to ultrasonication for 5 min. To 98 g of
films with good pH responsiveness were obtained by incorporating the above suspension, 2 g of sodium alginate powder (SA) was added.
natural pigment anthocyanin as pH-sensitive pigments in the above The mixture was stirred overnight to dissolve and obtain a CW/SA
mentioned two systems, CW/SA and CW/GE, by scraping and casting composite solution. Then to 21 g, 18 g, and 14 g of the above mixed
methods, respectively. The two films were also characterized by FT-IR, solution, 7 g, 12 g, and 14 g of 20 mg/mL LRM solution (the solvent is
SEM, automatic contact angle meter and differential scanning calorim­ pure water and the extractant is ethyl acetate) was respectively added to
eter to study the water solubility, thermal stability, pH sensitivity and obtain a mixed film-forming solution with anthocyanin content of 5, 8,
other physical characteristics of the two films. The optimal content of and 10 mg/mL (mass fraction of 0.5%, 0.8%, 1%, respectively). After
anthocyanins in both systems was also analyzed thermodynamically. In stirring for 10 min, 25 g of the film-forming solution was poured into the
addition, this study also analyzed the release process of anthocyanins in glass mold and soaked in a coagulation bath of 4 wt% CaCl2, and then
the two film systems through the Ritger–Peppas kinetic model, and 0.01 M HCl was added to the neutralize the NaOH residue in the coag­
studied the optimal addition amount of anthocyanins in the two com­ ulation bath and make the CW positively charged. The resultant
posite films. The two prepared colorimetric films have promising ap­ hydrogels were rinsed with ultrapure water for several times to remove
plications as milk/pork freshness monitoring and smart packaging excessive NaOH/HCl and salt to obtain CW/SA-LRM composite

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 1. The color (a) and UV absorption spectrum (b) of LRM at pH 3–12. the structure change of anthocyanin under different pH conditions(c). (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)

hydrogels, named CSL1, CSL2 and CSL3, respectively. The gel film is 5 kV.
fixed on the organic glass substrate with the aid of adhesive tape and According to the ASTM D7334 standard, use an automatic contact
then air dried. At the same time, CW/SA (without LRM) and SA/LRM angle meter (model SL200B, Kino Industry Co., Ltd., Boston, USA) to
(LRM content of 5 mg/mL) were prepared as a control group to observe measure the water contact angle (WCA) of the film sample at room
the performance of the test film. temperature. Each sample was measured at three different locations,
and the average contact angle was reported. In the process of measuring
2.3.2. Preparation of CW/GE series film the contact angle, a high-speed digital camera is used to capture digital
For preparation of CW/GE composite films, the pH of the above- images at the same time.
mentioned CW suspension was adjusted to 3.0–4.0 with 0.1 M HCl According to the ASTM D644-99 (ASTM, 2004) standard, the mois­
(where both CW and gelatin are positively charged), and the suspension ture content (MC) of the film is calculated by the mass change of the
was ultrasonicated for 5 min. To 98 g of the above suspension, 2 g of sample film (20 mm × 20 mm) dried in an oven at 105 ◦ C for 24 h. MC is
gelatins (GE) powder was added at 45 ◦ C and stirred for 30 min to calculated by formula (1) calculation:
accelerate the dissolution of the gelatin to obtain a CW/GE mixture.
M1 − M2
Then to 15 g, 12 g, and 9 g of the above mixed solution, 5 g, 8 g, and 9 g MC = × 100% (1)
M1
of 20 mg/mL LRM solution (the solvent is pure water and the extractant
is ethyl acetate) was respectively added to obtain a mixed film-forming Among them, M1 is the initial weight (g) of the film when it is stored
solution with anthocyanin content of 5, 8, 10 mg/mL (mass fraction is at 75%RH to reach moisture balance, and M2 is the final weight (g) of
0.5%, 0.8%, 1%, respectively). After stirring for 10 min, 15 g of the film- the film after drying at 105 ◦ C.
forming solution was poured into a petri dish and spread out. After The swelling rate and water solubility were determined, according to
drying, a CW/GE-LRM composite hydrogel film was obtained, which a method described in the literature (Al-Naamani, & Dobretsov, and
were named CGL1, CGL2, and CGL3. At the same time, CW/GE (without Dutta, 2016). Briefly, the sample film with a size of 20 mm × 20 mm was
LRM) and GE/LRM (LRM content of 5 mg/mL) were prepared as a dried in an oven at 70 ◦ C for 24 h, and weighed as the initial dry weight
control group to observe the performance of the test film. (W0). Then the film sample was immersed in a closed beaker containing
50 mL of pure water and soak for 24 h. After removing the film, wipe off
the water on the surface of the film with filter paper, weigh it to obtain
2.4. UV–vis spectra measurement
the wet weight (W1), and dry it again to obtain the final dry weight.
(W2). The swelling rate (SR) and water solubility (WS) of the sample film
The absorption spectrum of black wolfberry extract (LRM) solution
are calculated by formulas (2) and (3):
at different pH values was measured by UV–vis spectrophotometer. A
buffer solution of pH 3.0–12.0 was prepared with 0.2 M disodium W1 − W0
SR(%) = × 100% (2)
hydrogen phosphate, 0.1 M citric acid and 0.1 M sodium hydroxide. W0
Scanning to obtain the ultraviolet–visible spectrum of the above solution
in the range of 450–700 nm. WS(%) =
W0 − W2
× 100% (3)
W0

2.5. Characterization of films To determine the thermal stability of the films, a differential scan­
ning calorimeter (DSC) (TGA2/DSC3, Mettler-Toledo, Switzerland) was
Use Nicolet 5700 Fourier Infrared Spectrometer (NICOLET 5700 used in the temperature range of 100–300 ◦ C (change rate 10 ◦ C/min),
FTIR Spectrometer) to measure in the range of 4000-400 cm− 1. Zeiss under nitrogen conditions.
SIGMA (Carl Zeiss) scanning electron microscope was used to analyze The potential of the film for freshness indication is determined by
the surface microstructure of the film, and the acceleration voltage was measuring the responsiveness of the film to different pH buffer solutions.

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 2. FTIR spectra of different composite films. (CW= Chitin Whiskers, LRM = Lycium Ruthenicum Murr (extract anthocyanins from black wolfberry), SA= Sodium
Alginate, GE = Gelatin).

Fig. 3. Surface SEM micrographs (The scale bars are 5 μm) of CW/SA series films (a–e) and CW/GE series films(f-j).

Fig. 4. Schematic diagram of contact angle of SA/CW series films (a–e) and CW/GE series films (f–j), DSC graph of SA/CW series films (k) and CW/GE series films(l).

Prepare a buffer solution with a pH value of 3.0–12.0, and adjust the pH 2.6. Food freshness detection applications of the films
by 0.1 M NaOH and 0.1 M HCl, and then drop the buffer solution onto
the surface of the indicator film (10 mm × 10 mm), and let it stand for 5 2.6.1. CW/SA system film used for milk freshness detection
min to evaluate its color response, and record its color with a digital 20 mL of milk was poured into a sterilized petri dish at 25 ◦ C for 60 h,
camera. then the milk was dropped onto colorimetric film and waited for 10 min.
The color changes of the film were recorded every 12 h with a digital
camera and the corresponding ΔE value was calculated. The films were
stored under the conditions shown in Table S1, and the color of the

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Table 1 solution gradually became lighter, which turned to light purple at pH 7,

Water content, swelling rate and water solubility of CW/SA and CW/GE series and deeper purple at pH 8. Thereafter, the color of the solution gradually
film. changes from blue to yellow during pH range of 9–12. It can also be seen
Sample film Water content/% Swelling rate/% Water soluble/% in Fig. 1 (b), that the solution shows a maximum absorption near 525 nm
CS 9.73 ± 0.23 100.00 ± 0.47 21.25 ± 0.24
at pH 3, and the peak intensity decreases with the increase of pH,
SL 22.77 ± 0.61 145.33 ± 0.26 42.67 ± 0.72 especially at pH 5–6, the absorption almost disappeared completely;
CSL1 13.04 ± 0.45 71.83 ± 1.34 18.31 ± 0.44 while at pH 8–10, the maximum absorption peak showed a red shift of
CSL2 13.79 ± 0.38 54.93 ± 0.65 23.94 ± 0.56 about 580 nm, with intensity increased; finally, at pH 11–12, there was
CSL3 14.29 ± 0.26 93.88 ± 0.34 26.53 ± 0.84
no obvious characteristic peak during 450–700 nm. At this pH, it is likely
CG 8.54 ± 0.46 304.29 ± 1.21 48.57 ± 0.46
GL 21.52 ± 0.73 488.89 ± 0.57 50.00 ± 1.13 that anthocyanin degradation has occurred (Oroian & Escriche, 2015).
CGL1 14.10 ± 0.26 246.43 ± 0.18 26.79 ± 0.37 The color change trend is the same as the color change trend proposed
CGL2 14.58 ± 0.53 288.24 ± 0.36 30.88 ± 0.62 by Liu and Ma (Liu, Cui, Shang, & Zhong, 2021; Ma & Wang, 2016). As
CGL3 14.29 ± 0.34 263.64 ± 0.58 32.47 ± 0.48 Fig. 1 (c) shown, the pink color in an acidic environment is due to the
structural form of the flavylium cation. When the pH value of the so­
composite CW/SA series film was photographed once a day, and the lution rises to 6, the flavylium cation becomes colorless as methanol off
corresponding L*, a*, and b* parameters were recorded for 20 consec­ base due to the deprotonation of the hydroxyl group, and causes the
utive days. And calculate the color difference of the film according to the disappearance of absorption near 525 nm (Liu et al., 2021). In an
following formula, and determine the stability of the film according to alkaline environment, the anthocyanin structure molecule deprotonates
the color change of the film. △E and SRGB are calculated by formulas (4) for the second time to generate an anionic quinoid base and cause a dark
and (5): shift. When the pH value exceeds 10, due to the degradation of LRM in a
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ strong alkaline environment, the central ring of anthocyanins opens to
ΔE = (L* − L0 * )2 + (a* − a0 * )2 + (b* − b0 * )2 (4) produce chalcone, and the color of the solution changes to yellow
(Grajeda-Iglesias et al., 2016).
ΔR + ΔG + ΔB
SRGB = × 100% (5)
Ra + G a + Ba 3.2. Characterization of film structure and performance
Among them, the value of L* is the brightness, a* is from red to
green, b* is from yellow to blue, L0*, a0*, b0* are the initial values of the 3.2.1. Chemical structure
film color, and L*, a*, b* are after storage the color parameters of the Fig. 2 (a) shows the FTIR spectra of CW, SA, LRM, SA/LRM composite
film. △R = |Ra–Rb|, △G = |Ga-Gb|, △B = |Ba-Bb|, Ra, Ga, Ba are the film and CW/SA/LRM composite film. In the CW spectrum, it can be
initial values of red, green and blue, Rb, Gb, Bb are storage After the seen that two characteristic peaks at 1656 cm− 1 and 1630 cm− 1 are
chromaticity. attributed to the amide I band, which are C– – O stretching vibration and
− 1
N–H bending vibration; 1559 cm is attributed to amide II (Huang
2.6.2. CW/GE system film used for the detection of pork spoilage et al., 2015a; Watthanaphanit, & Supaphol, & Tamura, & Tokura, and
50 g of fresh pork was placed in a petri dish. the film was cut into a Rujiravanit, 2008a). In the LRM spectrum, it can be observed that the
size of 10 mm × 10 mm and fixed on the headspace of the petri dish, then characteristic peak at 1631 cm− 1 corresponds to the stretching vibration
the petri dish was placed at 25 ◦ C for 72 h. The colorimetry was recorded of C–O and C–O–C in aromatic compounds, and the peak at 1402 cm− 1
with a digital camera every day and the color change of the film is used corresponds to the C– – C stretching vibration of the aromatic ring. Two
to calculate the corresponding ΔE value. aromatic ring C–H deformation peaks at 1074 cm− 1 and 1035 cm− 1 and
a weak pyran ring (characteristic group of flavonoids) at 1270 cm− 1 (Li
3. Results and discussion et al., 2019). SA showed asymmetric and symmetric stretch carbonyl
groups corresponding to carboxylate ion (COO− ) at 1615 cm− 1 and
3.1. UV–vis spectrum analysis of LRM 1417 cm− 1. In the SA spectrum, the asymmetric stretching carbonyl
group of COO− at 1615 cm− 1 and the aromatic ring C–O group at 1631
In order to verify the feasibility of the LRM solution for pH detection, cm− 1 in the LRM spectrum overlap, shifting it to 1629 cm− 1 in the
the color change and UV absorption spectrum of the LRM solution at SA/LRM spectrum, and it can be seen that LRM was at 1040 cm− 1. The
different pH values were measured, and the results are shown in Fig. 1 characteristic peaks around indicate that LRM has been successfully
(a). When the pH increased from 3 to 12, the color of the LRM solution immobilized in the SA matrix. In the CW/SA spectrum, it can be
gradually changed from pink to yellow. At pH 3–6, the pink color of the observed that the characteristic peak of CW appears at 1627 cm− 1 and
the characteristic peak of SA appears at 1417 cm− 1, indicating that CW

Fig. 5. DSC graph of SA/CW series films(a) and CW/GE series films(b).

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 6. The color of LRM at pH 3–12(a). Color change of CW/SA system (b) and CW/GE system (c) at pH 3-12. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)

and SA have successfully mixed into a film, and the composite film 1656 cm− 1 has a low-frequency shift to 1632 cm− 1, and the amide A
shows 1317 cm− 1 The new peak may be the hydrogen bonding inter­ band is also shifted to 3437 cm− 1, It shows that the carbonyl group and
action between the alginic acid group of SA and the amino group of CW. the hydroxyl group form a hydrogen bond interaction (Huang et al.,
In addition, in the CW/SA/LRM spectrum with CW added, the charac­ 2015b; Watthanaphanit, Supaphol, Tamura, Tokura, & Rujiravanit,
teristic peak of CW around 1640–1540 cm− 1 can be observed, indicating 2008b). In addition, the characteristic peaks of the components can be
that CW was successfully mixed into a film. At the same time, the seen in each composite film, indicating that LRM has been successfully
composite membrane showed a new peak at 1320 cm− 1, which may be fixed in the matrix and has good compatibility with CW and GE.
due to the hydrogen bonding interaction between the alginic acid group
of SA and the amino group of CW or the hydroxyl group of LRM 3.2.2. Morphologies of the films
(Al-Naamani, Dobretsov, & Dutta, 2016). SEM can be used to analyze the influence of different mixtures and
Fig. 2 (b) shows the FTIR spectra of CW, GE, LRM, GE/LRM com­ their ratios on the membrane structure. Fig. 3(a–e) shows the surface
posite film and CW/GE/LRM composite film. The spectrum analysis of topography of the CW/SA series film. In the SL film (Fig. 3b), the sodium
CW and LRM is the same as above. GE shows a broad amide A band (O–H alginate gel film exhibits a groove-like morphology. After the addition of
stretch) around 3600-3300 cm− 1 (Ge et al., 2018; Sahraee, & Milani, & CW, the surface uniformity of the film was significantly improved and
Ghanbarzadeh, and Hamishehkar, 2017), and the bands at 1646 cm− 1 displayed a smooth and dense structure (Fig. 3a). When LRM was added
and 1535 cm− 1 correspond to amide I and amide II bands, respectively. in lower content, the compact texture was not altered (Fig. 3c&d). The
In the GE/LRM spectrum, the wide amide A bands of GE and LRM and main reason is due to the hydrogen bond interaction between the amino
the C–H deformation of the aromatic ring at 1074 cm− 1 and 1035 cm− 1 group in CW, the carboxyl group in SA and the hydroxyl group in LRM,
of LRM have high-frequency shifts, indicating that there may be accompanied by electrostatic attraction between ammonium ion (NH4+)
hydrogen bonds (Ge et al., 2018). In the CW/GE spectrum, the C– –O in CW and carboxylate (COO− ) ion in SA. But with further increase of
group of CW at 1656 cm− 1 has a low-frequency shift to 1646 cm− 1, and LRM content, the surface became rough and even cracks appeared on the
the amide A band also shifts to 3438 cm− 1. It shows that the carbonyl surface of the CSL3 (Fig. 3e, LRM mass fraction of 1 wt%) film. This may
group and the hydroxyl group form a hydrogen bond interaction (Huang be ascribed to the aggregation of anthocyanins at higher concentration
et al., 2015b; Watthanaphanit, & Supaphol, & Tamura, & Tokura, and during the drying process of the film.
Rujiravanit, 2008b). The characteristic peak of SA appeared at 1435 Fig. 3(f–j) shows the surface topography of the CW/GE series film. In
cm− 1, indicating that CW and GE were successfully mixed to form a film. the GL film, the gel film has some fibrous protrusions. After the addition
In addition, in the CW/GE/LRM spectrum, the C– – O group of CW at of CW, the film surface showed high uniformity with smooth and dense

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 7. The responsiveness of CW/SA series films to lactic acid (a-lactic acid response real-time image, b-film color difference △E change, c-film response sensitivity
SRGB change). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

structure. However, after introduction of LRM, the uniformity was still 2016; Li, et al., 2020; Yin, & Tang, & Wen, and Yang, 2007). But
slightly reduced, and with the increase of the LRM content, some blocky compared with GL film (contact angle WCA is 67.19◦ ), the contact angle
protrusions appeared on the surface of the film, which may be due to the of the composite film CGL1 (with the same LRM content) increased,
aggregation of anthocyanins at high content. indicating improved hydrophobicity by introduction of CW.
The SEM results show that the CW reinforced films exhibit relatively As shown in Table 1, when CW was added, the water content of the
homogeneous and dense surface, while no porous structure were CW/SA composite system film (CSL1) decreased from 22.77% of the
observed, which might contribute to the gas barrier property and original SA single system film (SL) to 13.04%, the swelling rate
moisture resistance of the films during their application as colorimetric decreased from 145.33% to 71.83%, and the water solubility decreased
labels However, higher content of anthocyanins might impair this ho­ from 42.67% to 18.31%. It may be due to the hydrogen bonding inter­
mogeneous structure by forming aggregates. action between the amino group in CW and the carboxylic acid group in
SA or the hydroxyl group in LRM, or it may be due to the electrostatic
3.2.3. Hydrophobic performance and thermal stability of the composite attraction of the ammonium ion generated by the protonation of the
films amino group in CW and the carboxylic acid ion in SA, which makes the
The food freshness detection process is usually in a humid environ­ system of the composite film more stable and reduces the binding with
ment, so the hydrophilicity and hydrophobicity of the film is very water molecules, based on this data it can be judged that the hydro­
important to the stability of the detection. As can be seen in Fig. 4(a–e), philicity of the film is suppressed and the hydrophobicity is improved,
since both SA and LRM are hydrophilic components, the thin films are which is consistent with the conclusion obtained from the contact angle
both hydrophilic and their contact angles are both lower than 90◦ . data. Similarly, the water content of the CW/GE composite system film
However, compared with the CS film, the water contact angle increased (CGL1) was reduced from 21.52% to 14.10% of the original GE single
after adding LRM, because LRM provides a large number of hydroxyl system film (GL), the swelling rate was reduced from 488.89% to
groups for the entire system, the probability of combining with water 246.43%, and the water solubility was reduced from 50.00% to 26.79%.
molecules is greatly increased, showing that the contact angle is reduced It may be due to the interaction of the amino group in CW and the
and the hydrophilicity is increased (Prietto et al., 2018). At the same carboxylic acid group in GE forming hydrogen bonds and the electro­
time, compared with the SL film, the contact angle of the film has static interaction of ammonium and carboxylic acid ions, which makes
increased after adding CW. This is mainly due to the hydrogen bond and the internal system of the composite film more stable and reduces the
electrostatic interactions between the amino group of CW, the carboxyl binding with water molecules, based on this data it can be judged that
group of SA and the hydroxyl group of LRM, which improved the the hydrophilicity of the film is suppressed and the hydrophobicity is
structure compactness of the composite film (consistent with the results improved, which is consistent with the conclusion obtained from the
in SEM) and contribute to the hydrophobicity of the film. It can be seen contact angle data.
from Fig. 4(f–j) that the CG film is hydrophobic. This is because strong As shown in Table 1, LRM is a hydrophilic component, so the water
hydrogen bonds and electrostatic attraction are formed between CW and content and water solubility of the films increased with the increase of
GE, which reduces the combination with water molecules. When LRM is LRM content (CSL1→CSL3:anthocyanin content 0.5%→0.8%→1%).
added, the hydrophilicity is increased and therefore the contact angles From the above table, it can be seen that the swelling rate of CS film is
decreased (Ahmad, & Nirmal, & Danish, & Chuprom, and Jafarzedeh, 100% and the swelling rate of SL film is 145.33%, while the addition of

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 8. The color change of the film of the milk sample liquid stored at 25 ◦ C for 60 h (a-Real-time image; c-Color difference change) and the pH value and TA value
change of the pig milk sample liquid during the spoilage process (b). (For interpretation of the references to color in this figure legend, the reader is referred to the
Web version of this article.)

both CW and LRM decreases the swelling rate of the film, indicating that the film (Compared with SL), that is, the thermal stability increased.
a strong interaction is formed between CW, SA and LRM, which inhibits This is related to the strong hydrogen bonding force and electrostatic
the interaction with water and therefore the dimensional stability of the attraction of CW and SA, which can also be explained in FTIR and SEM
film is enhanced. However, the CSL2 and CSL3 films exhibited increased surface topography. Fig. 5 (b) shows the DSC curves of chitin/gelatin
water solubility and swelling due to the excessive hydroxyl groups (CG), gelatin/black wolfberry extract (GL), and each composite film. It
introduced by the high content of anthocyanins, which increased their can be seen that there is a broad endothermic peak at 222 ◦ C in the film
binding to water molecules after prolonged immersion. Similarly, it can (CG) without added anthocyanin, which corresponds to the melting
be concluded that a large number of polyphenol molecules in LRM can temperature. After adding LRM, the peak shifts at low frequency. The
form hydrogen bonds with hydrophilic groups in CW and GE, which endothermic peak of CGL1, CGL2, and CGL3 film appeared at 199 ◦ C,
strengthen the cohesion of the system, so that CGL films exhibit lower 204 ◦ C, 207 ◦ C, respectively, but the peak of the membrane (GL) that is
water solubility and swelling than CG. And with the increase of antho­ relatively free of chitin shows A slight high-frequency movement in­
cyanin content (CGL1→CGL3:anthocyanin content of 0.5%→0.8%→ dicates that the addition of LRM reduces the melting temperature of the
1%), a large number of hydroxyl groups appear in the system, thus the film (Compared with CG), and the addition of CW increases the melting
hydrophilicity of anthocyanin occupies the main position, which in­ temperature of the film (Compared with GL), that is, the thermal sta­
creases the swelling rate and water solubility of the film. bility increases. This is related to the hydrogen bonding force of CW, SA
From the above data, it can be seen that the addition of both CW and and LRM. The higher the anthocyanin content (CGL1→CGL3: the change
LRM decreases the swelling rate of the film, indicating that a strong in anthocyanin content is 0.5%→0.8%→1%), due to the stronger inter­
interaction is formed between CW, SA/GR and LRM, which inhibits the action force, the melting temperature is further increased, from 199 ◦ C
interaction with water to some extent. to 207 ◦ C, that is, the thermal stability is further improved.
Fig. 5 (a) shows the DSC curves of CW/SA, SA/LRM, and each To this point, the composite membrane has multiple advantages over
composite film. It can be seen that the CS film without anthocyanin has a single-component membranes. Firstly, infrared spectroscopy proves that
sharp endothermic peak at 219 ◦ C, corresponding to the melting tem­ CW can form hydrogen bonds with SA/GE and LRM, which improves the
perature. After adding black wolfberry extract (LRM), the peak shifts at stability of LRM presence in the film and facilitates pH detection. sec­
low frequency, the endothermic peak of CSL1, CSL2 and CSL3 film ondly, SEM and contact angle prove that the addition of LRM decreases
appeared at 196 ◦ C, 200 ◦ C, and 210 ◦ C, respectively, indicating that the the smoothness of the single-component film of SA/GE, which is unfa­
addition of LRM reduced the melting temperature of the film (Compared vorable for individual pH detection, while the smoothness of the film is
with CS), and the addition of CW increases the melting temperature of improved after the addition of CW. It is beneficial to its application in

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

to study the release behavior of anthocyanins in two film systems. By

analyzing the standard curve of LRM in different solvents (pure water,
95% ethanol, 4% acetic acid) Fig. S1, the release curves and release
model diagrams of CW/SA/LRM system film and CW/GE/LRM system
film in different solvents can be obtained. Fig. S2 and Fig. S3. Through
analysis, it can be shown that the release behavior of LRM from the films
of the two systems is well in line with the characteristics of the
Ritger-Peppas kinetic model. It also shows that the release of LRM from
the membrane mainly follows the Fickian diffusion mechanism. And as
the content of LRM increases, the value of n gradually decreases, indi­
cating that low content of LRM is conducive to Fickian diffusion. For
specific analysis content, please refer to SI.

3.3. Application of film freshness indicator

3.3.1. Analysis of pH sensing performance of thin films

The films of both systems were placed in different pH environments
and the color change pattern was observed. Fig. 6 (a) shows the color
change of LRM solution at different pH environments. Fig. 6 (b) shows
the color change of the CW/SA system film with pH environments. The
color change with pH is very obvious in the acidic region., while the
main metabolite in milk spoilage process is lactic acid, therefore CW/SA
system is suitable for the detection of milk freshness. The color change of
the CW/GE system under different pH environments is shown in Fig. 4
(c). The color change of CW/GE system and LRM solution is not exactly
the same. Its color change with pH is more obvious in the alkaline re­
gion. The main metabolite in the decay of pork was ammonia, thus, CW/
GE system might be more appropriate for pork freshness detection. The
reason for this phenomenon is related to the preparation environment of
the two different system films. The CW/SA film is prepared under
alkaline conditions, which leads to its obvious discoloration under acidic
conditions. The CW/GE film is formed under acidic conditions, which
leads to its obvious discoloration under alkaline conditions.

3.3.2. CW/SA film used for milk freshness detection

Fig. 7(a–c) shows the responsiveness of CW/SA series films to lactic
acid, which can be used to simulate microbial spoilage caused by lactic
acid bacteria in the process of milk spoilage. The results showed that the
film showed obvious color response to lactic acid. The film color grad­
ually showed a red shift after exposure to lactic acid solution, and its ΔE
and SRGB values continued to increase within 2 h. The responsiveness of
the film was closely related to the concentration of LRM. The low-
concentration LRM film (CSL1) showed obvious red color at about 4
min, and the ΔE value exceeded 15, reaching 18.49; the CSL2 film
showed obvious red color after 2 min, with a ΔE value of 16.16; while
the highly concentrated LRM membrane (CSL3) became obvious red
within 1 min, and ΔE reached 21.28, SRGB reaching 27.23%. After that,
the redness of the film further deepened. When approaching the end of
the response time of 2 h, the ΔE value of the CSL3 film was 51.23 and the
SRGB value was 64.32%. Both visual observation and data results show
obvious difference of film color as pH changed, indicating that CW/SA
series films can be used to indicate the freshness of food with spoil
metabolite of lactic acid.
The pH of fresh milk is weakly acidic, probably between 6.6 and 6.8,
which is mainly related to the presence of citric acid, phosphate and
Fig. 9. The response of CW/GE series films to amine gases (a, b, c-ammonia; d, casein. When the milk leaves the refrigerated environment, the micro­
e, f-dimethylamine; h, i, j-trimethylamine). organisms in it begins to multiply, and lactose is decomposed by lactic
acid bacteria to produce lactic acid, which leads to the accumulation of
food packaging and freshness testing. lactic acid, so that the pH decreases and the quality of milk also drops
sharply. Therefore, the deterioration of milk can be detected by the pH
3.2.4. Analysis of the release properties of the two films colorimetric indicator film. As shown in Fig. 8(a–c), the pH value of milk
Existing research on the release of anthocyanins in films focused on decreased from the initial 6.71 to 5.56 within 60 h. After 12 h of storage,
simulation experiments (Wu et al., 2021; Liao et al., 2021; Singh, & the acidity of the milk increased from the initial 16.2 to 18. According to
Nwabor, & Syukri, and Voravuthikunchai, 2021; Zhou et al., 2021), but GB/T 5009.46-1996, the lactic acid degree of fresh normal milk should
this study used the classic release kinetic model Ritger-Peppas equation be no higher than 16–18◦ TA. Therefore, the milk became stale after
being stored at 25 ◦ C for 12 h. And in the process of milk spoilage, the

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

Fig. 10. The color change of the film of pork samples stored at 25 ◦ C for 72 h (a-Real-time image; c-Color difference change) and changes in pH and TVB-N value of
the pork sample during the spoilage process (b). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)

color of the indicator changed from blue-purple to light red, and ΔE difference changes within 1 min. The ΔE value of the CGL1 film was
reached 5.39–8.25 within 12 h. Among them, the discoloration of CSL1 5.39, the ΔE value of the CGL2 film was 6.4, and the ΔE value of the
was not obvious. By increasing the LRM content, it can be seen that the CGL3 film reached 7.81, and the color change stabilized in about 15 min.
ΔE of CSL3 at 12 h was 8.25, and it reached 25.34 after 60 h, showing a At this time, the ΔE value of CGL1 film was 19.95, the ΔE value of CGL2
clear color response that can be distinguished by naked eye observation film was 22.36, and the ΔE value of CGL3 film was 25.77, and its
and data, so it can be used to indicate the spoilage of dairy products. response sensitivity reached the maximum value of 30.69%. In sum­
mary, it can be seen that the composite film with high LRM content
3.3.3. CW/GE film is used for pork spoilage detection exhibited faster and more sensitive responsiveness to amines, and has
Fig. 9(a–j) shows the response results of CW/GE series films to amine the potential to be used as an indicator of meat freshness.
gases, which can be used to simulate the volatile nitrogen compounds The protein in meat is very susceptible to bacteria and molds, and
produced when meat is spoiled. The results show that ammonia and various volatile nitrogen-containing compounds such as ammonia and
water molecules combine to generate ammonium ions (NH4+) and hy­ amines are produced during the rotting process, which may change the
droxide ions (OH− ), which make the film form an alkaline environment, pH in the packaging atmosphere. Therefore, it is possible to establish a
causing color change of the composite film. At this time, the structure of connection between the change of pH and the degree of spoilage of the
anthocyanins is transformed into quinoid base or chalcone, which is food. Fig. 10(a–c) shows the color change of the CGL colorimetric sensor
reflected by that the film turns green (Choi, & Lee, & Lacroix, and Han, film applied to pork spoilage monitoring. It can be seen that the pH and
2017). The results indicate that the three membranes showed good TVB-N value of pork continued to increase during storage. The pH value
responsiveness to amine gases. In the ammonia response, the ΔE value of increased from 5.54 to 72 h to 7.6, and the TVB-N value increased from
the three kinds of membranes was greater than 5 within 1 min, that is, 8.33 mg/100g to 15.12 mg/100g within 24 h. According to the Chinese
the discoloration was obvious, and it became stable around 10 min. The standard GB/T 5009.44.2003, meat with TVB-N content below 15 mg/
ΔE value of CSL1 membrane reached 17.94 within 10 min, and the ΔE 100g is considered fresh meat, 15–25 mg/100g is in the deterioration
value of CSL2 membrane reached 10. The ΔE value in min is 21.21, and stage, meat higher than 25 mg/100g is considered fresh meat Not edible
the CSL3 film has the highest ΔE value, which is 24.68; and the film also meat (Khulal, & Zhao, & Hu, and Chen, 2017). It can be seen that the
shows obvious green. In the dimethylamine response, the ΔE value of pork samples are not fresh when stored at 25 ◦ C for 24 h. At 48 h, the
the CGL1 film was 4.12 within 1 min, and the ΔE value of the CGL3 film TVB-N value increased to 24.57 mg/100 g, and the pork began to
reached 7.14, and the color change became stable after 20 min. After 20 deteriorate. During this process, the film also showed a color change
min, the ΔE value of the CGL1 film is 18.00, the ΔE value of the CGL2 visible to the naked eye, which was reddish-brown at the beginning, and
film is 21.47, and the ΔE value of the CGL3 film reaches 25.16. In the then gradually changed to yellow-green within 72 h. The ΔE of the film
trimethylamine response, the three films also showed obvious color reached 7.55–11.83 at 24 h. At 48 h, the film showed an obvious yellow

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Y. Zheng et al. Food Hydrocolloids 127 (2022) 107517

color, and the ΔE reached 15.62–19.6. At this time, the pork was no soy protein isolate for food packaging applications. RSC Advances, 6(85),
82191–82204.
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Declaration of competing interest Kalpana, S., Priyadarshini, S. R., Maria Leena, M., Moses, J. A., &
Anandharamakrishnan, C. (2019). Intelligent packaging: Trends and applications in
food systems. Trends in Food Science & Technology, 93, 145–157.
The authors declare that they have no known competing financial Khulal, U., Zhao, J., Hu, W., & Chen, Q. (2017). Intelligent evaluation of total volatile
interests or personal relationships that could have appeared to influence basic nitrogen (TVB-N) content in chicken meat by an improved multiple level data
fusion model. Sensors and Actuators B: Chemical, 238, 337–345.
the work reported in this paper. Liao, M., Ma, L., Miao, S., Hu, X., Liao, X., Chen, F., et al. (2021). The in-vitro digestion
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Acknowledgements
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Linares, M. B., Peñaranda, I., Iniesta, C. M., Egea, M., & Garrido, M. D. (2022).
The authors acknowledge the usage of SEM, TEM, FTIR and XPS Development of edible gels and films as potential strategy to revalorize entire male
supported by the Open Subsidies for large-scale Equipment of Wuhan pork. Food Hydrocolloids, 123, 107182.
Liu, D., Cui, Z., Shang, M., & Zhong, Y. (2021). A colorimetric film based on polyvinyl
University (Grant No. LF20201282). It was funded by National Natural alcohol/sodium carboxymethyl cellulose incorporated with red cabbage
Science Foundation of China (Grant No. 51776143) and Wuhan Uni­ anthocyanin for monitoring pork freshness. Food Packaging and Shelf Life, 28,
versity postgraduate research credit course project of Intelligent Pack­ 100641.
Li, Y., Wu, K., Wang, B., & Li, X. (2021). Colorimetric indicator based on purple tomato
aging and Food Safety (Grant No. 1506/413100017). anthocyanins and chitosan for application in intelligent packaging. International
Journal of Biological Macromolecules, 174, 370–376.
Li, A., Xiao, R., He, S., An, X., He, Y., Wang, C., et al. (2019). Research advances of purple
Appendix A. Supplementary data
sweet potato anthocyanins: Extraction, identification, stability, bioactivity,
application, and biotransformation. Molecules, 24(21), 3816.
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org/10.1016/j.foodhyd.2022.107517. of edible packaging film prepared by soy protein isolate-eggshell membrane
conjugates loaded with Eugenol. International Journal of Food Engineering, 16(12).
Li, S., Yi, J., Yu, X., Wang, Z., & Wang, L. (2020b). Preparation and characterization of
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