polymers

Article
Development of Smart Bilayer Alginate/Agar Film Containing
Anthocyanin and Catechin-Lysozyme
Orapan Romruen 1 , Pimonpan Kaewprachu 2,3 , Thomas Karbowiak 4 and Saroat Rawdkuen 1,5, *

1 Food Science and Technology Program, School of Agro-Industry, Mae Fah Luang University,
Chiang Rai 57100, Thailand
2 College of Maritime Studies and Management, Chiang Mai University, Samut Sakhon 74000, Thailand
3 Cluster of Innovative Food and Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
4 Agrosup Dijon, UMR PAM A 02.102, Université de Bourgogne Franche-Comté, Esplanade Erasme,
21000 Dijon, France
5 Unit of Innovative Food Packaging and Biomaterials, School of Agro-Industry, Mae Fah Luang University,
Chiang Rai 57100, Thailand
* Correspondence: saroat@mfu.ac.th; Tel.: +66-53916739; Fax: +66-53916737

Abstract: Smart packaging can provide real-time information about changes in food quality and
impart a protective effect to the food product by using active agents. This study aimed to develop
a smart bilayer film (alginate/agar) with a cellulose nanosphere (CNs) from corncob. The bilayer
films were prepared using 1.5% (w/w) sodium alginate with 0.25% (w/v) butterfly pea extract
incorporated (indicator layer) and 2% (w/w) agar containing 0.5% (w/v) catechin–lysozyme (ratio
1:1) (active layer). The CNs were incorporated into the alginate layer at different concentrations (0, 5,
10, 20, and 30% w/w-based film) in order to improve the film’s properties. The thickness of smart
bilayer film dramatically increased with the increase of CNs concentration. The inclusion of CNs
reduced the transparency and elongation at break of the smart bilayer film while increasing its tensile
Citation: Romruen, O.; Kaewprachu,
strength (p < 0.05). The integration of CNs did not significantly affect the solubility and water vapor
P.; Karbowiak, T.; Rawdkuen, S.
Development of Smart Bilayer
permeability of the smart bilayer film (p > 0.05). The smart bilayer film displayed a blue film with a
Alginate/Agar Film Containing glossy (without CNs) or matte surface (with CNs). The developed bilayer film shows excellent pH
Anthocyanin and sensitivity, changing color at a wide range of pHs, and has a good response to ammonia and acetic
Catechin-Lysozyme. Polymers 2022, acid gases. The film possesses exceptional antimicrobial and antioxidant activities. The integration
14, 5042. https://doi.org/10.3390/ of CNs did not influence the antibacterial activity of the film, despite the presence of a higher level
polym14225042 of DPPH in film containing CNs. The smart bilayer film was effectively used to monitor shrimp
Academic Editor: Albert
freshness. These findings imply that smart bilayer films with and without CNs facilitate food safety
Linton Charles and increase food shelf life by monitoring food quality.

Received: 19 October 2022 Keywords: anthocyanin; antioxidant; antimicrobial; smart bilayer film; cellulose nanosphere; pH
Accepted: 17 November 2022
indicator; reduce waste generation; food loss
Published: 21 November 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
Recently, new food packaging concepts have emerged in response consumers’ in-
creased concern about the safety and quality of food products [1]. Detecting food freshness
now requires specialized testing equipment, which is out of reach for most consumers.
Copyright: © 2022 by the authors.
As a result, there is an essential need for a simple and practical approach to monitoring
Licensee MDPI, Basel, Switzerland.
changes in food freshness. “Smart packaging” refers to active and intelligent packaging
This article is an open access article
concepts that provide precise information regarding food conditions, thereby preserving
distributed under the terms and the food product with antioxidant and antibacterial chemicals [2,3]. When protein-rich
conditions of the Creative Commons foods degrade, they release volatile amines, causing apparent color changes in pH-sensitive
Attribution (CC BY) license (https:// indicators [4]. Despite the effectiveness of synthetic dyes, they are not suited for food
creativecommons.org/licenses/by/ applications due to their toxicity, which endangers the health of consumers and the en-
4.0/). vironment [5]. Many natural colorants, including anthocyanin, curcumin, and betalains,

Polymers 2022, 14, 5042. https://doi.org/10.3390/polym14225042 https://www.mdpi.com/journal/polymers

Polymers 2022, 14, 5042 2 of 18

have been used to produce freshness indicator films in substitute for synthetic dyes [6,7].
Anthocyanin is a non-toxic natural pH-sensitive compound that has recently been used in
laboratories to produce colorimetric indicator films [8]. Many sources of anthocyanin have
been reported to be useful as an indicator for the preparation of freshness indicator films,
such as red cabbage [9], sweet purple potato [10], blueberry [11], and butterfly pea [12]. Due
to the potential of anthocyanin from butterfly pea as a natural colorant and antioxidant, it
has been exploited in culinary and medical applications [13,14]. Butterfly pea anthocyanin
is light and temperature stable, and it exhibits remarkable color changes at neutral, acidic,
and basic conditions, making it a perfect candidate for color indicator films [15].
In order to achieve the purposes of smart packaging, the packaging should assist
in prolonging or extending a food product’s shelf life. As a result, films typically use
anti-microbials to improve their functional qualities and prolong the shelf life of food. Tea
catechins contain many biological properties, including antioxidant, antimicrobial, and
antifungal properties [16]. Lysozyme is an antibacterial protein in several biological tissues,
cells, and fluids [17]. In recent years, edible food packaging films/coatings containing
lysozyme have attracted the interest of many scientists [18]. According to Rawdkuen,
et al. [19], the combination of catechin and lysozyme enhances antibacterial activity over
either catechin or lysozyme alone.
New technologies for producing biodegradable packaging from renewable polymers
have been developed due to the rising customer demand for eco-friendly and high-quality
products generated from natural resources [20–22]. Agar is a polysaccharide derived
from the Rhodophyceae family of marine red algae [23]. As agar films are transparent,
biodegradable, and physiologically inert, they can readily interact with various bioactive
chemicals [24,25]. However, pure agar film has restricted applicability due to its weak
thermal stability, high water sensitivity, and water vapor permeability [26]. One of the
most common strategies used to circumvent these limitations is the combination of agar
with other substances, such as biopolymers, hydrophobic compounds, plasticizers, and
nanoparticles. Sodium alginate (SA), a naturally occurring linear polysaccharide in brown
algae, and which contains β-D-mannuronic acid and α-L-guluronic acid, is abundant and
safe. Its strong water solubility and film-forming characteristics make it popular in the
pharmaceutical and food sectors [27].
Bilayer film is a method for improving the properties of polymer films by combining
materials with different properties in order to produce a two-layer film [28]. The properties
of bilayer films are influenced by several factors, including preparation techniques, material
types, and layer thickness ratios [29,30]. “Casting” is a broad term used for the two-step
process of manufacturing bilayer film. Pouring and drying the first solution results in the
formation of the first layer of film. As a result, the formed film is covered with the second
film-forming solution, resulting in a bilayer film [31].
Nanocelluloses have recently received significant interest among the many nanomateri-
als for polymer reinforcement [32,33]. Wang, et al. [34] synthesized cellulose nanowhiskers
from mulberry pulp and integrated them into alginate films. Their results indicated that
the tensile strength of alginate films increased by 25%, although their water vapor per-
meability was unaffected. Roy, et al. [35] prepared smart films by incorporating cellulose
nanocrystals (CNC) obtained from onion peel and shikonin into CMC/agar film. The
addition of CNC considerably enhanced the mechanical, barrier, and optical properties
of the CMC/agar film. Salim, et al. [36] discovered that the addition of CNC from pea
pods improved the mechanical strength and thermal stability of chitosan films. Cellulose
nanospheres (CNs) are a new form of nanocellulose with a spherical shape and nanoscale
size [37]. The crystallinity and thermal stability of the polymer matrix can be enhanced by
adding CNs, as these have a higher potential than rod-shaped nanocellulose at the same
concentration [38]. According to our previous study (Romruen, et al. [39]), the utilization
of cellulose nanosphere from different agricultural byproducts. Corncob CNs exhibit better
properties than other materials. Therefore, it was used as a reinforcing material in order to
improve the properties of the film in this study. This study aims to develop a smart bilayer
Polymers 2022, 14, 5042 3 of 18

alginate/agar film containing anthocyanin and catechin–lysozyme film and study the effect
of CNs on film’s properties. In addition, application of the developed smart film for shrimp
freshness monitoring was also investigated.

2. Materials and Methods

2.1. Materials
Dried butterfly pea (Clitoria ternatea) flowers were purchased from a local market in
Chiang Rai, Thailand. Sodium alginate was obtained from Union Science Co., Ltd. (Chaing
Mai, Thailand). Agar was purchased from Krungthepchemi Co., Ltd. (Bangkok, Thailand).
Catechin hydrate (C1251) and lysozyme from chicken egg white (62971) were purchased
from Sigma–Aldrich (St. Louis, MO, USA). Escherichia coli TISTR 527 and Staphylococcus
aureus TISTR 746 were received from the Biological Laboratory (Mae Fah Luang University,
Chiang Rai, Thailand). All other chemicals employed in this study were of analytical grade.

2.2. Butterfly Pea Anthocyanin Extraction

The butterfly pea anthocyanin extract (BAE) was prepared using a modified version
of the method of Sai-Ut, et al. [40]. In brief, the dried butterfly pea flowers were ground
and mixed with 70% (v/v) ethanol at a 1:40 (g/mL) ratio and then kept in the dark at 4 ◦ C
for 18 h. The extracted solution was filtered and then concentrated at 40 ◦ C. Finally, the
concentrated extract was freeze-dried (Delta-2-24/LSC plus, Martin Christ, Osterode am
Harz, Germany) for 24 h. The BAE powder was stored at −20 ◦ C until used.

2.3. Fabrication of Smart Bilayer Film

First, an active layer of film-forming solution (FFS) was prepared with 2% (w/v) of
agar in distilled water and glycerol 50% (w/w) of based film. The FFS was stirred at 95 ◦ C
for 2 h. FFS was then cooled to 65 ◦ C and 0.5% (w/v) of catechin–lysozyme (ratio 1:1) was
added [41]. The FFS was continuously stirred for 30 min. Second, an indicator layer of FFS
was prepared using 1.5% (w/v) of sodium alginate and 50% (w/w) of based-film glycerol
in 100 mL of distilled water. BAE was used as a color indicator at concentrations of 0.5%
(w/v). Following that, corncob cellulose nanosphere (CN) obtained from our previous
study (Romruen, Kaewprachu, Karbowiak and Rawdkuen [39]) was added at various
concentrations (0, 5, 10, 20, and 30% w/w SA-based film). The FFS was continuously
agitated for 30 min and ultrasonically for 10 min. Finally, active FFS (5 g) was cast onto a
silicone plate (5 × 5 cm) and dried at room temperature for 18 h. The indicator FFS (6 g)
was then spread on top and allowed to dry at room temperature for 24 h. Before analysis,
the dried film was stored in an environmental chamber at 25 ± 0.5 ◦ C and 50 ± 5% RH
for 24 h.

2.4. Smart Bilayer Film Characterization

2.4.1. Thickness
The thickness of the film was measured using a thickness gauge (C112XBS, Mitutoyo
Corp., Kawasaki, Japan) at nine random positions [42].

2.4.2. Appearance and Color

The film’s appearance was recorded by a digital camera (Sony α6000, Sony Thai Co.
Ltd., Bangkok, Thailand). The color of the films was determined using a handheld chroma
meter (Konica Minolta Sensing Americas Inc., Williams Drive Ramsey, NJ, USA) [43].

2.4.3. Light Transmission and Transparency

The light transmission and transparency properties of developed films were inves-
tigated using the method of Jongjareonrak, et al. [44]. The test was performed at wave-
lengths ranging from 200 to 800 nm, using a UV–Visible spectrophotometer (G105 UV-
Polymers 2022, 14, 5042 4 of 18

VIS, Thermo Scientific Inc., Waltham, NJ, USA). Equation (1) was used to calculate the
film’s transparency:
−logT600
Transparency = (1)
X
where T600 is the transmittance at 600 nm and X is the thickness of the film (mm).

2.4.4. Morphology of Film

The microstructure of the developed film was investigated using a field emission
scanning electron microscope—FESEM (TESCAN MIRA, TESCAN, Brno, Czech Republic)—
at a 10 kV acceleration voltage. The image was captured using a magnification of 500× for
the upper surface and 1000× for the cross section.

2.4.5. Mechanical Properties

The mechanical properties, including tensile strength (TS) and elongation at break
(EAB), of the developed film were calculated using a Universal Testing Machine (Lloyd
Instrument, Hampshire, UK). The tests was performed with a 100 N load cell, and ten
samples (2 × 5 cm) with a 3 cm grip length were used to measure with the cross-head speed
at 30 mm/min [45].

2.4.6. Water Vapor Permeability (WVP)

The WVP of the smart bilayer film was determined using a modified version of the
method of ASTM [46]. The films were sealed onto a permeation cup containing silica gel
(0% RH). These cups were then placed in an environmental chamber with 50% RH at 25 ◦ C
and weighed at 1 h intervals for 8 h, after which the films’ WVP was estimated using
Equation (2) [47]:
W×X
WVP = (2)
A × t × ( P2 − P1 )
where W is the cup’s weight gain (g); X is the film’s thickness (m); A is the film’s area (m2 );
t is the gaining time (s); and (P2 − P1 )−1 is the vapor pressure difference throughout the
film (Pa). The WVP was represented in units of g m s−1 m−2 Pa−1 .

2.4.7. Film Solubility

The water solubility of the developed film was determined using the method published
by Gennadios, et al. [48]. A 2 × 2 cm dried film sample was weighed and dissolved into
10 mL of distilled water. Afterwards, it was shaken at 250 rpm in a shaker (Helidolth
Inkubator 10000, Schwabach, Germany) at 25 ◦ C for 24 h. After 20 min of centrifugation at
3000× g, 25 ◦ C, and undissolved particles were recovered. The pellet was weighed after
drying at 70 ◦ C for 24 h. The solubility of the film was computed using Equation (3):

W0 − W f
Filmsolubility(%) = × 100 (3)
W0

where W 0 is the dry matter weight of the film and Wf is the weight of the dry film residue
that had not been dissolved.

2.4.8. pH Sensitivity
The film’s pH sensitivity was assessed using a modified version of the method of
Pereira Jr, et al. [49]. Individual 2 × 2 cm pieces of film were immersed in different pH
buffer solutions (pH 2 to 12). The appearance changes of the films were photographed by a
digital camera after 5 min (Sony α6000, Sony Thai Co. Ltd., Bangkok, Thailand).

2.4.9. Response to Volatile Ammonia and Acetic Acid

The colorimetric reaction was evaluated in response to two pH stimuli solutions: acetic
acid (50%, v/v) and ammonia solution (0.1 M). Each test tube (16 × 150 mm) was filled
Polymers 2022, 14, 5042 5 of 18

with 5 mL of stimulus solution before the film being tested was wrapped over the tube
mouth. A digital camera (Sony 6000, Sony Thai Co. Ltd., Bangkok, Thailand) was used to
record the color shifts of the film over time.

2.4.10. Fourier Transform Infrared Spectroscopy (FTIR)

All films were clamped onto the FT-IR spectrometer mount for FTIR spectra analysis.
The films’ infrared spectra were captured at 25 ◦ C using a Fourier Transform Infrared
Spectrometer (Raman Spectrometer) (Thermo Fisher Scientific Inc., Waltham, MA, USA)
with 64 scans and a 4 cm−1 resolution [50].

2.4.11. Thermal Stability

Thermo-gravimetric analysis (TGA) and differential scanning calorimetry were con-
ducted in order to evaluate the thermal stability of the smart bilayer films (DSC). The
thermal stability of the films was measured using a thermogravimetric analyzer (Mettler
Toledo, Model 851e, Schwerzen-bach, Switzerland) according to the method of Kaewprachu,
et al. [51]. Experiments were conducted at 25–700 ◦ C with a heating rate of 10 ◦ C/min in a
nitrogen atmosphere (20 mL/min). As stated by Pelissari, et al. [52], the thermal properties
of the film were examined using a differential scanning calorimeter (DSC; TA-Instruments,
model 2920, New Castle, PA, USA). This test was conducted in a nitrogen atmosphere
(20 mL/min) with a temperature scan from 60 to 150 ◦ C at a rate of 10 ◦ C/min.

2.4.12. Bioactive Compounds and Antioxidant Properties

The film’s bioactive compound and antioxidant properties were determined according
to the methods of Tongnuanchan, et al. [53]. The film (30 mg) was extracted using distilled
water at 250 rpm for 3 h. The supernatant was utilized to assess the total anthocyanin
content (TAC), total phenolic content (TPC), ferric-reducing antioxidant power (FRAP), and
DPPH radical scavenging activity. According to the methods of Goodarzi, et al. [54], the pH
differential method was used to determine the TAC. At 510 and 700 nm, the absorbance of
supernatant by the pH 1.0 buffer and pH 4.5 buffer was determined. Calculated as cyanidin-
3-glucoside with an extinction value of 26,900 and a molecular weight of 484.83 g mol−1 .
Folin–Ciocalteu assay was utilized to determine TPC. The results are reported in terms of
mg GAE/g dry film. For FRAP, film extract solution was combined with FRAP solution
and incubated at 37 ◦ C for 30 min, and then the absorbance at 593 nm was measured.
The reducing power was expressed as µM ferrous sulfate/g dried film. By mixing film
extract solution with methanolic DPPH solution, the absorbance at 517 nm was determined
for DPPH. The DPPH activity was measured and reported in terms of mM Trolox/g of
dried film.

2.4.13. Antimicrobial Activity of the Film

The antimicrobial activity of films was determined following the methods of Rawd-
kuen, Suthiluk, Kamhangwong and Benjakul [41]. The films were evaluated for their ability
to inhibit Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus)
bacterial strains using the agar disc diffusion method. A bacterial strain concentration of
108 CFU/mL was employed. The films were cut into 5 mm circles, sterilized under UV
irradiation for 30 min, and then used to place on surface of agar with the test culture. The
test agar plates were incubated at 37 ◦ C for 18 h. The inhibition zone formed around the
film disc was utilized to determine the level of antimicrobial activity.

2.5. Application of Smart Bilayer Film for Monitoring Shrimp Freshness

The efficacy of smart bilayer films was tested in order to monitor the freshness of
shrimp. Two shrimps of similar size were placed in a circular PP tray, and the smart bilayer
film (2 × 3 cm) was affixed inside a PVC film wrap, tightly sealed, and stored at 25 ◦ C for
24 h. Changes to the pH and color of the film were observed.
Polymers 2022, 14, 5042 6 of 18

2.6. Statistical Analyses

In order to evaluate the analysis of variance, SPSS software (SPSS for Windows ver-
sion 26.0, SPSS Inc., Chicago, IL, USA) was used to conduct statistical analysis (ANOVA).
At a confidence level of 95%, Duncan’s multiple range tests were used to identify the
significance of difference between samples.

3. Results and Discussion

3.1. Thickness
The thickness of the smart bilayer films integrated with corncob cellulose nano-spheres
(CNs) at various concentrations are shown in Table 1. The thickness of the films was 0.082,
0.084, 0.091, 0.097, and 0.108 mm for 0% CNs, 5% CNs, 10% CNs, 20% CNs, and 30%
CNs, respectively. As the amount of CNs increased, the thickness of the smart bilayer
film increased drastically (p < 0.05). This increase in thickness may affect other properties
of the developed film, especially the mechanical and barrier properties. The increased
thickness might be characterized by accumulating a dense network between the sodium
alginate matrix (upper layer) and the CNs [55]. These findings were consistent with Salim,
Abdellaoui, Ait Benhamou, Ablouh, El Achaby and Kassab [36], who reported that the
thickness of chitosan films increased as the concentration of cellulose nanocrystals in pea
pod waste increased. Shankar and Rhim [56] also found that increasing the concentration
of nanocellulose thickened the agar film. The solid content is primarily responsible for the
rise in film thickness.

Table 1. Physical and mechanical properties of smart bilayer film incorporated with different concen-
trations of cellulose nanosphere (CNs).

CNs Concentration TS * EAB * WVP Film Solubility

Thickness (mm)
in Film (%) (MPa) (%) (×10−10 g m/m2 s Pa) (%)
0 0.082 ± 0.002 d 16.11 ± 1.32 c 61.63 ± 3.30 a 2.27 ± 0.06 a 58.65 ± 1.06 b
5 0.084 ± 0.002 d 18.26 ± 2.01 b 50.39 ± 7.19 b 2.11 ± 0.10 a 56.52 ± 1.32 bc
10 0.091 ± 0.001 c 18.63 ± 1.53 b 46.27 ± 4.97 bc 2.22 ± 0.05 a 53.87 ± 2.43 c
20 0.097 ± 0.001 b 19.02 ± 1.20 ab 41.86 ± 5.69 cd 2.34 ± 0.11 a 61.93 ± 1.72 a
30 0.108 ± 0.002 a 20.51 ± 2.06 a 40.44 ± 4.17 d 2.68 ± 0.08 a 63.43 ± 0.97 a
Values are given as mean ± SD, n = 3. * n = 10 for TS and EAB. Different superscripts in each column are
significantly different (p < 0.05).

3.2. Mechanical Properties

The mechanical characteristics of smart bilayer films with various concentrations of
CNs are shown in Table 1. After the addition of CNs, the mechanical characteristics of
the smart alginate/agar bilayer film were altered dramatically, and these qualities were
impacted by the amount of CNs added. The film’s tensile strength (TS) increased noticeably
(p < 0.05). On the other hand, incorporating CNs reduced the film’s flexibility (elongation
at break: EAB), inversely proportional to the TS direction. Similar findings were reported
by Roy, Kim and Rhim [35], who found that the incorporation of CNs obtained from
onion peel significantly improved the mechanical strength of a CMC/agar-based film.
As the concentration of CNs rose, the TS of the composite films gradually increased [34].
As nanocellulose has a strong tendency to self-aggregate, the major point of generating
nanocomposites with high mechanical performance is the interface compatibility between
the filler and the polymer matrix [57]. The CNs used in this study were 2–44 nm in size,
which is considered a small nanoparticle with a high self-aggregate. The EAB decreased
with increasing CNs concentration (from 61.63% for the neat smart bilayer film to 40.44% for
the film with 30% CNs), corresponding to a 34.38% decrease. These findings are consistent
with those of Salim, Abdellaoui, Ait Benhamou, Ablouh, El Achaby and Kassab [36], who
discovered that the addition of 10% nanocellulose was reduced the EAB value of chitosan
film by 37%. The strong interfacial adhesion between the polymer matrix and the CNs
can explain this performance, as it limits the matrix motion. Some authors have reported
Polymers 2022, 14, 5042 7 of 18

that adding CNs reduces the EAB values of polymer films [58,59]. Aside from interfacial
interaction/adhesion, the nanoparticle size influences interfacial/interphase properties [60].
Large nanoparticles exhibit poor interfacial/interphase characteristics and tensile strength
even at high interphase thickness, highlighting the importance of particle size.

3.3. Water Vapor Permeability (WVP)

The addition of CNs did not significantly affect the WVP of smart bilayer film (p > 0.05)
(Table 1). The WVP of the developed smart bilayer films with and without CNs ranged
from 2.11–2.68 × 10−10 g m m−2 s−1 Pa−1 . The WVP value of smart bilayer film decreased
when integrated with 5% and 10% CNs, but it increased when the CN concentrations were
increased to 20% and 30%. However, it was equivalent to the WVP value of CNs-free smart
bilayer films. These results were consistent with those of Oun and Rhim [61], who observed
that the WVP of CMC film was reduced after a small amount of cellulose nanofiber (CNF)
was applied and then steadily increased in conjunction with the CNF concentration. The
composite film’s CNs operate as an impermeable barrier to water vapor diffusion, resulting
in a more tortuous channel for water vapor diffusion and a lower WVP [62]. Meanwhile, the
rise in WVP at 20% and 30% CNs could be related to the discontinuous structure between
the CNs and polymer matrix caused by CNs agglomeration at higher concentrations [63],
as evidenced by FESEM results.

3.4. Film Solubility

The solubility properties of smart bilayer film reinforced with CNs are presented in
Table 1. The inclusion of CNs influenced the solubility of smart alginate/agar bilayer films
(p < 0.05). The solubility of the alginate/agar bilayer film decreased from 58.65% for 0%
CNs smart bilayer film to 53.87% for 10% CNs film, which increased with further CNs
inclusion. This result indicates that it reached maximum water resistance when 10% CNs
was added to the alginate/agar bilayer composite film.

3.5. Film Appearance and Color

The inclusion of CNs influenced the appearance and surface color of the smart bi-layer
film, as shown in Table 2. All film samples exhibited a blue color, which is representative of
the color of BAE. Delphinidin is the primary anthocyanin responsible for the deep blue to
purple color of butterfly pea flower [64]. The surface of the smart bilayer film was glossy
(without CNs) and matte (with CNs). After adding CNs, the lightness (L*) of the smart
bilayer films increased significantly (p < 0.05). The increase in film lightness could be
attributed to the white color of the CNs powder. Alternatively, the increasing concentration
of CNs decreased the greenness (−a*) and blueness (−b*) values of the smart bilayer film
(p < 0.05). These results indicate that the inclusion of CNs affected the smart bilayer film’s
lightness and greenness while not affecting its blueness.

3.6. Light Transmission and Transparency

The light transmission of a smart bilayer film with various concentrations of CNs
incorporation is shown in Figure 1. Smart bilayer films had 0.01 to 0.06% transmission
for UV light (200–280 nm), whereas those with and without CNs had transmissions of
1.78 to 87.78% and 1.55 to 70.92%, respectively, for visible light (350–800 nm). As a result
of the incorporation of CNs into the smart bilayer film, visible light transmission was
diminished. In addition, no UV light transmission was detected in any of the smart
bilayer films, regardless of whether or not CNs were present. This consequence could be
attributed to the presence of BAE in all of the film formulas. Polyphenol aromatic rings can
absorb UV light [65]. The CNs-free film had a light transmission value higher than 80%
at 700–800 nm, but the films with CNs added had light transmission values of less than
80% in the visible light spectrum, regardless of concentration. In the visible light spectrum,
the film was transparent to the human eye and had a light transmission value greater
than 80% [66]. As CNs concentration increased, the transparency of the smart bilayer film
 5 48.28 ± 0.23 b −9.28 ± 0.45 b −30.02 ± 0.64 a 2.44 ± 0.02 b

10 49.01 ± 1.06 ab −8.67 ± 0.05 b −29.89 ± 0.59 a 2.41 ± 0.05 bc

Polymers 2022,
2014, 5042 49.65 ± 0.81 ab −8.59 ± 0.19 b −29.65 ± 0.26 a 2.39 ± 0.01 c 8 of 18

30 50.07 ± 0.17 a −7.57 ± 0.24 a −29.51 ± 0.41 a 2.27 ± 0.01 d

reduced dramatically (p < 0.05) (Table 2). All of the smart bilayer film samples were proven
Values are given
to be more
Polymers 2022, 14, x FOR PEER REVIEW as mean
effective ± SD, n = 3.UV
at blocking Different superscripts inConsequently,
light transmission. each column arethe
significantly different
smart bilayer film
8 of 20
(p
can< 0.05).
reduce lipid oxidation in food packing, particularly for high-lipid meals.

3.6.
TableLight Transmissioncolor,
2. Appearance, and Transparency
and transparency of smart bilayer film incorporated with different
Table 2. Appearance, color, and transparency of smart bilayer film incorporated with different
The light transmission
concentrations
concentrations of cellulose of a smart
nanosphere
of cellulose nanosphere (CNs). bilayer film with various concentrations of CNs
(CNs).
incorporation is shown in Figure 1. Smart bilayer films had 0.01 to 0.06% transmission for
CNs
CNsConcentration
Concentration UV light (200–280 nm), L* whereas those with and without CNs had transmissions of 1.78 to
Appearance
Appearance L* a*
a* b*b* Transparency
Transparency (%)
(%)
in
inFilm
Film(%)
(%)
in Film (%) 87.78% and 1.55 to 70.92%, respectively, for visible light (350–800 nm). As a result of the
incorporation of CNs into cthe smart bilayercc film, visible light transmission aawas
00 44.97
44.97 ± 1.19 ccc −
± 1.19 10.33 ±
−10.33 ± 0.63
0.63 cc −34.17
−34.17± ± 0.66bbb b
0.66 2.50 ±
2.50 ± 0.01
0.01 aa
diminished. In addition, no UV light transmission was detected in any of the smart bilayer
films, regardless of whether or not CNs were present. This consequence could be
55 48.28
48.28
attributed to the presence ± of
± 0.23 b bbb
0.23
BAE in− −9.28 ±the
9.28of±
all bbbb
0.45film −30.02
−30.02± ±
formulas. 0.64aaa a
0.64
Polyphenol 2.44
2.44 ± 0.02
± bbbb
0.02 rings
aromatic
can absorb UV light [65]. The CNs-free film had a light transmission value higher than
1010 80% at 700–800 nm, but
49.01 ± the
49.01 1.06 films
± 1.06
ab abab with
ab − CNs
−8.67
8.67 ± added
± 0.05
0.05 bbbb had light
−29.89
−29.89 transmission
±±0.59
0.59aaa a values
2.41
2.41 ± 0.05ofbc
± 0.05 bcless
bc
bc

than 80% in the visible light spectrum, regardless of concentration. In the visible light
20
20
spectrum, the film49.65 was
49.65transparent
ab ab
± 0.81
± 0.81 ab
ab to
−
the ±human
−8.59
8.59 ± 0.19 bbbb eye and
−29.65
− had
29.65± ±
a light transmission
0.26aaa a
0.26 2.39
2.39 ±± 0.01 value
0.01 cccc
greater than 80% [66]. As CNs concentration increased, the transparency of the smart
bilayer film reduced dramatically a aa
(p < 0.05) (Table 2). All of the smart bilayer film samples
30
30 50.07
50.07 ± 0.17
± 0.17 −−7.57
7.57 ±± 0.24 aaa −29.51−29.51± ± 0.41aa a
0.41 2.27
2.27 ± 0.01 ddd
± 0.01
were proven to be more effective at blocking UV light transmission. Consequently, the
smart
Values
bilayer
Values
are given
filmascan
are given
as mean
reduce
mean
± SD,±nSD,
lipid oxidation
= 3. nDifferent
= 3. Different in infood
superscripts
superscripts
packing,
in eachare
each column
particularly
column for high-lipid
are significantly
significantly different (p <different
0.05).
meals.
(p < 0.05).

3.6. Light Transmission and Transparency

The light transmission of a smart bilayer film with various concentrations of CNs
incorporation is shown in Figure 1. Smart bilayer films had 0.01 to 0.06% transmission for
UV light (200–280 nm), whereas those with and without CNs had transmissions of 1.78 to
87.78% and 1.55 to 70.92%, respectively, for visible light (350–800 nm). As a result of the
incorporation of CNs into the smart bilayer film, visible light transmission was
diminished. In addition, no UV light transmission was detected in any of the smart bilayer
films, regardless of whether or not CNs were present. This consequence could be
attributed to the presence of BAE in all of the film formulas. Polyphenol aromatic rings
can absorb UV light [65]. The CNs-free film had a light transmission value higher than
80% at 700–800 nm, but the films with CNs added had light transmission values of less
than 80% in the visible light spectrum, regardless of concentration. In the visible light
spectrum, the film was transparent to the human eye and had a light transmission value
greater than 80% [66]. As CNs concentration increased, the transparency of the smart
bilayer film reduced dramatically (p < 0.05) (Table 2). All of the smart bilayer film samples
were proven to be more effective at blocking UV light transmission. Consequently, the
Figure
smart Light transmission
1. bilayer of smartlipid
film can reduce bilayer films incorporating
oxidation differentparticularly
in food packing, concentrations of high-lipid
for cellulose
nanosphere
meals. (CNs).

3.7. Morphology of Film

The microstructures of smart bilayer films with different CN levels are depicted in
Table 3. The CNs-free smart bilayer film had a smooth surface with no porous structures,
while the CNs-containing films had a rough surface without a porous structure, indicating
that the CNs particle films were well dispersed in the polymer matrix, though the degree of
roughness of the film surface was dependent upon the CNs concentration. This is primarily
due to CNs agglomeration in the polymer matrix at high concentrations [67]. Micrographs
show that the laminated layers (sodium alginate [SA] layers) were tightly bound with
the lower layer (agar [AG] layer) films due to surface interaction. Due to associative
interactions, phase separation could not be detected in all film samples, indicating high
compatibility and homogeneity between SA and AG layers [68].
 thatthat
thethe
CNs particle
CNs films
particle were
films well
were dispersed
well dispersed in the polymer
in the polymer matrix, though
matrix, thoughthethe
degree
degr
of roughness
of roughness
of roughness
of roughnessof the
of the
of filmfilm
of the
the
film surface
film
surface waswas
surface
was
surface wasdependent
dependent
dependent
dependent upon
uponupon
uponthethe
the CNs
the
CNs CNs
CNsconcentration.
concentration.
concentration.
concentration. This
This is
This
is
This
primarily
primarily
primarily due due
duedue
primarily to
to CNsto
CNs CNs
to CNs agglomeration
agglomeration
agglomeration
agglomeration in in
the
in the the polymer
polymer
polymer
in the polymer matrix
matrix
matrix
matrixat at
high
at high high concentrations
concentrations
concentrations
at high concentrations [67].
[67].[6
[6
Micrographs
Micrographs
Micrographs
Micrographs show
show show
show thatthat
the
thatthat the
thethe laminated
laminated
laminated
laminated layers
layers
layers (sodium
(sodium
(sodium
layers (sodium alginate
alginate
alginate [SA]
[SA]
alginate [SA] layers)
layers)
layers)
[SA] layers)were
were were tigh
tightly
tightly
were tigh
bound
bound
bound with
with the
with
the lower
the
lower layer
lower
layer (agar
layer
(agar [AG]
(agar
[AG][AG]layer) films
layer)
layer) films due
films
duedueto
to surface
to interaction.
surface
surface
bound with the lower layer (agar [AG] layer) films due to surface interaction. Due interaction.
interaction. Due
Due to
Due
to
Polymers 2022, 14, 5042 associative
associative interactions,
associative interactions,
interactions,
associative interactions, phase
phase
phase
phaseseparation
separation
separation
separation could
could
could
could not be
not
notnot
be bedetected
be detected
detected
detectedin all
in
in all film
all
9 of samples,
film
18
filmfilm
in all sampl
samples,
sampl
indicating
indicating high
indicating
high
indicating compatibility
high compatibility
compatibility
high compatibility andand
and homogeneity
and homogeneity
homogeneity
homogeneity between
between
between
betweenSA SA
SA andand
SA
and AGAG
and
AG layers
AG [68].
layers
layers [68].
layers [68].
[68].

Table
Table 3. Surface
Table
3.
Table Surface
3. andand
3. Surface
Surface
and cross-section
and
cross-section morphology
cross-section morphology
morphology
cross-section morphologyof smart
of smart
of bilayer
of smart
smart
bilayer films
bilayer
bilayer incorporating
films
films different
incorporating
incorporating
films different
different
incorporating different
Table 3. Surface and cross-section morphology of smart bilayer films incorporating different concen-
concentrations
concentrations
concentrations
concentrationsof cellulose
of
of cellulose nanosphere
cellulose
nanosphere
of cellulose (CNs).
nanosphere (CNs).
(CNs).
nanosphere (CNs).
trations of cellulose nanosphere (CNs).
CNs
CNs Concentration
CNs Concentration
Concentration
CNs Concentration
CNs Concentration Upper
Upper Surface
Upper Surface
Surface
Upper Surface Cross-Section
Cross-Section
Cross-Section
Cross-Section
ininFilm
in Film Film
in Film
Film
in Film
in
(%)
(%)(%)
(%)
(%)(%)
Upper Surface Cross-Section

0 00 00

5 55 55

10 10 10
10 10

20 20 20
20 20
Polymers 2022,2022,
Polymers 14, x14,
FOR PEER
x FOR REVIEW
PEER REVIEW 10 of1020o

30 30 30

TheThemagnifications
The magnificationsmagnificationsused
used for surface forcross-section
used
and surface andimages
for surface cross-section
and are 500× images
cross-section 1000are
and images 500×500×
×. are andand
1000×.
1000×.

3.8.3.8.
pH pH
3.8. pH SensitivitySensitivity
Sensitivity
TheThe
The response response
ofresponse
the BAEof the BAE
ofand
the BAEandand
smart smart bilayer
smart
bilayer films
bilayer
films with
films
with different
with
differentdifferentconcentrations
concentrations
concentrations of of CNsof C
to the
to pH
the buffer
pH bufferis displayed
is displayed in Table
in 4.
Table When
4. When the
CNs to the pH buffer is displayed in Table 4. When the pH was raised from 2 to 12, BAE pH
the was
pH raised
was raisedfrom from2 to2 12,
to BAE
12, BA
changed
changed from from pinkpinkto greenish
to greenish yellow.
yellow.The BAE
The BAE
changed from pink to greenish yellow. The BAE was pink when the level of acidity waswas pink
was when
pink whenthe level
the of
level acidity
of was
acidity w
high (pH
high 2),
(pH and
2), it
and turned
it turnedto purple
to purple(pH 3),
(pH purplish-blue
3), purplish-blue
high (pH 2), and it turned to purple (pH 3), purplish-blue (pH 4–5), and blue (pH 7) as the (pH 4–5),
(pH and
4–5), andblue (pH
blue 7)
(pH as
7) the
as t
level
level changed. changed.
level changed.
At At pH
pH levels levels
Atranging
pH ranging
levelsfromranging from
9 to 9the
from
12, to9color
12, the
to 12, color
ofthe of the
thecolor
BAE of BAE
the
shifted BAE shifted
from shifted
bluefromfrombluebl
to green.toRawdkuen,
green.
to green.Rawdkuen,
Faseha, Faseha,
Rawdkuen, Faseha,
Benjakul Benjakul
andBenjakul andand
Kaewprachu Kaewprachu
[12] also[12]
Kaewprachu alsoalso
[12]
reported reported
reported
that thatthat
butterfly butterfly
butter
peapeaanthocyanin
pea anthocyanin anthocyanin extracts
extracts exhibited exhibited
extracts samethe
exhibited
the same
the
color samecolor
changes changes
color
and wereand
changes were
and were
successfully successfully
used as a used
successfully as
used
a pHa indicator
pH indicator in gelatin
in film.
gelatin The
film. color
The of
color the
of developed
the developed
pH indicator in gelatin film. The color of the developed smart bilayer film changed from smart
smartbilayer
bilayerfilm changed
film changed from fro
purple
purpleat pH
at 2–3
pH to
2–3 purplish-blue
to purplish-blue at pH
at 4–5,
pH blue
4–5, at
blue pH
at
purple at pH 2–3 to purplish-blue at pH 4–5, blue at pH 6, bluish-green at pH 7–8, green at 6,
pH bluish-green
6, bluish-green at pH
at 7–8,
pH green
7–8, gre
pH 9–10,atand
pH
at pH9–10, andand
9–10,
greenish-yellow greenish-yellow
greenish-yellow
at pH 11–12. at Although
pH
at pH11–12.
11–12.Although
the Although
color the the
change color change
color
shown change
by theshownshown
film by bythe t
filmfilm
depends
depends upon uponthe the
pHpH levels, the the
levels, results were
results werein line with
in line the the
with color change
color change of BAE
of BAE at
the the
samesamepHpH values.
values. TheThecolor change
color change in response
in response to pH levels
to pH of all
levels of smart
all smart bilayer films
bilayer film
is the same
is the color,
same indicating
color, indicating thatthat
the the
addition
addition of CNs
of CNs diddid
notnotaffect the the
affect pHpH sensitivity
sensitivityof
the the
smart
smartbilayer
bilayerfilm. These
film. Theseresults indicate
results indicatethatthat
the the
color change
color changeof aofsmart
a smart bilayer filmfi
bilayer
containing
containing all concentrations
all concentrations of CNsof CNsin acidic
in acidicandandalkaline solutions
alkaline solutions remains
remains visible to the
visible to t
 changed
changed
changed
changed changed
changed
changed
changed
changed from
from
changed
from
from
from from from
pink
pink
pink from
pink
from from
pink pink
pink topink
toto to greenish
pink
greenish
pink
to greenish
to to
togreenish
greenish
to greenish
to greenish
greenish
greenish yellow.
yellow.
greenish
yellow.
yellow. yellow.
yellow. The
yellow.
yellow.
yellow. TheThe
yellow.
The The The
BAE
BAE
BAE
The The
BAE
The
BAE BAE
Thewas
BAE BAE
was
was
wasBAE
BAE
was was
pink was
pink
pink
pink
was was
was
pinkpink
when
pink pink
when
when
pink
when
pink
whenwhen when
the
the
when the
when
when
thethe the
level
level
level
the the
levellevel
ofof
the
the
level of
levellevel
of
levelof
acidity
acidity
level
ofacidity
of of
ofacidity
acidity
acidity
of was
was was
acidity
acidity
acidity
acidity waswas was
was was
was
was
high
high
high high
high
high
high
high (pH
high
(pH
(pH
(pH
(pH
high (pH
high
high
(pH
high
high
(pH 2),2),
(pH
2),
2), 2),
2),(pH
2),
2),
(pH andand
and
(pH
and
(pH
(pH
andand
2),
and
2),
and 2),
it itturned
and
itand
2),
it
2),
2),it turned
it
turned
and
and turned
andit turned
itturned turned
turned
and
turned
it it
turneditit tototo
turned
to
itturned
it to to to
to
turnedpurple
purple
to
purple
turnedpurple purple
to
purple
purple
purple
to toto
to(pH
purple
purple (pH
(pH (pH
purple
purple
(pH(pH
purple
purple
(pH(pH 3),3),
(pH
3),
3),
3),
3),
(pH 3),
3),
(pHpurplish-blue
purplish-blue
3), purplish-blue
purplish-blue
(pH
(pH 3),
3),
purplish-bluepurplish-blue
purplish-blue
purplish-blue
(pH 3),purplish-blue
purplish-blue
3), 3), purplish-blue
purplish-blue
purplish-blue (pH
(pH
purplish-blue (pH
(pH
(pH(pH
(pH
(pH 4–5),
(pH
4–5),4–5),
(pH
4–5),
4–5), (pH
4–5),
4–5),
(pH (pH
(pH
4–5), and
4–5),
and
and
andand
4–5), and
4–5),
and
4–5),
blue
4–5),
and blue
and
blue
4–5), blue
blueand
and
blue
blue
and and
and
blue(pH(pH
blue
(pH
(pH
(pH
blue (pH
blue
(pH 7)
blue
blue
blue
(pH 7)7)as
(pH
7)
7)
(pH 7)
as
7)as7)
(pH
as
(pH
7)asas
7)
(pH
as
(pH
as the
the
the
the
thethe
as
7)7)
the
7)
as the
as
7)as
the as the
asthe
the the
the
level
level
level level
level
level
levellevel level
changed.
changed.
changed.
level
level
changed.
changed.
changed.
level
changed.
level changed.
changed. AtAt
changed.
At
changed.
At
changed.
changed. At
At
At pH pH
pHpH
pHAtpH
pH
AtAt At
levels
levels
AtpH pH
levels pH
levels
levels
levels
At
levels
pH pHpH
levels levels
ranging
ranging
ranging
levels
levelsrangingranging
ranging
ranging
levels
levels
ranging ranging
from
from
ranging from
from
ranging
from
ranging
ranging from
fromfrom99
from9
9 from
9
toto
from
to
9
to
from
9from to
to12,
12,
912,
12,
to912, 9
to
12,9
12,
the
9toto
the
the
the
9 the
to tothe
12,
the
to
12,12, 12,
color
color
12,
the
color
color
12, the
color
the
color
color
the the
the of
ofof
color
of
of
color color
of
of the
the
color
the
color
the
color
thethe
of
theBAE
ofof
BAE
the
BAE
BAE
of of of
BAE
theBAE
BAEthe
the
the
the BAE
shifted
shifted
shifted
BAE BAE
shifted
shifted
BAE shifted
BAE
shifted
BAE shifted
from
fromfrom
shifted
shifted from
from from
shifted
shifted
shifted from blue
from from
blue
blue
from
blue
blue blue
from
blue
from from blue
blue blue
blue
blue
blue
to
to toto
to
to to green.
green.
green.
to
green. to
green.
green.
green.
to toto
green. Rawdkuen,
Rawdkuen,
Rawdkuen,
toRawdkuen,
green.
green.
green. Rawdkuen,
green.
green.
Rawdkuen, Rawdkuen,
Rawdkuen,
Rawdkuen,
Rawdkuen, Rawdkuen,
Rawdkuen,
Rawdkuen, Faseha,
Faseha,
Faseha,
Faseha, Faseha,
Faseha,
Faseha,
Faseha, Benjakul
Faseha,
Faseha, Benjakul
Benjakul
Faseha,
Faseha, Benjakul
Faseha,
Benjakul
Benjakul and
and
Benjakul
Benjakul
Benjakul
Benjakul and
Benjakul
and
Benjakul
Benjakul andand
and Kaewprachu
and Kaewprachu
Kaewprachu
and
and
Kaewprachu
Kaewprachu
Kaewprachu
and
Kaewprachu
and and Kaewprachu
Kaewprachu
Kaewprachu
Kaewprachu
Kaewprachu
Kaewprachu [12]
[12] [12]
[12]
[12] [12]
[12] also
also
[12]also
also
also [12]
[12]
also
also
[12] [12]
[12] reported
also reported
reported
also
also
reported
reported
reported
also
reported
also also that
that
reported
reported
reported that
reported
that
reported
reported thatthat
that butterfly
thatbutterfly
butterfly
that
that
butterfly
butterfly
butterfly
that
butterfly
that that butterfly
butterfly
butterfly
butterfly
butterfly
butterfly
pea
peapea
pea
pea
pea
pea pea pea
anthocyanin
anthocyanin
pea
anthocyanin
anthocyanin
anthocyanin
pea peapea anthocyanin
anthocyanin
anthocyanin
anthocyanin
pea anthocyanin
anthocyanin
anthocyanin
anthocyanin
anthocyanin extracts
extracts
extracts
extracts
extracts
extracts extracts
extracts exhibited
exhibited
extracts
extractsexhibited
extracts
extracts
exhibited
extracts
extracts exhibited exhibited
exhibited
exhibited the
the
exhibited
exhibited
the
exhibited
exhibited
exhibited
exhibited the
thethe
the same
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samesame
the
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same color
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changes
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color
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color
color color
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color
color changes
and
and
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changes and
changes
changes
changes and
changes
changes
and
andand were
were
and
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were
were
and and
and
were
and
and
and
were were were
successfully
successfully
were
successfully
were
successfully
weresuccessfully
were
were successfully
successfully
successfully
successfully
successfully
successfully
successfully
successfully
successfully used
used used
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used
used
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used
as
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Polymers 2022, 14, 5042 aaaapH
pHapH
aapHpH
pH
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a pHindicator
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purple
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at atat
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pHpH
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pH
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at
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2–3
at 2–3
pH
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at2–3
2–3
pH pH to
pH
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to toto
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to
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2–3 to
to purplish-blue
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to purplish-blue
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2–3
2–3 to
to
purplish-blue purplish-blue
purplish-blue
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to to purplish-blue
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pH
at
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pH pH
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at4–5,
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4–5,
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at pH pH
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pH 6,6,
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atbluish-green
pH 6,bluish-green
bluish-green
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monitor
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fermentation.
fermentation.
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fermentation.
fermentation.
of fermentation.
Table 5. Response to volatile ammonia and acetic acid of smart bilayer films incorporating different
Table
Table
5.5. 5. Response
5.
Response Responseto volatile
to ammonia
volatile ammonia and acetic
and acid
acetic of
acid smart
of bilayer
smart films
bilayer incorporating
films incorporating
Table
Table
Table
Table
Table
Table
Table
Table 5.5.
Table
5. Response
5.
Response
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Response
Response
5.5.
concentrations ofto
Response
Response
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Response to
to
to volatile
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cellulose ammonia
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3.10.
3.10. 3.10.
3.10.
3.10.
3.10.
3.10. 3.10.
FTIR
3.10. FTIR
FTIR
FTIR
FTIR
FTIR FTIR
FTIR
FTIR
3.10. FTIR
The
The
The The
The
The The
The FTIR-evaluated
FTIR-evaluated
The FTIR-evaluated
FTIR-evaluated
FTIR-evaluated
FTIR-evaluated
FTIR-evaluated
FTIR-evaluated
FTIR-evaluated differences
differences
differences
differences
differences
differences
differences
differences
differences between
between
between
betweenbetween
between
between
between
between the
the
thethe
the
the chemical
chemical
the
chemicalchemical
chemical
the chemical
chemical
chemical
the chemical structures
structures
structures
structures
structures
structures
structures of
structures
structures ofof
of of
ofsmartsmart
smart
of
smart
of
smart
smart
of smart
smart
smart bilayer
bilayer
bilayer bilayer
bilayer
bilayer
bilayer
bilayer
bilayer
The FTIR-evaluated differences between the chemical structures of smart bilayer
films
films
films
films
films
films films
are
films
films are
are
are
areare are
depicted
are depicted
depicted
depicted
depicted
depicted
depicted
are depictedin
depicted in
in
ininFigure
in
in in
Figure
in
Figure
Figure Figure
Figure
Figure
inFigure 2a.
Figure
Figure 2a.
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2a. All
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All
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film samples
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displayed a dominant
a dominant bandband 3250
at
cm
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cm−1−1
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3250 and
cm
and
−1
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and
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−12925
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2925
and
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2925
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2925 cm
−1
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and
and C-H
and
and
the C-H
C-H
and
C-H
C-H
C-H C-H
C-H
O-H stretching
stretching
C-H
stretching
stretching
stretching
stretching
stretching
stretching
and C-H vibrations,
vibrations,
stretching vibrations,
vibrations,
vibrations,
vibrations,
vibrations,
vibrations,
vibrations,
stretching which
which
which
which
which which
which
which
which
vibrations, corresponds
corresponds
corresponds
corresponds
corresponds
corresponds
corresponds
corresponds
corresponds
which
to
toto
to to
to the
the
the
tothe
to
the
the the aliphatic
aliphatic
the
aliphatic aliphatic
aliphatic
aliphatic
aliphatic
aliphatic moieties
moieties
moieties
moieties
moieties moieties
moieties
moieties in
inin
in in
in inpolysaccharides
polysaccharides
in polysaccharides
polysaccharides
polysaccharides
polysaccharides
polysaccharides
polysaccharides [69].
[69].
[69].
[69].
[69].
[69]. The
The
[69].
The
corresponds to the aliphatic moieties in polysaccharides [69]. The peaks at 1407 cm1293
to the aliphatic moieties in polysaccharides [69]. The
[69].
The
The The peaks
peaks
The
peaks
peaks
peaks
The at
peaks
atat
peaks
peaks at
peaks at
1407
at 1407
1407
at
1407
at
1407
1407
at cm
1407
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1407
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1407 cm
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−1
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and
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and cm
−1−1
−1−1 −1
−1−1−1

1293 cm−1 correspond to the O-H and C-H expansion and sway vibration absorption bands,
respectively. Asymmetric and symmetric COO stretching vibrations were assigned to the
absorption bands at 1597 cm−1 [70]. All of the film samples observed a peak at 2930 cm−1 ,
corresponding to C-H aliphatic stretching vibration [71]. The presence of C-H aliphatic
stretching vibration represents the phenolic compounds that may have resulted from the
incorporation of BAE and catechin. All of the samples had similar diffraction spectra,
indicating that the addition of CNs at any concentration did not affect the functional group
of the smart alginate/agar bilayer film.

3.11. Thermal Stability

The thermal stability of smart bilayer/CNs composite films is shown in Figure 2b. The
weight-loss regions in the TGA of smart bilayer films were distinct. The initial weight loss
of the smart bilayer film occurred at a temperature between 40 and 120 ◦ C, corresponding
to water and volatile compound evaporation and accounting for 9–10% of the weight
loss [72]. The second degradation occurred at temperatures ranging from 160 to 300 ◦ C,
caused by thermal degradation of the biopolymer chain. Up to the second stage of thermal
deterioration, CNs-containing films had slightly higher thermal stability than 0% CNs
smart bilayer films, but this difference was not statistically significant. The higher thermal
Polymers 2022, 14, x FOR PEER REVIEW 12 of 20

Polymers 2022, 14, 5042 12 of 18

correspond to the O-H and C-H expansion and sway vibration absorption bands,
respectively. Asymmetric and symmetric COO stretching vibrations were assigned to the
stability of CNs-containing
absorption bands at 1597 cmfilms was
−1 [70]. All associated
of the film with the observed
samples presence aofpeak
crystalline-form
at 2930 cm −1,
CNs. The CNs to
corresponding composite films had
C-H aliphatic considerably
stretching vibrationlower
[71].residue left after
The presence of final thermal
C-H aliphatic
decomposition ◦ C than the 0% CNs smart bilayer film. The char of the 0% CNs at
at 700represents
stretching vibration the phenolic compounds that may have resulted from the
700 ◦ C was 37.33%, but it decreased to 34.55–35.48% depending on the CNs concentration.
incorporation of BAE and catechin. All of the samples had similar diffraction spectra,
The findings were
indicating that the consistent
additionwith thoseatofany
of CNs Reddy and Rhim [73],
concentration did who
not reported
affect thethat adding
functional
mulberry pulp CNC resulted in less residue at 600 ◦ C than neat agar film.
group of the smart alginate/agar bilayer film.

Figure2.2.FTIR
Figure FTIR spectra
spectra (a),
(a), TGA
TGA (b),
(b), and
and DSC
DSC thermograms
thermograms (c)
(c) of smart bilayer films incorporating
differentconcentrations
different concentrationsofofcellulose
cellulosenanosphere
nanosphere(CNs).
(CNs).

3.11.The calorimetric
Thermal Stability thermogram for all film samples is depicted in Figure 2c. The en-
dothermic peaks that occur in DSC curves are typically associated with the breaking of
The thermal stability of smart bilayer/CNs composite films is shown in Figure 2b.
hydrogen bonds [74]. The DSC thermogram of the smart bilayer film and the CNs compos-
The weight-loss regions in the TGA of smart bilayer films were distinct. The initial weight
ite film samples revealed that the transitions occur over a wide temperature range. The
loss of the smart bilayer film occurred at a temperature between 40 and 120 °C,
addition of CNs did not affect the melting temperature of the smart bilayer film. After
corresponding to water and volatile compound evaporation and accounting for 9%–10%
the addition of CNs, the melting temperature of the smart bilayer film was reduced from
of the weight loss [72]. The second degradation occurred at temperatures ranging from
104.2 ◦ C to 98.83–103.11 ◦ C. This result agreed with Chen, et al. [75], who found that the
160 to 300 °C, caused by thermal degradation of the biopolymer chain. Up to the second
melting temperature decreases as the nanocellulose concentration increases.
Polymers 2022, 14, 5042 13 of 18

3.12. Bioactive Compounds and Antioxidant Properties

The antioxidant properties are the common essential functional role of the active films.
They have the ability to effectively prevent oxidative deterioration in packaged foods. Since
CNs have no superior antioxidant properties, their incorporation alone does not affect
the antioxidant properties of the film. As a result, CNs are frequently used in films as a
synergist with the polymer matrix or other active ingredient additives in order to enhance
the antioxidant and antibacterial properties [76]. The bioactive compounds and antioxidant
properties of smart bilayer films with varying concentrations of CNs are presented in
Table 6. The inclusion of CNs had no significant effect on the total anthocyanin and TPC of
smart bilayer film (p > 0.05). The anthocyanin content in smart bilayer films ranges from
2.19 to 2.42 mg/100 g db. The TPC of smart bilayer film ranges between 46.40 and 51.88 mg
GAE/g db. The increase in CNs content had a minor effect on the TPC of smart bilayer film.
The presence of the phenolic compound in the smart bilayer film resulted from the BAE and
catechin in the film formulation. Catechins have been reported to be excellent antioxidant
agents [51]. Kaewprachu, Osako, Rungraeng and Rawdkuen [51] also reported that films
incorporating catechin–Kradon extract had higher antioxidant activity than exclusively
Kradon extract-added fish myofibrillar protein films. All of the film samples had the same
concentration of BAE and catechin, but the addition of CNs increased the total weight of
the film-forming solution, causing the bioactive compounds’ intensity to decrease.

Table 6. Antioxidant and antimicrobial properties of smart bilayer films incorporating different
concentrations of cellulose nanosphere (CNs).

CNs Bioactive Compounds Antioxidant Properties Antimicrobial Activity (mm)

Concentration Anthocyanin TPC (mg FRAP (mM Fe DPPH (mM
in Film (%) E. coli S. aureus
(mg/100 g) GAE/g) (II)/g) Trolox/g)
0 2.30 ± 0.24 a 46.58 ± 1.14 a 575.09 ± 1.18 a 628.25 ± 8.50 c 11.67 ± 0.58 a 11.67 ± 0.58 a
5 2.33 ± 0.23 a 46.54 ± 1.08 a 551.67 ± 13.97 ab 911.54 ± 51.68 b 10.67 ± 0.58 a 10.83 ± 0.76 a
10 2.19 ± 0.12 a 45.66 ± 1.12 a 525.96 ± 17.23 bc 943.04 ± 35.17 b 11.17 ± 0.29 a 11.67 ± 0.29 a
20 2.42 ± 0.08 a 45.29 ± 0.16 a 522.83 ± 26.47 bc 1020.98 ± 15.39 a 10.67 ± 0.58 a 10.67 ± 0.58 a
30 2.38 ± 0.21 a 45.36 ± 0.08 a 518.79 ± 2.52 c 1033.87 ±9.31 a 10.83 ± 0.29 a 10.00 ± 0.00 a
Values are given as mean ± SD, n = 3. Different superscripts in each column are significantly different (p < 0.05).

The antioxidant properties of smart bilayer film samples, including ferric-reducing

antioxidant power (FRAP) and DPPH radical scavenging activity, are shown in Table 6. All
of the film samples had antioxidant properties ranging from 518.79 to 575.09 mM Fe (II)/g
db. for FRAP and 628.25 to 1033.87 mM Trolox/g db. for DPPH. The inclusion of CNs had
the effect of decreasing the FRAP values of the film (p < 0.05). Conversely, as the concen-
tration of CNs increased, the DPPH values of the smart bilayer film increased (p < 0.05).
Furthermore, CNs can improve antioxidant properties by interacting directly with the film
matrix. Mujtaba, Akyuz, Koc, Kaya, Ilk, Cansaran-Duman, Martinez, Cakmak, Labidi and
Boufi [55] revealed that the incorporation of cellulose nanofiber (CNF) greatly enhanced
the antioxidant and antibacterial properties of mucilage films by enhancing their releasing
behavior and solubility. Nanocellulose, as a controlled release agent and stabilizer, is an
excellent filler for enhancing the antioxidant and antibacterial characteristics of films. These
findings imply that the developed smart bilayer films, both with and without CNs, have
excellent antioxidant properties and high phenolic contents. The smart bilayer film with
high antioxidant activity can be used for active packaging in order to preserve food quality
and extend food shelf life by preventing food oxidation.

3.13. Antimicrobial Activity

The antibacterial activity against Escherichia coli and Staphylococcus aureus as measured
by the diameters of the clear zones of smart bilayer films is presented in Table 6. All of the
smart bilayer films displayed 10.00–11.67 mm diameter inhibition zones against S. aureus
Polymers 2022, 14, 5042 14 of 18

and 10.67–11.67 mm diameter inhibition zones against E. coli. All of the film samples
exhibit outstanding antibacterial activity against Gram-positive and Gram-negative bac-
teria, regardless of the CNs concentration (p > 0.05). Due to CNs having no obvious
antimicrobial activity, their incorporation into the film does not affect its antimicrobial
properties. The antimicrobial properties of smart bilayer film are attributed to the presence
of catechin–lysozyme in the film formulation. For all film samples, the same concentrations
of catechin–lysozyme were used. As a result, there was no difference in the antimicrobial
activity of all smart bilayer film samples. The mechanical and antimicrobial properties of
lysozyme consist of hydrolysis of peptidoglycan layers in bacteria’s cell wall and mem-
brane disruption [77]. Rawdkuen, Suthiluk, Kamhangwong and Benjakul [41] reported the
antimicrobial activity of gelatin film incorporating catechin–lysozyme at different concen-
trations (0.125 to 0.5%, w/v). The added catechin–lysozyme film inhibited all of the tested
microorganisms: S. cerevisiae, S. aureus, E. coli, and L. innocua. Another study indicated
that lysozyme combined with nanocellulose had the strongest antibacterial effects against
S. aureus and E. coli [78]. Tea polyphenol has excellent antibacterial properties as a natural
active substance with a high antioxidant capacity [79]. According to Jamróz, et al. [80],
incorporating green and black tea extracts into the furcellaran–gelatin film can increase
its antibacterial efficacy against S. aureus and E. coli. Consequently, smart bilayer films
containing all concentrations of CNs inhibit the growth of microorganisms in food during
storage, resulting in the prolonged shelf-life of food.

3.14. Application of Smart Bilayer Film for Monitoring Shrimp Freshness

Due to high concentrations of total volatile basic nitrogen (TVB-N), spoilage of meat,
fish, and poultry items causes an increase in pH from 5.8 to 7.8 [81]. Therefore, shrimp
was used to evaluate the use of smart bilayer film containing various concentrations of
CNs for monitoring freshness at 25 ◦ C (accelerated conditions). After 24 h of storage, as
a pH-detecting indication (untouched by the shrimp), all of the film samples exhibited
Polymers 2022, 14, x FOR PEER REVIEW
consistent color changes (Figure 3). In addition, after 24 h, the film’s color changed15from
of 20
blue to green, and the pH changed from 6.74 to 7.65 (Figure 3). Consequently, smart bilayer
films, both with and without CNs, could be used as intelligent food packaging films in
orderintoorder
films monitor the freshness
to monitor of seafood
the freshness or other
of seafood orfresh
othermeat
freshproducts through
meat products direct
through
visual inspection.
direct visual inspection.

Figure 3. Cont.
Polymers 2022, 14, 5042 15 of 18

Figure
Figure 3.
3. Application
Application of
of aa cellulose
cellulose nanosphere
nanosphere (CNs)-containing
(CNs)-containing smart
smart bilayer
bilayer film
film for
for monitoring
monitoring
changes in shrimp quality at 25
changes in shrimp quality at 25 C.°C.
◦

4. Conclusions
4. Conclusions
The addition
The addition ofof butterfly
butterfly peapea anthocyanin
anthocyanin extract
extract and
and catechin–lysozyme
catechin–lysozyme to to film
film
formulations resulted
formulations resulted in in the
the successful
successful preparation
preparation of of aa smart
smart bilayer
bilayer film.
film. The
The smart
smart
bilayer film
bilayer film has
has aa blue
blue film
film color
color and
and has
has the
the ability
ability to
tochange
change colors
colors depending
depending on on the
the pH
pH
level. Furthermore, it has the potential to change color when exposed to
level. Furthermore, it has the potential to change color when exposed to volatile ammonia volatile ammonia
and acid.
and acid. The
Thesmart
smartbilayer
bilayerfilm
filmisishighly
highlyantioxidant
antioxidantand andantimicrobial.
antimicrobial. Corncob
Corncobcellulose
cellulose
nanospheres (CNs) can improve the mechanical properties of smart
nanospheres (CNs) can improve the mechanical properties of smart bilayer films withoutbilayer films without
affecting their
affecting their chemical
chemical properties.
properties. TheThe smart
smart bilayer
bilayer film,
film, containing
containing allall concentrations
concentrations
of CNs, was successfully used to monitor the freshness of the shrimp. After the shrimp
spoiled, the color of the smart bilayer film changed from blue to green. These findings
imply that smart bilayer films with and without CNs can be used in food packaging systems
to monitor spoilage without causing damage and to extend the shelf life of food.

Author Contributions: Conceptualization, O.R. and S.R.; methodology, O.R. and S.R.; formal analysis,
O.R.; writing—original draft preparation, O.R. and P.K.; writing—review and editing, O.R., P.K.
and S.R.; visualization, P.K., T.K. and S.R.; supervision, S.R.; project administration, S.R.; funding
acquisition, S.R. All authors have read and agreed to the published version of the manuscript.
Funding: The Thailand Research Fund financed this study through the Royal Golden Jubilee Ph.D.
scholarship program, grant number PHD/0218/2560.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors would like to thank the Thailand Research Fund for providing
financial support to Orapan Romruen through the Royal Golden Jubilee Ph.D. scholarship program
(Grant No. PHD/0218/2560). Mae Fah Luang University, Chiang Rai, Thailand is also acknowledged
for providing full chemical and instrument support.
Polymers 2022, 14, 5042 16 of 18

Conflicts of Interest: The authors declare no conflict of interest.

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