Food Packaging and Shelf Life 23 (2020) 100439

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Food Packaging and Shelf Life

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Fabrication of high stability active nanofibers encapsulated with T

pomegranate peel extract using chitosan/PEO for meat preservation
Duraiarasan Surendhirana, Changzhu Lib, Haiying Cuia,*, Lin Lina,b,*
a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
b
Department of Bioresource, Hunan Academy of Forestry, Changsha 410007, China

A R T I C LE I N FO A B S T R A C T

Keywords: Pomegranate peel extract (PE) immobilized electrospun active nanofibers was fabricated using Chitosan/Poly
Chitosan (ethylene oxide) (CS/PEO). Various blending ratio of 4 % (w/v) CS/PEO as 00/100, 50/50, 60/40, 70/30 and
E.coli O157:H7 80/20 with PE was added and electrospun. The average diameter of nanofibers ranged between 227 and 406 nm.
Punica granatum The beaded nanofibers were attained for the blends of 70/30/PE and 80/20/PE due to high content of CS which
Electrospinning
increased its viscosity. Addition of Pluronic F-127 (1 v%) notably reduced the beads in nanofibers which was
Nanofibers
confirmed by SEM. The nanofibers were characterized by FTIR and tensile strength, elongation at break, swelling
rate and TGA/DSC were analyzed. The active nanofibers strongly preserved beef by reducing the population of
E.coli O157:H7 up to 2.96 and 5.80 log CFU/g at 4 and 25 °C respectively. In conclusion, nanofibers showed
brilliant physical, mechanical and thermal properties that exhibited excellent potential for food packaging ap-
plications.

1. Introduction failure (Miryala & Ramaiah, 2018).

Meat and its products, especially beef, is one of the main nutrient
food resources in human diet worldwide. Beef acts as an ideal medium
for food pathogens due to high level of proteins, fat and water, thereby
resulting in spoilage and subsequent food borne diseases (Lin, Gu, &
Cui, 2018). Microbial food spoilage is not only a risk factor for human
life but also a concern over huge economic loss for food industries in-
volved in the manufacture of packed foods (Bondi, Lauková, de
Niederhausern, Messi, & Papadopoulou, 2017; Lin, Liao, & Cui, 2019).
For instance, recently in USA, Cargill Meat Solutions, one of the pio-
neering meat processing industries has recalled about 25,288 pounds of
beef products on August 22, 2018 due to the contamination of E.coli
O157:H7 (https://www.fsis.usda.gov/wps/portal/fsis/topics). E.coli
O157:H7 is one of the most virulent food pathogens that causes
265,000 sicknesses and 30 death cases each year in USA (https://
www.cdc.gov) and is a big challenge to the meat industries since it
easily contaminates meat during butchering and packaging (Miryala &
Ramaiah, 2018). The pathogenicity of E.coli O157:H7 is due to secre-
tion of Shiga toxin (Stx) and biofilm formation which shows high re-
sistance to many antibiotics (Cui, Bai, Yuan, Surendhiran, & Lin, 2018;
Miryala & Ramaiah, 2018). E.coli O157:H7 causes enteric infection with
symptoms of bloody diarrhea, abdominal pain, fever and, in severe
cases, renal failure and hemolytic uremic syndrome leading to kidney
Toxic nature of synthetic chemicals as preservatives motivated re-
searchers to explore isolation of natural alternatives from plant sources
(Nazari et al., 2019; Jridi et al., 2019; Wang, Lim, Tong, & Thian,
2019). Punica granatum (Pomegranate) belongs to Punicaceae family
was recently termed as nature’s power fruit due to its wide and sig-
nificant biological activities including acting as antioxidant, anti-dia-
betic, anti-obesity, anti-hypersensitive, anti-mutagenic, anticancer,
anti-inflammatory and broad spectrum antiviral along with its anti-
bacterial properties. In Chinese and Indian traditional medicines po-
megranate are used to treat intestinal disorders like diarrhea, dysen-
tery, gastritis and ulcers (Alexandre et al., 2019; Kharchoufi et al.,
2018). Recent reports suggests that its extract could work against sev-
eral food pathogens and can be used as natural food preservatives
(Licciardello, Kharchoufi, Muratore, & Restuccia, 2018; Pagliarulo
et al., 2016; Wafa et al., 2017). However, appropriate carrier is required
to deliver the natural bioactive compounds through controlled release
on food surfaces which are prime sites of infection. Encapsulation of
natural antimicrobial agents using electrospun nanofibers which is one
of the most popular and attractive methods due to its large surface area
and excellent physical and mechanical properties (Tu et al., 2019).
Moreover, electrospinning process is very simple, consumes less cost,
more flexible, possible to operate in commercial scale, heat labile
compounds can be encapsulated since the equipment runs at ambient

⁎
Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China.
E-mail addresses: cuihaiying@ujs.edu.cn (H. Cui), linl@ujs.edu.cn (L. Lin).

https://doi.org/10.1016/j.fpsl.2019.100439
Received 15 March 2019; Received in revised form 6 November 2019; Accepted 12 November 2019
Available online 19 November 2019
2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
D. Surendhiran, et al.
conditions and will able to spin several biopolymers blends with that of
the synthetic polymers (Zhu, Cui, Li, & Lin, 2019; Cui, Bai, Rashed, &
Lin, 2018; Lin, Gu, & Cui, 2019; Wen, Wen, Zong, Linhardt, & Wu,
2017).
Chitosan (CS) is a cationic polysaccharide obtained from crustacean
shells or some fungi and has incredible applications because of its
biocompatibility and biodegradability (Haiying Cui, Wu, Li, & Lin,
2017). In addition, it showed a good antimicrobial activity, excellent
film capacity and gas and aroma barrier property (Lin, Xue,
Duraiarasan, & Haiying, 2018; Tripathi, Mehrotra, & Dutta, 2009)
which makes it an ideal candidate to fabricate active nanofibers for
food packaging applications. However, electrospinning of biopolymer
alone is still quite challenging due to the formation of gel via hydrogen
bond at the tip of the needle. Hence, in this study, a water soluble and
biodegradable synthetic polymer called Poly(ethylene oxide) (PEO) was
blended with CS to facilitate its spinnability.
To the best of our knowledge no report has been published on en-
capsulation of pomegranate peel extract (PE) using electrospun nano-
fibers for food preservation applications. In this sense, the aim of this
study was to fabricate active nanofibers using CS/PEO blends with
immobilized PE to inhibit the proliferation of E.coli O157:H7 and the
prepared nanofibers were analyzed for its preservation capacity on
meat at various temperature and time.
2. Materials and methods
2.1. Materials and bacterial culture
Gallic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), Poly(ethylene
oxide) (PEO) (Mv ∼900,000) and chitosan (molecular weight
100,000–300,000 Da) were purchased from Sigma-Aldrich (Saint Louis,
USA). Ascorbic acid was purchased from Zhengzhou Bainafo
Bioengineering Co., Ltd. (Henan, China). E.coli O157:H7 was obtained
from China Center of Industrial Culture Collection (Beijing, China) and
was maintained at 4 °C prior to use, subcultured in nutrient broth and
incubated for 24 h at 37 °C in an orbital shaker. The test meat, beef, was
purchased from local market.
2.2. Pomegranate peel extraction
Pomegranate was purchased from supermarket located in the
Jiangsu University campus. The pomegranate peels were separated
manually, cut, dried at 50 °C for 12 h and was powdered. The ground
material was passed via 60 mesh sieve and treated with methanol to
peel ratio of 10:1 for 72 h at room temperature in shaker. Then the
extracts were centrifuged at 3500 rpm for 10 min. The supernatant was
then collected and filtered via Whatman No.1. Finally, the filtrate was
concentrated using rotary evaporator (R206, SENCO, China) at 40 °C,
freeze dried using a lyophilizer (FD-1A-80, YMNL, China) and stored at
4 °C for further analysis.
2.3. Biological activities of pomegranate peel extract (PE)
2.3.1. Determination of total phenolic content (TPC) and antioxidant
activity
TPC of PE was estimated according to Yehia, Elkhadragy, and Abdel
Moneim (2011)) using Folin–Ciocalteau reagent. In brief, 1 mL of 10 %
Folin–Ciocalteau reagent was added to 0.4 mL of PE (1 mg/mL in me-
thanol) in a test tube and allowed for 5−8 min at room temperature.
Then 0.8 mL of Na2CO3 (7.5 %) was added along with distilled water to
the mixer to bring it to 10 mL. After 2 h, absorbance was measured at
765 nm. The TPC was calculated and expressed as mg gallic acid (GA)
equivalents/g sample using GA standard curve.
The total antioxidant activity was determined by radical scavenging
activity (%) using 2, 2- Diphenyl-1-picrylhydrazyl (DPPH) (Pagliarulo
et al., 2016). In brief, different concentrations of working stock of PE
2
Food Packaging and Shelf Life 23 (2020) 100439
were prepared (10–100 μg/mL with an interval of 10) and 1 mL of
0.16 mM DPPH methanolic solution was added into each concentration.
The mixture was then vortexed for 1 min and left to stand at room
temperature for 30 min under dark conditions, and the absorbance was
read at 517 nm using UV-Spectrophotometer (UV-4100, Shimadzu
Corporation, Tokyo, Japan). The following equation was used to cal-
culate the antioxidant activity of PE:
Antioxidant activity (%) = 1-(Asample ˗ Asample blank/Acontrol) × 100
(1)
Where, Acontrol is the absorbance of the control, Asample is the absor-
bance of the test sample (PE), and Asample blank is the absorbance of the
sample only (sample without DPPH solution). Ascorbic acid was used as
the positive control.
2.3.2. Determination of minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) of PE
Different concentrations of PE were prepared (5−50 mg/mL) in test
tubes containing sterile nutrient broth. Then the test bacterial culture
E.coli O157:H7 (105-6 CFU/mL) was inoculated into each test tube and
incubated at 37 °C for 24 h in shaker incubator. The lowest concentra-
tion of PE showing no visible growth of bacteria was recorded as MIC
and MBC was determined as the lowest concentration of PE showing no
bacterial colonies on agar plate.
2.3.3. Time-kill curve of PE
Time kill curve assay was performed to check rate of killing of E.coli
O157:H7 by plate colony counting method (Lin, Xue et al., 2018). In
brief, MIC and MBC concentrations of PE were added into the test tube
with nutrient broth containing 106 CFU/mL test bacterium and in-
cubated. During incubation at different time intervals (0, 2, 4, 8, 12 and
24), 100 μL of sample was taken from the culture tube and serially di-
luted and plated to calculate CFU/mL. The killing rate curve was de-
termined by plotting CFU/mL against incubated time.
2.4. Preparation of electrospinning solutions and characterization
Electrospinning solution was prepared by dissolving 4 % (w/v) of
CS and PEO separately using 2 % acetic acid (v/v). Then, CS and PEO at
different blending ratios of 50/50, 60/40, 70/30 and 80/20 were pre-
pared (total volume 20 mL) and kept under constant stirring at room
temperature for 24 h to get homogenous solutions. PE (MBC, mg/mL)
was added into the blending solution which was corresponding to the
total volume. The electrical conductivity was measured by a portable
multi-parameter analyzer (DZS-718, Shanghai Precision Science
Instrument Co., Ltd, China). Viscosity of different concentrations of
blending solutions was measured by a rotational rheometer (DHR-1,
Waters Technologies Co., Ltd, China) with plate-plate sensor. For
measurement, the shear rate range varied between 0.1-200 s-1.
2.5. Fabrication of CS/PEO/PE nanofibers
The active nanofiber was fabricated using lab scale electrospinning
machine (SNAN-01, Electrospinning Setup, MECC Co., Ltd., Fukuoka,
Japan). Each prepared solution was pumped using 5 mL syringe into a
20 gauge needle with a tip of 0.50 mm internal diameter. The flow rate,
applied voltage and the distance between the needle and the collector
were 0.5 mL/h, 20 kV and 15 cm, respectively. The fabricated nanofi-
bers were collected on aluminum foil placed on the collector.
2.6. Physicochemical and mechanical properties of nanofibers
2.6.1. Scanning electron microscopy (SEM) analysis of nanofibers
The morphological features and size of nanofibers were analyzed by
Scanning Electronic Microscope (SEM) (Model-JSM-7001 F, JEOL,
D. Surendhiran, et al.
Japan) with an acceleration voltage of 10 kV for nanofibers from blends
and pure PEO. The mean diameter of nanofibers was calculated by
measuring random points using ImageJ software.
2.6.2. Fourier transform infrared (FTIR) spectroscopy
Functional groups and the molecular interactions of CS/PEO/PE
nanofiber were analyzed by FTIR (Thermo Nicolet Corporation, USA).
The dried nanofibers were mixed with KBr powder was pressed to a disc
using a hydraulic press and then subjected to FTIR spectral measure-
ment with the wavelength ranging between 4000 and 400 cm-1. The
used resolution and number of scans were 4 and 32 respectively.
2.6.3. Analysis of nanofiber moisture content
To determine the moisture content, the known quantity of nanofiber
mat was weighed before and after drying in an oven at 120 °C until the
equilibrium weight was achieved. Each experiment was carried out in
triplicate to get exact results. The final moisture content of nanofibers
was calculated by the following equation (Lin, Gu et al., 2018).
M0 − Mi
Moisture content= × 100%
M0 (2)
Where, M0 is the mass of initial sample (mg) and M1 is the mass of dried
sample (mg).
2.6.4. Analysis of swelling property
Swelling ability of nanofibers was measured by immersing the pre-
weighed nanofibers in distilled water for 24 h. Then the excess water
was removed by blotting with filter paper and weighed. The swelling
rate was calculated by the following formula, where Sw is the swollen
weight and Dw is initial dry weight of nanofibers.
S w − Dw
Swelling property (%) = × 100
Dw (3)
2.6.5. Water solubility test
The dried nanofibers were weighed (W1) and immersed in distilled
water for 24 h under shaking condition. Then the undissolved nanofi-
bers was separated and dried in vacuum oven at room temperature for
24 h to attain stable weight. Subsequently the dried nanofibers were
weighed (W2). The water solubility of each nanofiber was calculated by
the following equation (Lin, Gu et al., 2018):
Water solubility (%) = (W1-W2)/W1 × 100 (4)
2.6.6. Tensile strength and elongation at break
Stability of the fabricated nanofibers were assessed by analyzing the
mechanical behavior of the nanofibers namely the tensile strength (TS)
and elongation at break (EAB) using a TA-XT2i Texture Analyser (Stable
Microsystems, UK) by the ASTM method (ASTM, 2001). The fibers were
cut into 80 mm x 20 mm long and were fixed between the grips with an
initial separation of 50 mm. Tensile load was set to 20 g and fibers were
stretched to elongate at a constant speed of 0.5 mm/s until the fibers
broke apart. TS were calculated by the following equation:
F
TS=
L× M (5)
Where, F is the axial tensile force (N), M is the membrane thickness
(mm), L is the width of the membrane (mm).
EAB was expressed as percent of the initial height as follows:
H−H0
EAB= × 100%
H0 (6)
Where, H0 and H are the initial and final height of membrane after
deformation, respectively.
3
Food Packaging and Shelf Life 23 (2020) 100439
2.7. Thermal analysis of CS/PEO/PE nanofibers
Weight loss and thermal stability was conducted by
Thermogravimetric analyzer (TGA) and Differential scanning calori-
metry (DSC) using STA 449 F3 Netzsch apparatus (Germany). The
temperatures maintained between 25 and 500 °C with a heating rate of
10 °C/min with a flow rate of nitrogen at 20 mL/min.
2.8. Application of CS/PEO/PE active nanofibers to preserve beef
Meat preservation experiment was carried out according to Nychas,
Manohar, Prabhawathi, Sivakumar, and Doble (2015)) with slight
modification. The fresh beef was cut into small pieces weighing 1 g,
dipped into E.coli O157:H7 culture (103-4) and kept for few minutes.
Then samples were wrapped in aluminium foil consisting of CS/PEO/PE
active nanofibers and tested against control without nanofibers. One set
of wrapped beef samples were stored at 4 °C for 10 days and another set
of samples were stored at 25 °C for 7 days. Following the preservation
period, the beef samples were taken and homogenized in sterile dis-
tilled water. After serial dilution, about 0.1 mL of the suspension was
transferred onto the nutrient agar, spread and incubated at 37 °C for
24 h. After incubation, number of viable colonies were recorded by
plate colony counting method and expressed as colony forming units
(CFU/g of beef). Finally, the sensory evaluation of preserved raw beef
was then evaluated as described by Sani, Ehsani, and Hashemi (2017)).
2.9. Statistical analysis
All experiments were performed in triplicates and the results were
evaluated using one way ANOVA. P value < 0.05 was considered as
statistically significant.
3. Results and discussions
3.1. Total phenolic content and antioxidant activity of PE
Total phenolic content (TPC) of PE was investigated using methanol
was found to be 346.24 ± 3.1 mg GAE/g with an antioxidant activity
of 72.64 ± 0.06 %. Several literatures revealed that methanol is the
most efficient solvent to extract polyphenols from plant materials
showing highest DPPH scavenging activity (Arun, Jayamurthy, Anusha,
Mahesh, & Nisha, 2017; Kaderides, Goula, & Adamopoulos, 2015).
3.2. Antibacterial activity and time killing analysis of PE
In this study, E.coli O157:H7 was selected due to its ability to cause
food spoilage even at refrigerated conditions which is a major concern
globally (Gullon, Pintado, Pérez-Álvarez, & Viuda-Martos, 2016).
Minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of PE was found to be 15 and 20 mg/mL respec-
tively. However, different MIC and MBC values have been recorded in
the literatures from different researchers for PE against bacteria. For
instance, Voravuthikunchai et al. (2004) reported the MIC and MBC
value of PE against E.coli O157:H7 as 0.39 and 3.12 mg/mL. In another
study done by Navarro, Villarreal, Rojas, and Lozoya (1996)) and re-
corded MIC value of 10 mg/mL against E.coli ATCC with methanolic
extract of PE. Recently, Gullon et al. (2016) recorded the MIC and MBC
value of PE against E.coli as 50 and 60 mg/mL. These differences in the
antibacterial activity of PE against same group of bacteria might be
different cultivars of pomegranate, cultivated in different seasons at
different locations, variations in phenolic content of extracts and
methods of extraction (Al-Zoreky, 2009; Gullon et al., 2016).
Time kill study was also performed to determine the time required
for PE to deteriorate and destroy E.coli O157:H7 completely. As shown
in Fig. 1, viable cells of E.coli O157:H7 started decreasing progressively
from 6.62 to 2.86 log CFU/mL at 8th h of incubation time and further
D. Surendhiran, et al.
Fig. 1. Time-kill analysis of PE on E.coli O157:H7.
reduced the bacterial population to 1.24 log CFU/mL at 24th h when
the cells treated with MIC value (15 mg/mL) showing bacteriostatic. At
the same time, the PE concentration was increased to 20 mg/mL (MBC),
complete elimination of E.coli O157:H7 was detected after 8th h of
incubation time and obtained 99.9999 % reduction in population
(CFUmL) due to the bactericidal effects. Whereas, in the control, no
bacterial growth inhibition was observed instead growth was constantly
increasing (CFU/mL).
This finding confirmed the significant antimicrobial activity of PE
on the growth rate of surviving E.coli O157:H7 and revealed that con-
centration of the natural bioactive compounds and treatment time en-
compasses great influence on antimicrobial effects. Many researchers
reported that PE effectively killing both Gram positive and negative
bacteria due to its broad spectrum antibacterial activity. The presence
of tannins, such as anthocyanins and polyphenols, especially punica-
lagin and ellagic acid, found in PE has been reported as the main factor
behind its antibacterial activity (Rosas‐Burgos et al., 2017; Gullon et al.,
2016). The mechanism of action of PE takes place probably due to the
interactions of OH group in phenolic compounds with bacterial cell
membrane; this may induce membrane instability causing leakage of
cytoplasmic inclusions which ultimately leads to cell death (Alexandre
et al., 2019; Gullon et al., 2016; Mohammed, Hassanien, El, Afify, &
Mohammed, 2016).
3.3. Characterization of electrospinning polymer blends
Viscosity and electrical conductivity of the polymer blends are very
crucial parameters of electrospinning process to obtain high-quality of
nanofibers (Serodio et al., 2019). Viscosity and electrical conductivity
of different electrospun blending solutions of CS/PEO/PE are shown in
Table 1. CS alone cannot be electrospun due to strong hydrogen bond
between NH2 and OH making them rigid and highly viscous (Yuan,
Jenkins, DiGeorge Foushee, Jockheck-Clark, & Stahl, 2016). As shown
in the Table 1 pure CS solution showed higher viscosity (731.4 ± 2.2,
mPas) than that of PEO blended CS. Addition of PEO favored the
electrospinning process by reducing the viscosity (Safi, Morshed,
Hosseini Ravandi, & Ghiaci, 2007; Yuan et al., 2016) which is attributed
to the change in inter and intramolecular interactions of CS chains. PEO
molecules bound onto CS backbone disrupt the self-association of
Table 1
Different blending ratio of CS/PEO/PE and their viscosity and electrical conductivity.
Parameters Different ratios of electrospun solution CS/PEO(v/v) with 500 mg of PE (w/v)
100/00 00/100
a b
Viscosity (mPas) 731.4 ± 2.2 189.0 ± 1.6
Electrical conductivity (mS/cm) 4.8 ± 0.1a 1.2 ± 0.2b
a–e
with different lowercase letters in the same row are significantly different (p < 0.05).
Food Packaging and Shelf Life 23 (2020) 100439
chitosan chains forming additional hydrogen bonds between its hy-
droxyl groups and water molecules. However, when reducing PEO as
60/40/PE, 70/30/PE and 80/20/PE in blends, it resulted in increased
viscosity as 572.1 ± 1.3, 601.3 ± 2.1 and 644.0 ± 2.6 (mPas) re-
spectively, which corroborated well with the earlier report by Safi et al.
(2007). Polymers with very less viscosity are not entangled, easily drop
out of needle and shun formation of jets occur, while high viscosity
hinders the spinning ability of polymers producing beads (Amariei,
Manea, Bertea, Bertea, & Popa, 2017).
In addition, electrical conductivity during electrospinning is crucial
which controls the tendency to form nanofibers by increasing the
elongation capacity of the blends and does not produce sprayed nano-
drops. The blends devoid of conductivity cannot be electrospun
(Amariei et al., 2017). The maximum electrical conductivity of blends
was recorded as 4.8 ± 0.1 mS/cm for pure CS since it was cationic and
lowest conductivity was observed for pure PEO with 1.2 ± 0.2 mS/cm
followed by 50/50/00 (CS/PEO/PE) with 2.7 ± 0.1 mS/cm. Electrical
conductivity of blending solutions was decreased by increasing PEO
ratio as reported by Saquing et al. (2013). Moreover, higher con-
ductivity leads to more electrostatic repulsive force, which could hinder
electrospinning process and inhibit the fiber formation (Lin, Gu et al.,
2018; Saquing et al., 2013). Therefore, fabricating fine nanofibers,
viscosity and electrical conductivity ought to be optimized.
3.4. SEM analysis of nanofiber
The morphology of fabricated nanofibers is represented in Fig. 2.
Pure PEO nanofiber was prepared as control and compared with fibers
produced from CS/PEO/PE blends. As shown in Fig. 2A, the PEO na-
nofibers appeared as thin and uniform thread like fibers without any
beads with size distribution ranging between 100−400 nm and mean
diameter as 227 ± 16 nm (Fig. 2A1). However, the diameter of na-
nofibers showed an increase upon blending CS and PE (50/50 and 50/
50/PE) (Fig. 2B,C), with average diameters of 253 ± 23 and
306 ± 21 nm respectively (Fig. 2B1,C1). The increase in fiber diameter
after encapsulation of natural antimicrobial agents within electrospun
fibers had been documented earlier (Lin, Xue et al., 2018). It is to be
noted that the average diameter of 60/40/PE blend was found to be
increased substantially to 347 ± 23 nm (Fig. 2D,D1). However, when
CS was increased to 70/30/PE and 80/20/PE, uneven beaded nanofi-
bers were obtained (Fig. 2 E,F), which could be ascribed to the increase
in viscosity. This finding was in agreement with Safi et al. (2007), who
obtained uniform fibers with no beads by increasing the content of
synthetic polymer PVA to sodium alginate. Hence, it is inferred that the
reduced CS content yielded uniform beads electrospun nanofibers with
average diameter. Also FDA approved surfactants could be used for
creating smooth nanofibers. Pluronic F-127, is one such choice due to
its non-toxic nature unlike Triton-X 100 (Bonino et al., 2011). As shown
in SEM images, Fig. 2G,H, addition of Pluronic F-127 (1 v%) effectively
reduced the beads in the nanofibers from blends of 70/30/PE and 80/
20/PE (CS/PEO/PE) with average diameter of 370 ± 26 nm (Fig. 2G1)
and 406 ± 15 nm respectively (Fig. 2H1). Uniform and unbeaded na-
nofibers were due to decrease in viscosity and surface tension after
adding Pluronic F-127 (Bonino et al., 2011).
50:50 50:50/PE 60/40/PE 70/30/PE 80/20/PE
c cd d e
512.6 ± 2.1 524.6 ± 2.1 572.1 ± 1.3 601.3 ± 2.1 644.0 ± 2.6e
2.4 ± 0.1c 2.7 ± 0.1cd 3.1 ± 0.1d 3.4 ± 0.2de 3.8 ± 0.4de
4
D. Surendhiran, et al. Food Packaging and Shelf Life 23 (2020) 100439

Fig. 2. SEM images of nanofibers with histograms of diameter at different ratio of CS/PEO and CS/PEO/PE. (A) Pure PEO; (B) 50/50; (C) 50/50/PE; (D) 60/40/PE;
(E) 70/30/PE; (F) 80/20/PE; (G) 70/30/PE + 1 % Pluronic F-127; (H) 80/20/PE + 1 % Pluronic F-127.

5
D. Surendhiran, et al.
Fig. 3. FTIR spectra of pure PE, CS, PEO and CS/PEO/PE nanofibers.
3.5. FTIR analysis
The molecular interactions of CS, PEO and PE in the nanofibers were
confirmed by FTIR study by monitoring perturbations in their re-
spective functional groups (Fig. 3). The observation of strong peaks at
1644 and 2873 cm-1 for CS corresponds to the stretching vibration of
C]O and C–H groups respectively. The bands situated at 1154, 1103
and 1065 cm-1 are ascribed to the CeOeC groups in CS while the peaks
at 1362, 1342 and 1065 cm-1 revealed CH deformation of methyl group
(Chen, Xin, Saha, & Kim, 2017). The wide band appeared at 3346 cm-1
belonged to stretching vibration of NeH and OeH groups and the peak
positioned between 1046 to 1800 cm-1 could be attributed to the pre-
sence of some aliphatic and aromatic functional groups of phenolic
compounds in PE including CH, C]O, eC]CeC]O, eC]Ce and
eCeCe (Shahbazi & Shavisi, 2018). The bands between 2868 and
2935 cm-1 corresponded to asymmetric C–H bond stretching of alkane
molecules and 1116 and 1065 cm-1 were quite visible in PE, CS, PEO
and nanofiber.
3.6. Physical and mechanical properties of nanofibers
Physical properties. The maximum moisture content was 17 ± 0.23
% for pure PEO nanofiber mat and lowest was observed for 80/20/PE
ratio of CS/PEO/PE electrospun blends. It is noted that increasing the
content of CS leads to reduction in moisture content. Moreover, the
polyphenols from PE also added little moisture due to its hydrophilicity.
Swelling behavior of active nanofibers was analyzed by immersing in
distilled water. The maximum swelling rate was found to be
88.34 ± 1.8 % for pure PEO nanofibers and lowest was recorded as
30.1 ± 2.4 % for 80/20/PE (CS/PEO/PE). It was observed that in-
creasing the content of PEO increased water holding or uptake capacity.
Likewise, an increase in CS reduced water uptake as shown in Table 2,
Table 2
Different ratios of CS/PEO/PE and their physical and mechanical properties.
Properties Electrospun blend ratio CS/PEO/PE (v/v/w)
00/100/00 50/50/00
a b
Moisture content (%) 17 ± 0.23 12 ± 0.18
Swelling rate (%) 88.34 ± 1.8a 60.40 ± 1.2b
Water solubility (%) 92.24 ± 0.17a 50.32 ± 0.14b
TS (MPa) 2.40 ± 0.56a 4.31 ± 0.86b
EAB (%) 68.56 ± 2.34f 46.86 ± 2.14b
a–f
with different lowercase letters in the same row are significantly different (p < 0.05).
Food Packaging and Shelf Life 23 (2020) 100439
which was consistent with the previous data published by Jeong et al.
(2010). In addition, higher moisture content also increased swelling
rate (Mendes et al., 2018).
The nanofiber mat prepared from pure PEO solution immediately
shrunk and became transparent when immersed in water, almost
completely solubilized and lost its weight about 92.24 ± 0.17 % in the
water after 24 h incubation under shaking conditions (Table 2). This is
due to high water solubility of PEO. Çay and Miraftab (2013) also ex-
perienced that pure synthetic polymer PVA dissolved and disintegrated
around 94 % after 24 h of incubation. However, the lowest water so-
lubility was recorded as 17.63 ± 0.10 % for the nanofiber fabricated
from 80/20/PE (CS/PEO) blend. It was noted that water solubility
decreased as the CS content in the nanofibers increased. Though, both
PEO and CS are hydrophilic, CS is insoluble in water due to its neutral
property and their ability to form inter-molecular hydrogen bonds un-
like PEO (Martinová & Lubasová, 2008). Therefore, in this study higher
blending ratio of CS/PEO was used in order to enhance the anti-water
property for commercial food packaging applications. Moreover, high
water resistance nanofiber mats would be preferred for food packaging
application in order to reduce the water vapor permeability (Zivanovic,
Li, Michael Davidson, & Kit, 2007) that could minimize the growth of
pathogens which require more water as an ideal medium for their co-
lonization.
3.6.1. Mechanical properties
When the ratio of CS to PEO is increased, the tensile property of the
mat was increased and decreased in elongation at break as shown in
Table 2. The highest TS value was recorded as 10.80 ± 2.2 (MPa) for
80/20/PE (CS/PEO/PE) due to the stiff nature of CS and at the same
time its EAB was reduced to 18.44 ± 2.30. The highest EAB was ob-
tained for pure PEO nanofibers; however, it is highly water sensitive
and not suitable for prolonged meat preservation. Generally EAB is ir-
reversibly proportional to TS i.e. higher TS results in a lower EAB
percentage (El-hadi, 2017). Production of pure CS nanofibers could be
possible by immersing the CS/PEO fibers in methanol overnight to re-
move PEO from the mat. Nevertheless, due to the high rigidity and
brittleness of pure CS nanofibers resulted in poor food wrapping ap-
plication. Hence, biodegradable synthetic co-polymer PEO is allowed to
retain with CS to maintain elongation and flexibility of the nanofiber
mat (Fazli & Shariatinia, 2017). Therefore, CS could improve physical
and mechanical properties and decreased water solubility of PEO which
could produce more flexible nanofiber mats.
3.7. TGA and DSC
Fig. 4 shows TGA and DSC curves of CS, PEO and nanofibers. The
weight loss of CS occurred at two phases; first an endothermic peak at
130 °C due to the moisture loss in CS and second at 260 °C due to the
decomposition of CS (Bizarria, d’Ávila, & Mei, 2014; Tripathi et al.,
2009). For PEO, the thermal degradation started at only 280 °C with
maximum weight loss peak at about 360 °C. Thereafter, it remained
steady until the end of the test at 500 °C. This final stability is possibly
due to the inert environment (nitrogen flow) in which the test was
50/50/PE 60/40/PE 70/30/PE 80/20/PE
c d e
13 ± 0.09 12 ± 0.56b 11 ± 0.27 10 ± 0.33de
62.22 ± 2.0c 53.2 ± 2.6d 39.8 ± 1.4e 30.1 ± 2.4f
51.48 ± 0.11bc 40.15 ± 0.16d 29.18 ± 0.04e 17.63 ± 0.10f
4.06 ± 1.2bc 6.60 ± 1.7d 8.20 ± 1.24e 10.80 ± 2.2f
47.08 ± 2.3bc 30.6 ± 1.86d 21.30 ± 2.26e 17.44 ± 2.30a
6
D. Surendhiran, et al. Food Packaging and Shelf Life 23 (2020) 100439

Fig. 4. Typical curves illustrating the effect of

CS/PEO blending on thermal properties of na-
nofibers at the temperature range from 25 to
500 °C with heating rate of 10 °C/min. (A) TGA
thermograms derived weight loss (%/°C) of CS,
PEO and CS/PEO/PE nanofibers; (B) DSC
thermograms showing endothermic peaks and
heat flow (mW/mg) of CS, PEO and CS/PEO/
PE nanofibers.

Fig. 5. Antimicrobial effect and preservation capacity of CS/PEO nanofibers containing PE against E.coli O157:H7 in raw beef by CFU counting method stored at 4 °C
(A) and 25 °C (B) for 10 and 7 days respectively.

Fig. 6. Illustrate the release feature of PE from CS/PEO nanofibers and its in vitro antibacterial effect against E.coli O157:H7. (A) Control nanofiber without PE
produce no zone of inhibition; (B) CS/PEO nanofiber incorporated with PE (20 mg/mL) produce zone of inhibition.

conducted (Bizarria et al., 2014). In case of nanofibers blends with CS,
PEO and PE, thermograms were observed at three stages. Firstly, a
slight weight loss was observed between 170 and 240 °C which was due
to decomposition of some phytochemicals in PE. A sharp peak was
observed on second stage at about 285 °C which is higher thermal
stability than pure CS. This behavior is due to the strong interactions
and intermolecular hydrogen bonding between CS and PEO during
blends, leading to higher thermal stability of nanofibers (Fazli,
Shariatinia, Kohsari, Azadmehr, & Pourmortazavi, 2016). Third loss
was equivalent to pure PEO.
DSC thermograms of CS, PEO and CS/PEO/PE nanofiber are shown
in Fig. 4B. DSC thermogram of CS showed sharp exothermic peak at
307 °C without showing any melting temperature (Tm). Generally, CS
and some other polysaccharides, suffer thermal degradation without
melting, even under extreme temperatures (Bizarria et al., 2014). PEO’s
exothermic peak was only at 372 °C. Exothermic peak of CS was shifted
from 307 to 378 °C showing more stability when it was blended with
PEO. When CS reacts with PEO it resulted in higher thermal stability
due to molecular interaction between them increasing crystallinity (Hu,
Gong, & Zhou, 2015). TGA and DSC results indicated that the

7
D. Surendhiran, et al. Food Packaging and Shelf Life 23 (2020) 100439

Table 3
Effects of CS/PEO/PE nanofiber mat on sensory assessment of preserved beef at 4 and 25 °C.
Temperature (°C) Parameters Fresh beef (0th day) Control (10 days) CS/PEO/PE nanofiber (10 days) Sodium nitrite (10 days)

a b
4 Appearance 9.00 ± 0.00 5.34 ± 0.56 7.52 ± 0.32 7.77 ± 0.51b
Color 9.00 ± 0.00 5.11 ± 0.26a 7.38 ± 0.60b 7.69 ± 0.44b
Odor 9.00 ± 0.00 4.62 ± 0.30a 6.32 ± 0.51b 6.04 ± 0.12b
Overall 9.00 ± 0.00 5.02 ± 0.37a 7.07 ± 0.47b 7.16 ± 0.37b
Acceptability

Temperature (°C) Parameters Fresh beef (0th day) Control (7 days) CS/PEO/PE nanofiber (7 days) Sodium nitrite (7 days)

25 Appearance 9.00 ± 0.00 1.92 ± 0.23a 5.14 ± 0.24b 5.38 ± 0.21b

Color 9.00 ± 0.00 2.11 ± 0.12a 5.16 ± 0.20b 5.27 ± 0.18b
Odor 9.00 ± 0.00 1.20 ± 0.33a 5.18 ± 0.12b 5.49 ± 0.24b
Overall 9.00 ± 0.00 1.74 ± 0.28a 5.16 ± 0.18b 5.38 ± 0.27b
Acceptability

Data based on 1–9 point scale (1: extremely dislike; 2-4: dislike; 9: extremely like; 5-7: highly acceptable).
a, b
with different lowercase letters in the same row are significantly different (p < 0.05).

electrospun nanofibers from CS/PEO blends showed excellent thermal 4. Conclusions

stability up to 400 °C.
3.8. Bacterial reduction and sensory evaluation at storage
Effect of CS/PEO/PE nanofibers against E.coli O157:H7 inoculated
in beef sample at two different temperatures is shown in Fig. 5. The
initial counts of E.coli O157:H7 was about 3.60 log CFU/g for all the
samples. Bacterial counts increased at studied temperatures in control.
Even though CS possesses innate antimicrobial properties, it did not
exhibit its antimicrobial effects in control. This is due to that CS mo-
lecules were mostly trapped with PEO particles during electrospinning
process; as a result they could not release out and react on bacterial
cells (Wang et al., 2019). Recently, a similar result was obtained by
Wang et al. (2019). Moreover, the antimicrobial properties of CS are
greatly dependent on its physical characteristics, most notably mole-
cular weight. An increase in molecular weight leads to a decrease in the
antimicrobial activity of chitosan (Benhabiles et al., 2012). The bac-
terial growth on beef was slower at 4 °C than at 25 °C. During psy-
chrophilic storage the total bacterial count reached 6.60 log CFU/g,
whereas for test sample it reaches only 2.96 log CFU/g at 10 days of
storage period. Likewise, during mesophilic preservation conditions,
the maximum bacterial population attained 9.22 log CFU/g in control
and 5.80 log CFU/g for test meat after 7 days (Fig. 5). These results
indicated that PE containing active nanofibers significantly restricted
the proliferation of tested bacterium on beef. The inhibitory effect was
less pronounced at higher temperatures (25 °C). This finding corrobo-
rated with Hayrapetyan, Hazeleger, and Beumer (2012)). It is due to the
fact that bacterial cells are more active and reproduce at higher tem-
perature but could resist antimicrobial agents. However, this can be
solved by increasing PE concentration into the nanofibers during elec-
trospinning process. For instance, Licciardello et al. (2018) reported
that significant reduction of P.putida was attained with increasing
concentration of PE from 0.072 to 0.361 g in the packaging film pro-
duced from CS during shrimp storage. In vitro leaching effect of PE from
nanofibers and its action on E.coli O157:H7 is shown in Fig. 6.
Sensory assessment and the results pertaining to PE preserved beef
sample with control groups are represented in Table 3. From the sta-
tistical analysis it was clear that there were significant differences
(P < 0.05) of the test sample when compared to the control at 4 °C and
25 °C. Control group score was poor due to low quality of beef sample.
Preservation capacity of PE immobilized nanofiber mat was compared
with standard preservative sodium nitrite and found that PE performed
almost equivalent to the sodium nitrite within the acceptable range
(score 5–7). This might be attributed that PE possesses both anti-
microbial and antioxidant activities (Hayrapetyan et al., 2012).
Electrospun nanofibers from CS are explored as a potential re-
placement for synthetic polymers. Herein, CS based active nanofibers
were produced with PEO at different concentrations and encapsulate
natural antimicrobial agent PE. Characterization of fiber morphology
revealed that beaded structure could be changed into uniform nanofi-
bers even at a high content of CS by adding FDA approved surfactant
Pluronic-F127. Swelling and water solubility studies showed that in-
creasing content of CS in the blends produces more water resistant
nanofibers mat which would be highly suitable for the food industry.
The TGA/DSC results exhibited that nanofibers composed of CS/PEO
shows weight loss only at above 200 °C and have excellent thermal
stability up to ∼400 °C, compared to pure individual compounds. The
present study demonstrated that CS was an ideal choice for electro-
spinning nanofibers since it was simple, economic, and possible to
operate in commercial scale. Moreover, CS/PEO/PE active nanofibers
effectively inhibited E.coli O157:H7 growth on beef sample stored at 4
and 25 °C for 7 and 10 days respectively than control. Hence, PE im-
mobilized nanofiber mat is an excellent food wrapping material to
preserve and extend the shelf life of meat without losing their sensory
properties.
Declaration of Competing Interest
None.
Acknowledgements
This study was financially supported by Hunan Science and
Technology Major Project (Grant no. 2016NK1001-3), National Natural
Science Foundation of China (Grant no. 31972172), Natural Science
Foundation of Jiangsu Province (Grant no. BK20170070), Jiangsu
Province Research Fund (Grant no. NY-013 and JNHB-131) and Jiangsu
University Research Fund (Grant no. 11JDG050).
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