Food Packaging and Shelf Life 42 (2024) 101269

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Food Packaging and Shelf Life

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

Agar-altered foaming bacterial cellulose with carvacrol for active food

packaging applications
Anita Chandra Kusuma a, Yu-Chieh Chou b, Chen-Che Hsieh c, Shella Permatasari Santoso d,
Alchris Woo Go e, Hong-Ting Victor Lin f, I.-Lun Hsiao g, Shin-Ping Lin b, g, h, i, *
a
Master Program in Food Safety, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
b
Ph.D. Program in Drug Discovery and Development Industry, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
c
Institute of Biotechnology, National Taiwan University, 1 Roosevelt Rd., Section 4, Taipei 10617, Taiwan
d
Department of Chemical Engineering, Widya Mandala Surabaya Catholic University, Kalijudan 37, Surabaya 60114, Indonesia
e
Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 10607, Taiwan
f
Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 20224, Taiwan
g
School of Food Safety, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
h
TMU Research Center for Digestive Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan
i
Research Center of Biomedical Device, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan

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

Keywords: Bacterial cellulose (BC) is a biodegradable nanoscale film produced by microorganisms, which possesses great
Bacterial cellulose mechanical properties and biocompatibility. In this study, we produced foaming BC (FBC) utilizing foam medium
Foaming bacterial cellulose with different concentrations of agar. In addition, carvacrol was loaded into the agar/FBC to establish active
Agar
packaging material. Evaluation by characterization tests indicated that agar/FBC provided lower crystallinity
Carvacrol
Sea bass
(59.8%~71.4%) and a high swelling ability (94.7%~97.9%) compared to BC (92.2% and 96.1%). The 1.5%
Food packaging agar/FBC with carvacrol group exhibited the significantly highest antimicrobial properties against Escherichia
coli and Staphylococcus aureus. Moreover, release measurements showed that greater agar addition contributed to
higher protein and carvacrol release extents out of the film. Finally, a sea bass packaging test indicated that
carvacrol with 1.5% agar/FBC presented a higher antimicrobial ability against Shewanella putrefaciens, great
inhibition of lipid oxidation (67.5%), and a slower spoilage degree (49.8%) which suggested that agar/FBC with
carvacrol has potential for active food packaging.

1. Introduction
In recent times, significant progress in the realm of food packaging
technology, often referred to as smart food packaging, has brought about
transformative impacts on the sector, further underscoring its essential
contribution to maintaining benchmarks of food safety (Dainelli et al.,
2008). Active packaging is a packaging method that integrates "active
compound" additives, either into the packaging material or within the
packaging container. These active compounds have direct impacts on
perishable items which they contact (Wilson et al., 2018). Numerous
research studies have concentrated on advancing active packaging with
antimicrobial compounds to ensure the safety of packaged foods
(Bolumar et al., 2011). Antimicrobial food packaging films play vital
roles in preventing recontamination and extending shelf lives by
creating a protective barrier between food and the environment (Beig­
mohammadi et al., 2016). This type of packaging reduces, retards, or
inhibits the growth of spoilage and harmful bacteria through in­
teractions with the packaged food or the package’s airspace. Antimi­
crobial properties can be incorporated into a polymer matrix to create
films with such functionality (Lomate et al., 2018). Common antimi­
crobial agents used in active packaging include chitosan, essential oils,
potassium sorbate, nisin, and silver-substituted zeolite.
Bacterial cellulose (BC) is a natural polymer that is distinguished by
its excellent physical and chemical properties. The notable purity of BC
further ensures its suitability for various applications in material sci­
ence, tissue engineering, medicine, and food technology, especially food
packaging. BC was proven to be beneficial through much research on
active food packaging, such as combined with postbiotics of lactic acid

* Corresponding author at: Ph.D. Program in Drug Discovery and Development Industry, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan.
E-mail address: splin0330@tmu.edu.tw (S.-P. Lin).

https://doi.org/10.1016/j.fpsl.2024.101269
Received 5 September 2023; Received in revised form 20 February 2024; Accepted 2 March 2024
Available online 12 March 2024
2214-2894/© 2024 Elsevier Ltd. All rights reserved.
A.C. Kusuma et al.
bacteria (Yordshahi et al., 2020), whey protein as an electrospray
coating (Paximada et al., 2020), hydrochloric acid hydrolysis for coating
apples (Zhai et al., 2020), and silver nanoparticles (Salari et al., 2018),
making it a potential option as a polymer. However, to expand the use of
BC in active food packaging film, the absorptive ability of BC fibers still
needs to be improved by utilizing modifications.
To enhance BC’s capabilities, there are two types of modifications: in
situ modification, performed during BC formation, and ex situ modifi­
cation, carried out after BC has formed. According to a study by Rühs
et al. (2018), foaming BC (FBC) is attained by saturating the oxygen
requirement of bacteria during cellulose growth, directly stabilizing air
bubbles. The xanthan and cremodan were used as stabilizer and foaming
reagent mixed with nutrient to generate the stable foaming environment
for BC production. This enhances the overall surface-to-air ratio. The
same technique was also applied by Lin et al. (2023), by optimizing the
foaming agent concentration and integrating several additives such as
microcrystalline cellulose, carboxymethylcellulose, and chitosan to
create unique porous modifications of FBC.
Agar is a linear polysaccharide with an excellent film-forming abil­
ity, biocompatibility, and biodegradability that can improve water ab­
sorption. In a study conducted by (Han et al., 2013), it was discovered
that varying agar levels in both foaming and drop-in-oil techniques led
to alterations in the structure, pore size, and distribution of ceramic
foams. That experiment revealed that higher agar contents in ceramic
foams resulted in a narrower range of macropore sizes due to enhanced
foam stability and faster gelation, which facilitated the controlled
release of additives. The incorporation of agar, combined with a foaming
system, allowed for modification of its inherent properties, yielding
novel characteristics suitable for tailored functional packaging needs.
As to additive agents, carvacrol has garnered significant scientific
interest for its distinct qualities and versatile potential uses. For
instance, Shemesh et al. (2016) demonstrated its incorporation into
polyamide as a means to reduce decay and extend shelf lives, notably
against Penicillium expansum and Aspergillus niger as cherry tomato,
lychee, and grape coatings. Other research from Ochoa-Velasco et al.
(2021) investigated its antibacterial effects in edible starch films against
Colletotrichum gloeosporioides as mango and papaya coatings. Further­
more, Tao et al. (2022) examined carvacrol and glycerol concentrations
in soy protein isolate films. Thus, carvacrol has the potential for
enhanced food safety as an active packaging agent.
Due to existing limitations of BC systems, the main objective of this
study was to explore impacts of incorporating agar into a BC foaming
system. Additionally, this research aimed to evaluate agar/FBC films by
incorporating carvacrol, addressing requirements for functional
packaging.
2. Materials and methods
2.1. Materials
The corn steep liquor (CSL) -fructose culture medium underwent
sterilization at 121 ◦ C for 15 min. The foaming medium was altered with
xanthan (Danisco, Copenhagen, Denmark) and cremodan (Danisco) as
foaming agents, alongside technical agar (Biolife Italiana, Viale Monza,
Italy) and carvacrol (Sigma Aldrich, St. Louis, MO, USA) as additives.
These components were blended with cremodan and xanthan, followed
by foaming using a commercial milk frother (unbranded, China).
2.2. Preparation of the culture strain
The strain used in this study was based on Lin et al. (2023). Koma­
gataeibacter xylinus ATCC 700178 was procured from the Bioresource
Collection and Research Center (BCRC; Hsinchu City, Taiwan), and
stored at a temperature of − 80 ◦ C. To culture a new stock, a frozen cell
suspension was combined with 50 mL of CSL-fructose medium and
cultivated under static conditions at 28 ◦ C for 5 days. The resulting BC
2
Food Packaging and Shelf Life 42 (2024) 101269
pellicle was subjected to cellulase hydrolysis (Sigma Aldrich) for 6–8 h
at 28 ◦ C. Subsequently, centrifugation (CF15RXII, Hitachi, Tokyo,
Japan) at 104 ×g for 10 min was performed to consolidate the cell
biomass. This cell pellet was then resuspended in a 20% glycerol solu­
tion and preserved as a stock solution at − 80 ◦ C.
2.3. Production of FBC with carvacrol
Modified FBC was produced based on Rühs et al. (2018). The
foaming medium (CSL-fructose medium with selected concentrations of
glucose and CSL and 0.2% xanthan and 1% cremodan) was mixed with
different concentrations of agar (0%~2%) and 2% inoculation. Aeration
was conducted using a commercial milk frother at its maximum speed
for 10 min, followed by static incubation at 28 ◦ C for 5 days. Subse­
quently, samples were rinsed twice with 0.1 N NaOH for 30 min, under a
continuous stream of water overnight, and subsequently freeze-dried the
following day. The dried BC was then soaked in a DMSO solution con­
taining 10 mg/mL of carvacrol for 2 h and subsequently subjected to
another round of freeze-drying.
2.4. Characterization of the agar/FBC film
Fourier transform infrared (FTIR) spectral absorbance was assessed
utilizing a Spectrum 100 FTIR Spectrometer (Perkin Elmer, Wellesley,
MA, USA). Measurements were performed using air as the background
and freeze-dried BC as the specimen. The spectral range for each sample
spanned 4000 to 1000 cm− 1, comprising 30 scans at a resolution of 1
cm− 1.
Scanning electron microscopy (SEM) was employed to capture high-
energy electrons emitted from the surface of freeze-dried BC specimens,
which were coated with platinum particles utilizing a Q150R rotary
pumped coater (Quorum, Laughton, East Sussex, UK). The morphology
was examined by scanning at an acceleration voltage of 5 kV (Hitachi S-
4800 field emission SEM) with an image magnification of 15,000 ×g.
Image processing software (ImageJ, National Institute of Mental Health,
Bethesda, MD, USA) was used to measure fiber sizes.
A thermogravimetric analyzer (Q50, TA Instruments, New Castle,
United Kingdom) was utilized to perform a dynamic weight loss
assessment. Freeze-dried BC was subjected to a thermal degradation
analysis under an N2 purge (40 mL/min), employing a temperature
range of 90 to 600 ◦ C, with an increment rate of 10 ◦ C/min.
To detect the crystallinity, freeze-dried BC samples were ground into
a powder to avoid preferred orientation (French, 2014) using plant tis­
sue grinding equipment sourced from Lawson Scientific (Ningbo,
China). Subsequently, x-ray diffraction (XRD) patterns were generated
employing an x-ray powder diffractometer. Scans were conducted at a
rate of 4◦ /min across the range of 5◦ to 30◦ 2θ. The extent of crystallinity
was determined by analyzing the peak through a deconvolution method
(Park et al., 2010).
The water content was calculated using the formula:
(Wo − Wt )
× 100%;
Wo
where Wo represents the weight before drying, and Wt represents the
weight after drying.
Freeze-dried BC was soaked in double-distilled (dd)H2O for 24 h to
obtain its wet weight. Afterward, BC underwent freeze-drying again to
determine its dried weight, allowing the calculation of the swelling rate
using the water content formula. This process was repeated for the initial
swollen BC sample to determine the re-swelling rate.
2.5. Antimicrobial property of the agar/FBC-carvacrol film
The disk diffusion method was employed to assess the antimicrobial
activity of BC film with carvacrol sections measuring 1 cm2. Sterile
A.C. Kusuma et al.
nutrient agar (NA) was prepared, and agar was inoculated with Escher­
ichia coli (E. coli) ATCC 25922 and Streptococcus aureus (S. aureus) ATCC
6538 P cultures. After allowing the agar surface to dry, the BC film was
placed on the agar plate. A maximum of six disks could be positioned in a
9-cm Petri dish. The BC-loaded agar plates were incubated for 24 h, and
the diameter of the inhibition zone was measured the following day.
2.6. BSA/carvacrol release analysis
For carvacrol release activity, a dried film measuring 1 cm2 was
introduced into a glass beaker containing 10 mL of a carvacrol solution
with a concentration of 100 mg/mL. On the other hand, to measure the
protein release activity, the dried film was dipped in 10 mL of a bovine
serum albumin (BSA) protein solution at a concentration of 500 mg/mL.
The mixture was incubated at room temperature with mild stirring.
Subsequently, a sample was extracted and analyzed using a UV spec­
trometer at a wavelength of 293 nm for carvacrol and 595 nm for pro­
tein. Measurements were taken at intervals of 2, 4, 8, 12, 24, and 36 h
for all samples.
2.7. Application in active food packaging
Fish was prepared by removing the skin, bones, and intestines,
leaving only the flesh, which was then sliced into uniform-weight pieces.
These 9-cm2 fish slices were inoculated with 105 colony-forming units
(CFU)/mL of Shewanella putrefaciens BCRC 10596 and enclosed in plastic
bags, forming three groups of a control; S. putrefaciens addition, bac with
FBC; and bac, FBC with carvacrol. Samples were stored in a refrigerated
environment at 4 ◦ C. Sampling and analysis occurred on days 0, 3, 6, 9
and 12.
The total viable count (TVC) was calculated by cutting a segment
measuring 3 cm2 extracted from each fish sample. Subsequently, sam­
ples were mixed with 40 mL of 0.1% pancreatic digest gelatin and
subjected to squeezing in a puncher machine for 5 min. This was fol­
lowed by serial dilution ranging from 10− 2 to 10− 7. A volume of 0.1 mL
from the solution was extracted and spread onto TSA agar, and then
incubated for 24 h. Values of the CFU/mL of samples were quantified
using the following formula:
TVC(CFU total dilution factor
mL ) = no. of colonies × the volume of culture plated in ml.
Total volatile bases of nitrogen (TVBN) was measured by cutting a 3-
cm2 section from each fish sample, which was then blended with 20 mL
of 2.2% trichloroacetic acid (TCA). The resulting solutions underwent
centrifugation and filtration. A volume of 1 mL of a boric acid solution
(consisting of 1 g boric acid, 20 mL ethanol, 70 mL deionized water,
0.5 mL bromocresol green indicator, and 0.5 mL methyl red, and diluted
to 100 mL with deionized water) was placed in the central compartment
of a Conway dish. Additionally, 1 mL of the filtered sample was added to
the outer compartment, along with 1 mL of saturated potassium car­
bonate. The dish was covered with a glass plate and maintained at 37 ◦ C
for 90 min. The central section of the Conway dish was titrated with
0.01 N HCl. The TVBN level of the sample was then quantified using the
following formula:
(
mg
) the agar concentration increased, similar peak patterns with greater or
vol extract
TVBN 100g = Vs − Vb × 14 × sample volume × weight;.
where Vs represents the HCl titration volume of the sample, and Vb is
the HCl titration volume of the blank.
A thiobarbituric acid reactive substance (TBARS) analysis was con­
ducted by extracting a segment of fish sample measuring 1 × 3 cm and
mixing it with 30 mL of a 7.5% TCA solution (which included 0.1%
EDTA-Na and 0.1% propyl gallate). The resulting solutions were filtered.
Subsequently, 5 mL of the filtered sample was combined with 5 mL of a
0.02 M TBA reagent and placed in a boiling water bath for 40 min,
followed by rapid cooling in cold water. Samples were then analyzed
with a UV spectrometer at a wavelength of 538 nm.
3
Food Packaging and Shelf Life 42 (2024) 101269
2.8. Statistical analysis
Experimental results were subjected to statistical analysis using SPSS
Statistics 22 (IBM, Armonk, NY, USA). Differences between different
experimental groups were determined using a one-way analysis of
variance (ANOVA) with Tukey’s honest significant difference (HSD) as
the post-hoc test (p < 0.05).
3. Results and Discussion
3.1. Optimization of agar/FBC production
Three different factors of the carbon source, nitrogen source, and
agar addition were used to modify BC production. As seen in Figs. 1a and
1b, different concentrations of carbon and nitrogen sources were tested
to find the highest yield of BC production. With carbon source modifi­
cation, a 2% nitrogen source concentration was used as the normal
composition based on a previous experiment (Lin et al., 2023). After
finding the best carbon source concentration (12.5% carbon source with
a significant result for dry weight of around 7.9 ± 0.5 g/L), that con­
centration was used for nitrogen source modification. The highest ni­
trogen source was obtained with a 0.5% nitrogen source with a
significant result of dry weight of around 6.05 ± 0.77 g/L. As to nitro­
gen source modification, after adding a higher nitrogen source concen­
tration, softer and breakable BC films were found.
Using the chosen concentrations of the carbon (12.5%) and nitrogen
sources (0.5%), BC production continued with agar addition. Various
agar concentrations were used (0%, 0.5%, 1%, 1.5%, and 2% agar/FBC).
FBC production results after 5 days of incubation are shown in Fig. 1c
below.
As the agar concentration increased, the color of FBC changed to
yellow (Fig. 1d). The structure of FBC also became denser, especially at
2%, which exhibited soft and weak BC foamation. This concentration
needs to be considered when applied to food packaging applications.
Based on the dry weight results in Figs. 1c, 1.5% agar/FBC had the
highest dry weight and with no significant results at 1% agar/FBC.
3.2. Material property analyses
3.2.1. FTIR analysis
FTIR results of the normal BC, FBC, and FBC groups with different
agar concentrations are shown in Fig. 2b. All of the FBC samples showed
two strong peaks at 2918 and 2851 cm− 1; these peaks were probably due
to xanthan and sodium alginate from cremodan. The same results were
also shown in Lin et al. (2023) experiment. The large peaks seen in all
films in the 3600–3000 cm− 1 range were caused by O-H stretching vi­
brations and intermolecular or intramolecular H-bonding (Nisar et al.,
2018). There was a small difference in the peak at around 1648 cm− 1
that was contributed by agar addition to FBC. This refers to stretching
vibrations of conjugated peptide bonds in agar. In addition, the peak
around 1375 cm− 1 refers to the ester sulfate group present in agar
(Ghosh et al., 2010). The peak at 1151 cm− 1 was caused by vibrations of
ester-sulfate bonds of agar, and the peak around 889 cm− 1 referred to
the C-H residual carbon of β-galactose of agar (Roy & Rhim, 2019). As
lower intensities were seen and were most likely results of physical in­
teractions such as van der Waals forces and hydrogen bonds between the
joint and matrix polymer (Roy & Rhim, 2021).
3.2.2. TGA analysis
The addition of agar to BC influenced the thermal degradation
behavior as observed through the TGA. However, the specific effects of
agar addition on the TGA results of BC varied depending on the exper­
imental conditions and the concentration of agar used. In Fig. 2b, BC and
FBC with different agar concentrations almost had the same percentage
weight degradation, except for FBC with a significant difference
A.C. Kusuma et al.
Fig. 1. Optimization of FBC production with different concentrations of (a) the carbon source, (b) nitrogen source, and its production with different amounts of
added agar (c), and the morphology of agar/FBC samples (d). Different letters on the error bars indicate a significant difference (p < 0.05) relative to the control.
compared to the others. FBC had a higher weight loss temperature peak
(387 ◦ C) compared to normal BC (368 ◦ C) due to more ingredients
combined inside BC that created more than one peak. Three steps of the
peak of FBC films were observed. An initial mass loss was found at
around 213 ◦ C, 217 ◦ C, not found, not found, 244 ◦ C, and second mass
losses occurred at around 280 ◦ C, 285 ◦ C, 280 ◦ C, 304 ◦ C, and 323 ◦ C for
FBC, 0.5% agar/FBC, 1% agar/FBC, 1.5% agar/FBC, and 2% agar/FBC,
respectively, that may be attributed to xanthan, cremodan, and agar
addition (Lin et al., 2023; Ouyang et al., 2018). The first peak from 1%
and 1.5% agar/FBC were not found which may be due to the overlapping
with its widely second peak. A third thermal degradation was found in
FBC, 0.5% agar/FBC, 1% agar/FBC, 1.5% agar/FBC, and 2% agar/FBC
at temperatures of 387, 385, 382, 376, and 366 ◦ C, respectively, due to
the thermal decomposition of the polymer (Reddy & Rhim, 2014).
Rhim et al. (2013) indicated that the major thermal decomposition
peak of agar was 306 oC. However, no similar peak existed in the
agar/FBC, which suggested that the agar concentration might be too
lower to be detected or he agar will interact with FBC instead of existing
independently. Interestingly, the second peak of 0.5% and 1% agar/FBC
were similar to FBC group, and 1.5% and 2% agar/FBC were signifi­
cantly increased to 304 ◦ C, and 323 ◦ C, which may be caused by
increasing agar addition to influent the structure of FBC or its formation.
The 2% agar/FBC cannot form the complete solid state, which also
supported that the FBC formation was inhibited resulting to lower cel­
lulose content. This can be used to explain why the major peak from
cellulose (366 oC) of 2% agar/FBC is the lowest compared to other
agar/FBCs. From these results, agar addition to FBC produced shifts in
the degradation temperatures, indicating alterations of hydrogen bonds
between agar and FBC in the thermal behavior of the composite (Wang
4
Food Packaging and Shelf Life 42 (2024) 101269
et al., 2018).
3.2.3. XRD analysis
The XRD analysis of BC with agar addition was performed to inves­
tigate the effects of agar on the crystallinity of BC. From the XRD
analysis, it was observed that all BC samples exhibited four distinct
peaks appearing at diffraction angles of 14◦ , 16◦ , 22◦ , and 34◦ . These
peaks were consistently present in all diffractograms, indicating the
presence of a native cellulose Iα crystalline structure. Another peak,
located at approximately 21.5◦ , was identified as representing the
amorphous portion of the samples (Lin et al., 2023). Fig. 2c shows that
as agar was added to BC, the crystallinity index (CI) of the material
decreased (the CI is shown in Fig. 2c). This was evidenced by the lower
intensity that showed lower peaks and flattening of the XRD peaks in
agar-modified BC samples compared to the BC control. Furthermore, the
decrease in CI became more pronounced at higher agar concentrations.
One of the main reasons was interference by agar of the formation and
alignment of cellulose chains during the biosynthesis of BC. Agar mol­
ecules can disrupt the regular arrangement of cellulose microfibrils,
leading to a less-ordered and more-amorphous structure. This interfer­
ence can hinder the formation of crystalline regions in BC and result in a
lower CI (Nicolas et al., 2021). Additionally, agar is known to possess a
relatively amorphous structure compared to cellulose. When added to
BC, the amorphous regions of agar can mix with the amorphous regions
of cellulose, reducing the overall CI of the composite material (Lin et al.,
2016). The concentration of agar in FBC also played a role in reducing
the CI. Higher agar concentrations provide a greater extent of interfer­
ence with cellulose chains and a higher number of amorphous regions,
resulting in a more-pronounced decrease in crystallinity.
A.C. Kusuma et al.
Fig. 2. Characterizations of agar/foaming FBC samples. (a) FTIR, (b) TGA, (c) XRD, (d) water content ability, and (e) SEM.
3.2.4. Water content swelling and re-swelling ratio analysis
The addition of agar to FBC can impact the water content and
swelling ratio of the composite material. Compared to (Lin et al., 2023)
experiment, between the BC and FBC groups, FBC seemed to have higher
results for the swelling and re-swelling ratio. However, in our experi­
ment, FBC had a lower result of the swelling ratio. This may have been
due to the different structure of FBC. As can be seen in Fig. 2d, FBC had
more-compact fibrils that may have reduced the water intake and space
5
Food Packaging and Shelf Life 42 (2024) 101269
inside FBC. Increasing the agar concentration not only significantly
increased the water content but also increased the swelling and
re-swelling ratio. The highest water content was obtained with normal
BC (99.15%) followed by 2% agar/FBC (99.09%), 1.5% agar/FBC
(98.65%), 1% agar/FBC (98.44%), 0.5% agar/FBC (98.33%), and FBC
(98.04%). The same results are also presented in Fig. 2d of the swelling
and re-swelling ratio analysis with the lowest percentage obtained with
FBC (91.93% and 89.98%). Due to agar’s hydrophilic characteristics
A.C. Kusuma et al.
that have the ability to attract water, agar molecules can absorb and
hold water, adding to the composite material’s overall water content
(Cheng et al., 2009). In addition, since agar is a hydrocolloid, the
addition of agar increased the water content of BC (da Costa et al.,
2020). Hydrocolloids are compounds that have a high water-absorption
capacity. When agar was mixed with BC, a network of hydrated chains
emerged that trapped water molecules. However, percentages in all
treatments (BC, FBC, and FBC with different agar concentrations) were
still considered to be high water contents.
3.2.5. SEM analysis
SEM was employed to investigate the morphological characteristics
of BC and FBC formation at different agar concentrations, as depicted in
Fig. 2e. The BC control sample exhibited a typical nanofiber BC network,
with an average fiber size (FS) of 588 nm. After introducing agar addi­
tives, not only did average FSs change to 647, 744, 541, 977, and
591 nm for 0%, 0.5%, 1%, 1.5%, and 2% agar/FBC, respectively, but it
also altered the distribution of BC FSs. Following the addition of agar,
particularly at a 2% agar/FBC concentration, most of the observed
morphologies exhibited large and loosely packed fibrils with a homo­
geneous and interconnected structure. This phenomenon can be attrib­
uted to the flocculating properties of agar, as it functioned as a binding
agent for BC fibrils (Lee et al., 2014).
3.3. Antimicrobial property of agar/FBC-carvacrol
From the antimicrobial activity analysis, different films and agar
concentrations exhibited different results of diameter inhibition. E. coli
and S. aureus were used as they are gram-positive and gram-negative
bacteria, respectively, in order to test the ability of carvacrol in inhib­
iting a broad spectrum of bacterial strains. In addition, the type of
bacteria can also influence the antibacterial efficacy (Wei et al., 2011).
From Table 1 below, the antimicrobial activity of BC had higher diam­
eter inhibition compared to FBC with E. coli. The film thickness could be
the reason. Thus, normal BC allowed carvacrol to more easily diffuse
(Darvish & Ajji, 2021). As the agar concentration increased from 0% to
1.5%, greater diameter inhibition was observed, and it decreased in both
E. coli and S. aureus with 2% of agar addition. Different agar concen­
trations can contribute to different film structures, networks, in­
teractions, and porosities. Increasing concentrations of agar can result in
the formation of a more-extensive gel network, resulting in increased
porosity (Varshney, 2007). However, as the agar-agar concentration
increased, the film matrix became thicker and less permeable. The
higher agar content resulted in a more-compact framework with less
porosity, which limited the migration of carvacrol compounds through
the film. This decrease in porosity limited carvacrol release into the
surrounding medium, decreasing the observed carvacrol flow (Rehman
et al., 2014). Interrupting the phospholipid bilayer structure that causes
leakage of cell contents can significantly contribute to inhibition of
overall cell growth (Wijesundara et al., 2022).
Table 1
Antimicrobial ability of agar/FBC films with carvacrol.
Film with carvacrol* Average inhibition zone (mm)
Escherichia coli Staphylococcus aureus
BC 26.67 ± 8.08ab 42 ± 2a
FBC 22.67 ± 6.11bc 33 ± 2bc
0.5% agar/FBC 22.67 ± 2.52bc 26.33 ± 3.21b
1% agar/FBC 22.33 ± 3.79bc 26.67 ± 3.06b
1.5% agar/FBC 31.67 ± 4.93ab 36.33 ± 3.21acd
2% agar/FBC 29.67 ± 1.53ab 30.67 ± 5.03bd
*
The concentration of carvacrol was 10 mg/mL. Data are expressed as the
mean±standard deviation (n = 3). Values in a row with different superscript
letters significantly differ (p < 0.05).
6
Food Packaging and Shelf Life 42 (2024) 101269
3.4. BSA/carvacrol release measurement
It is crucial to investigate food simulations in active packaging
development to achieve the gradual, controlled release of active com­
pounds (Adilah et al., 2018) The release of additives can be influenced
by the film composition and structure. As shown in Fig. 3, we used two
different additives of carvacrol as an essential oil for food packaging and
BSA to mimic enzymes or complex molecules. In addition, we used
different types of solvents with these two components.
Fig. 3a provides release measurements using BSA protein as the
facilitator. Giving the same results, a higher agar concentration pro­
duced a higher protein concentration released from the film. As can be
seen at 36 h, amounts of protein released were 134.47, 139, 158.16,
172.3, 169.8, and 173.58 µg/mL for the normal BC, FBC, 0.5% agar/
FBC, 1% agar/FBC, 1.5% agar/FBC, and 2% agar/FBC groups, respec­
tively. Higher agar concentrations increased the film’s water-holding
capacity, facilitating the release of hydrophilic proteins. The increased
water content enhanced protein solubility, resulting in a higher con­
centration of proteins released from the film. Particles such as protein
molecules can interact with packaging materials through processes like
adsorption, absorption, or migration (Chen et al., 2023). On the other
hand, Fig. 3b shows that released concentrations of carvacrol were
around 32.34, 93.07, 132.84, 126.29, 191.6, and 250.8 µg/mL for the
BC, FBC, 0.5% agar/FBC, 1% agar/FBC, 1.5% agar/FBC, and 2%
agar/FBC groups, respectively, with 12 h of incubation. It was found
that higher agar addition contributed to a higher carvacrol concentra­
tion released from the film. With the same concept in the previous
antimicrobial analysis, higher agar concentrations increased water
retention in the film (Arham et al., 2016), which enhanced carvacrol
solubility and promoted its release. Additionally, increased agar con­
centrations caused greater film swelling upon contact with a liquid
medium, creating more space and facilitating carvacrol release. How­
ever, 2% agar addition provided faster release of carvacrol, resulting in
lower concentrations which had decreased to 208.89 and 172.59 µg/mL
by 24 and 36 h, respectively.
3.5. Active food packaging application in sea bass
3.5.1. TVC analysis
The TVC analysis is important because it helps determine the mi­
crobial contamination level of samples. Theoretically, the amount of
bacterial growth increases as the storage time increases (Sarika et al.,
2012). This result was also similar to our experiment. As can be seen in
Table 2, the number of bacteria increased from days 0 to 9. The amount
of bacterial growth in the control group (with bacteria inoculation but
no packaging treatment) was 5.97 log10 CFU/mL on day 0 and increased
to 8.98, 11.49, and 15.25 log10 CFU/mL on days 3, 6, and 9, respec­
tively. These results also had the same traits in every group such as that
of S. putrefaciens with film, and that of S. putrefaciens with film and
carvacrol. However, from days 3 to 6, S. putrefaciens with film and
carvacrol had the lowest microbial counts followed by the control and
S. putrefaciens with film. As seen on day 0, the microbial count was
4.99 log10 CFU/mL and increased to 6.7 and 10.08 log10 CFU/mL on
days 3 and 6, respectively. These results proved that the addition of
carvacrol to 1.5% agar/FBC film significantly inhibited bacterial
growth, resulting in lower microbial counts (Mastromatteo et al., 2009).
Interestingly, the antimicrobial ability of carvacrol seemed to have
slightly decreased after day 9. This may have been due to the volatility of
essential oils caused by carvacrol released from the film during storage.
Typical TVC values ranged 102 to 106 CFU/mL, which are considered
safe, and higher levels of ≥ 107 CFU/mL are typically linked to sensory
rejection, indicating spoiled or degraded fish (Esteves & Aníbal, 2021).
On day 9 and later of storage time, almost all of the results had no sig­
nificant difference due to the limited serial dilution (10− 11 serial dilu­
tion is still considered high microbial growth). Thus, the number of
bacteria was too numerous to count (TNTC), and samples were
A.C. Kusuma et al. Food Packaging and Shelf Life 42 (2024) 101269

Fig. 3. Measurement of (a) BSA release and (b) carvacrol release.

exhibited significant differences in results between days 0 and 3. How­

Table 2
ever, from days 6 to 12, they all had the same traits of carvacrol
TVC analysis from different groups in sea bass fish filet for 12 days of storage.
exhibiting the lowest and the S. putrefaciens group the highest levels.
Distinct letters (p < 0.05) indicate the presence of significantly different values.
Carvacrol addition to the 1.5% agar/FBC film showed a significant dif­
Group Total viable count (log10 CFU/mL) ference in TBARS results with amounts of around 3.38, 5.22, and
day 0 day 3 day 6 day 9 day 5.99 µg/mL, respectively. In addition, the group with added bacteria
12 reached the highest TBARS results which were around 6.08, 6.69, and
Control 5.97 8.98 11.495 15.25 TNTC 7.52 µg/mL, with no significant difference from each other. These re­
± 0.06b ± 0.08b ± 0.28a ± 0.04b sults showed that carvacrol addition to the film contributed to a sig­
Agar/FBC 5.86 7.82 11.128 15.04 TNTC
nificant deceleration of lipid peroxidation leading to a prolongation of
± 0.12b ± 0.03c ± 0.3a ± 0.05a
Agar/FBC- 4.99 6.70 10.08 14.96 TNTC
the shelf life of fish to prevent spoilage.
Carvacrol ± 0.59c ± 0.2d ± 0.36b ± 0.03a
3.5.3. TVBN analysis
CFU, colony-forming units; the conc. of Agar/FBC is 1.5%; TNTC, too numerous
The total amount of volatile nitrogenous compounds found in a food
to count. Data are expressed as the mean±standard deviation (n = 3).
sample mainly originates from the decomposition of proteins and amino
acids, particularly when microbial spoilage occurs. When fish or seafood
considered to have spoiled.
spoils, the protein contents degrade, leading to the emission of these
volatile nitrogenous compounds (Zhuang, Hong, Zhang, & Luo, 2021).
3.5.2. TBARS analysis
Idakwo et al. (2016) provided a suggested range of 20 to 30 mg N/100 g
In food spoilage analysis, TBARSs are used to assess the extent of
as an acceptable limit of TVBNs for fish with a higher upper limit of 30 to
lipid peroxidation in samples. This analysis is predominantly employed
40 mg N/100 g. Fig. 4b provides the change in TVBN of sea bass from
to assess the extent of secondary lipid oxidation, serving as a common
days 0 to 12 of storage. On days 0 and 3, there were no significant dif­
method to evaluate the formation of malondialdehyde (MDA) during the
ferences in results in any groups. However, after storage for 3 days, sea
oxidation process (L. Wang et al., 2023). In all groups in Fig. 4a, as the
bass fillets began to reach the upper limit of TVBN in all groups, which
storage time increased, TBARS amounts also increased. Although there
indicated poor quality or spoilage of the fish. On days 6 and 9 of storage
might have been a slight decrease in some treatments, the results still
time, the group with carvacrol addition to 1.5% agar/FBC film
revealed no significant differences at different storage times. No groups

Fig. 4. TVB-N (a) and TBARS (b) values of sea bass fillets wrapped in different sample groups. Different letters on the error bars indicate a significant difference
(p < 0.05) relative to the control.

7
A.C. Kusuma et al.
maintained TVBN amounts which were around 95.07 and 90.59 TVBN
mg/100 g, respectively. Moreover, carvacrol addition also showed a
significant difference in TVBN results among the other groups on day 9
and even on day 12 of storage time. From these results, we might
conclude that 1.5% agar/FBC film can provide a good release of
carvacrol from day 6 on.
4. Conclusions
Optimal proportions for producing FBC with a maximum dry weight
were a 12.5% carbon source and a 0.5% nitrogen source. FBC was
investigated with different agar concentrations of 0.5% to 2%. FTIR,
TGA, and SEM analyses revealed that additives were integrated into
FBC, shown by new peaks and changes in the main peak. Agar incor­
poration reduced the crystallinity, increased the water content, and
improved the swelling ratio. Agar/FBC with carvacrol met the criteria
for active food packaging, particularly 1.5% agar/FBC displayed strong
antimicrobial properties against E. coli (31.67 mm) and S. aureus
(36.33 mm). Moreover, 1.5% agar/FBC exhibited gradual carvacrol
release. This material is suitable for fish food packaging. Using TVC,
TVBN, and TBARS analyses, fish freshness was evaluated. Carvacrol in
1.5% agar/FBC inhibited microbial growth on days 3 and 6. It mini­
mized nitrogen compounds from protein decomposition after day 6.
Carvacrol in 1.5% agar/FBC slowed lipid peroxidation from day 6 on­
ward. This validates 1.5% agar/FBC with carvacrol as effective active
food packaging for sea bass fillets. However, the limitation of Agar/FBC
lies in the inability to control the generation of larger pore sizes through
agar addition. Future work will involve creating agar nanoparticles and
carvacrol-loaded particles to control FBC pore sizes and reduce carvacrol
evaporation. Exploring diverse additives and food samples to enhance
agar/FBC properties is important. Scaling up production while main­
taining quality and reducing costs is a forthcoming challenge in the
industry.
CRediT authorship contribution statement
Alchris Woo Go: Methodology. Chen-Che Hsieh: Investigation,
Methodology. Shella Permatasari Santoso: Methodology. Anita
Chandra Kusuma: Data curation, Methodology, Writing – original
draft. Yu-Chieh Chou: Methodology. Shin-Ping Lin: Conceptualiza­
tion, Funding acquisition, Methodology, Supervision, Writing – original
draft, Writing – review & editing. Hong-Ting Victor Lin: Methodology,
Supervision. I-Lun Hsiao: Methodology.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
This work was financially supported by Taipei Medical University
and the Ministry of Science and Technology, Taiwan, through a research
grant with contract no. MOST110-2221-E-038-003-MY3. The authors
acknowledge the academic and science graphic illustration services
provided by the TMU Office of Research and Development.
8
Food Packaging and Shelf Life 42 (2024) 101269
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