Food Chemistry 401 (2023) 134081

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effects of natural wax types on the physicochemical properties of starch/

gelatin edible films fabricated by extrusion blowing
Yue Cheng a, 1, Xiaosong Zhai a, 1, Yuhao Wu a, Cheng Li a, Rui Zhang a, Chanchan Sun b,
Wentao Wang a, *, Hanxue Hou a, *
a
Department of Food Science and Engineering, Shandong Agricultural University, Tai’an 271018, China
b
Key Laboratory of Food Nutrition and Safety (Tianjin University of Science &Technology), Ministry of Education, Tianjin 300457, China

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

Keywords: To address the limitation of strong hydrophilicity of edible films, starch/gelatin (S/G) films incorporated with
Starch natural waxes (beeswax (BW), candelilla wax (CL), and carnauba wax (CB)) were fabricated by extrusion
Gelatin blowing. Rheological analysis demonstrated that the incorporation of natural waxes reduced storage modulus
Edible film
and complex viscosity of S/G blends. BW and CL weakened molecular interactions among film components,
Natural wax
whereas CB did not. CB exhibited the strongest crystalline behavior after film formation. The degree of starch
Extrusion blowing
gelatinization and water vapor barrier property of films depended on wax type. The presence of waxes increased
the water resistance and surface hydrophobicity of the films. However, CL and BW addition decreased the tensile
strength of films. The highest water contact angle (102.6◦ ), strongest thermal stability, and lowest water vapor
permeability were found in S/G-BW film, which could be the optimal choice to produce highly hydrophobic
edible films.

1. Introduction
Starch, as a polysaccharide with good film-forming properties, has
attracted considerable attention in the field of edible and biodegradable
packaging because of its low cost, easy availability, and rapid biode­
gradability (Podshivalov, Zakharova, Glazacheva, & Uspenskaya, 2017).
However, the poor mechanical properties and strong hydrophilic char­
acteristic of starch films, such as high water permeability and high
sensitivity to moisture, restrict their application in the field of food
packaging. Blending starch with gelatin improved these performance of
films, but they still presented a high tendency to absorb moisture
(Cheng, Wang, Zhang, Zhai, & Hou, 2020), which greatly limited their
applications to packaging foods with high moisture content.
Some hydrophobic substances, such as fats, oils, natural waxes, have
been shown to reduce their hydrophilicity when added to bio-based
edible films for food packaging (Haq et al., 2016; Muscat et al., 2013;
Zhang et al., 2019; Zhang et al., 2022). Among natural waxes, beeswax
(BW), candelilla wax (CL), and carnauba wax (CB) exhibit great appli­
cation potential due to their non-toxicity and high hydrophobicity.
Although composed of hydrocarbons, wax esters, alcohols, and fatty
acid, the different composition ratios between them significantly affect
their physical properties, such as melting points, hardness, and hydro­
phobicity. In particular, their melting points determines their size and
related state in matrixes during film processing (Xiao et al., 2016). The
melting point of waxes above or below the film-forming temperature
affects their deformability and distribution in matrixes, which is closely
related to the construction of the hydrophobic surface structure (Zhang
et al., 2020). Furthermore, hydrophobic characteristics of waxes usually
affect their affinity to film-forming substrate. Waxes with higher hy­
drophobicity generally result in a higher hydrophobicity of the film, but
a weaker affinity to the hydrophilic polymer. Therefore, these properties
make waxes exhibit different behavior when added to the film matrix,
which affected the physicochemical properties of films. CL is usually
used to prepare super-hydrophobic coatings of films due to its high
hydrocarbon content, but this may deteriorate the mechanical proper­
ties of films (Liu, Xue, An, Jia, & Xu, 2019). BW is a natural animal-
derived wax with high inertness and plasticity produced by bees. It ex­
hibits good viscoelasticity and a low melting point due to the presence of
fatty acids. In contrast, CB exhibits a high melting point and hardness,
which can be used to develop durable coating (Gupta, Ivvala, & Grewal,

* Corresponding authors.
E-mail addresses: wangwt@sdau.edu.cn (W. Wang), hhx@sdau.edu.cn (H. Hou).
1
These authors contributed equally to this work and are co-first authors.

https://doi.org/10.1016/j.foodchem.2022.134081
Received 25 December 2021; Received in revised form 25 July 2022; Accepted 29 August 2022
Available online 7 September 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
Y. Cheng et al.
2021).
Starch films incorporated with the above three waxes as hydrophobic
agents have been developed and the properties of these films are very
dependent on the type of wax (Muscat et al., 2013). However, these films
made of high amylose starch and wax are extremely brittle. Meanwhile,
the preparation method is based on solution casting that is difficult to
apply in commercial production. Moreover, due to the long drying time
of solution casting process, waxes tend to crystallize and aggregate,
which destroy the uniformity of the film, deteriorating its performance
(Haq et al., 2016). For this reason, emulsifiers are commonly incorpo­
rated to combine hydrocolloids with lipids. In previous research, poly­
sorbate 80 (Tween 80) and/or other synthetic emulsifiers were usually
applied, which have adverse effects on human health (Dickinson, 1993),
and also increased the water sensitivity of films due to their high
hydrophilicity.
Gelatin may combine starch with waxes to form uniform and stable
films since it is amphipathic. In addition, the hydrophobic character of
gelatin can further enhance the overall hydrophobicity of combined
films. Extrusion blowing is the most common commercial method to
produce films as an alternative to solution casting to achieve industrial-
scale film production (Cheng et al., 2021; Cheng et al., 2022). Thus,
adding appropriate waxes into starch/gelatin (S/G) films prepared by
extrusion blowing may be a viable option to obtain highly hydrophobic
edible films with great commercial potential for food packaging, how­
ever, little information is available. Herein, S/G edible films with the
three natural waxes as hydrophobic agents were prepared by extrusion
blowing, and the structure and properties of S/G films were examined.
2. Materials and methods
2.1. Materials
Modified cassava starch (hydroxypropyl distarch phosphate) was
provided by Puluoxing Starch Co., ltd (Hangzhou, China). The starch
contained 12.5 wt% moisture, 3.1 % hydroxypropyl group, and 22.5 wt
% amylose. Cow skin gelatin (200 Bloom value) was obtained from
Fuyuan Gelatin Co., ltd (Zhoukou, China). BW (melting points of 62–64
℃), CL (melting points of 67–71 ℃), and CB (melting points of 82–86
℃) were provided by Likang Weiye Technology Co., ltd (Beijing, China).
Glycerol was purchased from Kaitong Chemical Reagent Co., ltd (Tian­
jin, China).
2.2. Preparation of S/G-natural wax films
Starch (2000 g) and gelatin (1000 g) were mixed for 10 min in an
SHR50A mixer (Hongji Machinery, Zhangjiagang, China). Natural
waxes (i. e. BW, CL, and CB, 180 g) were mixed with glycerol (900 g) and
deionized water (250 g), respectively, and homogenized for 5 min at
20000 rpm, and then added to S/G mixture, and mixed for another 10
min. The samples with BW, CL, and CB were coded as S/G-BW, S/G-CL,
and S/G-CB, respectively. The control was prepared without the addi­
tion of natural waxes following the abovementioned procedure. The
resulting mixtures were compounded with an SHJ-20B laboratory twin-
screw extruder (Giant Machinery, Nanjing, China) with a screw diam­
eter (D) of 21.7 mm and length of 40 D. The barrel temperatures from the
feed zone to the die zone were set to 85, 95, 100, 105, 95, and 85 ◦ C. The
screw speed was set to 130 rpm. The extrudates were air-cooled and cut
into pellets. S/G films were prepared with an SCM-50 laboratory film-
blowing extruder (Lianjiang Machinery, Zhangjiagang, China) with a
screw diameter (D) of 25 mm, length of 30 D, and die diameter of 30
mm. The screw speed was set to 25 rpm, and the extrusion temperatures
were set to 80, 105, 120, 120, 115, and 95 ◦ C. The thickness of the final
film was controlled at 75–85 μm.
2
Food Chemistry 401 (2023) 134081
2.3. Rheological measurements of three natural waxes and S/G-natural
wax blends
The pellets made in Section 2.2 were hot-pressed into 1 mm thickness
film samples for rheological test. The rheological behavior of three
natural waxes and all S/G pellets with or without wax were measured by
an MCR 102 modular intelligent rheometer (Anton Paar, Austria) at a
fixed temperature of 120 ◦ C. The gap distance between the parallel
plates is set to 1 mm, and the upper parallel plate is used as the rotor. The
complex viscosity (|η*|), storage modulus (G′ ), and loss modulus (G’’)
were determined in a controlled deformation mode (CD) with 1 % strain
and a frequency range of 0.01–100 rad/s.
2.4. Characterization of S/G-natural wax films
2.4.1. Scanning electron microscopy (SEM)
Cross-sectional and surface morphologies of films were observed
using a Quanta FEG 250 scanning electron microscope (FEI, Oregon,
USA) operating at 5.0 kV. Cross-sectional samples were produced by
cryogenic fracture after submerging in liquid nitrogen.
2.4.2. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR)
spectroscopy
ATR-FTIR spectra of films were recorded using a Nicolet iS5 spec­
trometer attached with iD5 ATR sampling accessory (Thermo Fisher
Scientific, MA, USA). The absorption spectra were acquired over a
wavenumber range of 4000–550 cm− 1 with 32 scans and a resolution of
4 cm− 1 a resolution against the background.
2.4.3. X-ray diffraction (XRD)
The crystal structure of film samples was measured using a D8
Advance X-ray diffractometer (Bruker–AXS, Karlsruhe, Germany) with a
diffraction angle (2θ) ranging from 5◦ to 40◦ at a scanning rate of 0.02◦ /s
at room temperature. The relative crystallinity of the samples was
calculated as follow:
Ac
Relativecrystallinity(%) = × 100,
Aa + Ac
where Aa and Ac are the amorphous and crystalline areas, respectively.
2.4.4. Thermogravimetric analysis (TGA)
TGA was realized in a DTG-60AH thermogravimetric analyzer (Shi­
matzu, Tokyo, Japan). Measurements were conducted from 25 ℃ to 600
℃ at 10 ℃/min under nitrogen flow of 50 cm3/min. Differential form
(DTG) was obtained by taking the first derivative of the TGA values.
2.4.5. Dynamic contact angle (DCA)
DCAs of films were tested using a JC-2000C1 contact-angle goni­
ometer (Zhongchen Digital Technology, Shanghai, China). A drop of
deionized water was placed onto film surface using a precision micro­
syringe. The contact angle between the film surface and the tangent of
the droplet was calculated, and the change of the contact angle within
150 s was recorded as a DCA curve.
2.4.6. Water vapor permeability (WVP)
The WVP of films was measured by a PERME W3/030 WVP tester
(Labthink Instruments Co., ltd, Jinan, China) according to the standard
testing method of ASTM E96/E96M-16 (2016). The specimens were
placed under specific conditions (53 % Relative humidity (RH) and
23 ◦ C) for at least 72 h before testing. The samples under inspection were
cut into round pieces (80 mm in diameter) using a special sampler and
then fixed to a circular permeation cell. The test was carried out at 90 %
RH and 38 ℃, with a preheating time of 4 h and a weighing interval of 2
h.
Y. Cheng et al. Food Chemistry 401 (2023) 134081

2.4.7. Moisture content (MC), swelling degree (SD), and water solubility
(WS)
The films equilibrated at 23 ◦ C and 53 % RH for 72 h were cut into
squares (2 cm × 2 cm), weighed (W0), and then dried to constant weight
(W1) at 105 ℃. The dried samples were submerged in deionized water at
25 ℃ for 24 h. The films were then taken out and were weighed after
removal of the surface water (W2). Then, the samples were dried again at
105 ℃ to constant weight (W3). The MC, SD, and WS values of S/G films
were determined as follows (Ahammed et al., 2021; Wu et al., 2019;
Zhang et al., 2019):
W0 − W1
MC(%) = × 100
W0
W2 − W1
SD(%) =
W1
× 100
W1 − W3
WS(%) = × 100
W1
2.4.8. Mechanical properties measurement
Tensile strength (TS), elongation at break (EAB), and Young’s
modulus (YM) were obtained using a XLW auto tensile tester (Labthink
Instruments Co. ltd., Jinan, China). Films were cut into strips (150 mm
× 15 mm) and equilibrated at 23 ◦ C and 53 % RH for at least 72 h before
testing. Then, the strips were fixed between the grips with a 100 mm
initial separation. Testing was performed at a speed of 100 mm/min
traction extension. All measurements were repeated at least six times.
2.5. Statistical analysis
The statistical significance among samples was determined by
analysis of variance (ANOVA) using SPSS 20.0. Duncan’s multiple range
test (p < 0.05) was used to compare the means to determine the sig­
nificant difference between the samples.
3. Results and discussion
3.1. Rheological properties
Rheological behavior plays an important role in polymer processing,
especially in extrusion blowing, which requires an appropriate melt
strength and fluidity (Bian et al., 2014). A lower |η*| value of the wax is
beneficial for its flow during extrusion processing, making it tend to
break into wax droplets under shearing. Previous studies have reported
that the wax droplets with smaller particle sizes can be more evenly
distributed in the film matrix, which is conducive to the water vapor
barrier properties of starch-based films (Oliveira Filho et al., 2020). The
measured viscosities of the three natural waxes at 120 ℃ are shown in
Fig. 1a. The viscosity of BW distinctly decreased with the increase of
shear rate, suggesting that BW presented shear-thinning behavior and
belonged to the non-Newtonian fluids. CL showed weaker shear-
thinning behavior, and the viscosity of CB showed a slight change
with increasing shear rate. These results indicated that higher shear
reduced the viscosity of BW to a greater extent. In addition, under the
same shearing conditions, CB presents the highest |η*| value, followed
by CL and BW.

Fig. 1. Viscosity of three natural waxes (a) and rheological behavior of S/G, S/G-BW-P, S/G-CL-P and S/G-CB-P blends: (b) complex viscosity (|η*|), (c) storage
modulus (G’), and (d) loss modulus (G’’).

3
Y. Cheng et al.
Changes in |η*|, G’, and G“ values for S/G and S/G-natural wax
blends are plotted as functions of angular frequency in Fig. 1b, c, and d.
The |η*| values of the S/G blends with natural waxes are closely related
to its fluidity in the matrix during melt extrusion. As shown in Fig. 1b,
the addition of natural waxes reduced the |η*| values of S/G blends,
which was due to the lubrication effect of the natural waxes during
extrusion. From the perspective of the entire angular frequency range
shown in Fig. 1c and d, the G’ and G” values of all samples increased
with an increase in frequency, and the dependencies were relatively
strong. Furthermore, the magnitudes of the G’ values were systemati­
cally higher than those of the G“ values over the frequency regions,
suggesting that the studied blends exhibited gel-like characteristics. In
addition, the G’ values of all S/G-natural wax blends were lower than
that of the control, indicating that the addition of natural waxes reduced
the melt strength of S/G blends. The above phenomenon could be owing
to the lubrication effect of natural waxes, which facilitated polymer
movement and even resulted in phase separation of starch and gelatin
(Hassan, Murray & Dickinson, 2009). In addition, the G’ values of the
blends were related to the phase state of waxes. The liquid wax droplets
formed a flow-able dispersed phase in the blends and could be easily
deformed, which lead to lower G’ value. The natural waxes with lower |η
*| values flowed easily and changed into smaller droplets under high
temperature and shear, which promoted their distribution and lubrica­
tion effect in the film matrix. Thus, compared with the S/G-CB and S/G-
CL blends, the S/G-BW blend presented the lowest |η*| and G’ values.
This finding was beneficial to the processing of blends under certain
conditions.
3.2. Morphology
The distribution of natural waxes in film matrix and the interfacial
interaction with the substrate are directly related to the performance of
composite films. Fig. 2 provides the cross-sectional and surface mor­
phologies of all films. The scattered wrinkles in the cross section of the
S/G film was attributed to the conformation and association of gelatin
(Peng et al., 2022). Many “round zones” wrapped in the gelatin phase
appeared owing to the inadequate gelatinization of starch granules
(Cheng, Wang, Zhang, Zhai, & Hou, 2020). These results demonstrated
the occurrence of phase separation between starch and gelatin.
The addition of wax significantly affected the microstructure of
films, that is, less starch granules were observed in S/G-BW and S/G-CL
films than that in the control, indicating the better gelatinization of
starch. This was related to the promoting effect of waxes on the melting
of film matrixes during processing (Zhang et al., 2019). The better
Fig. 2. SEM images of S/G, S/G-BW, S/G-CL and S/G-CB films.
4
Food Chemistry 401 (2023) 134081
gelatinization of starch was conducive to interfacial adhesion between
starch and gelatin molecules due to the exposure of starch hydrophilic
groups, forming a denser internal structure. More starch granules existed
in the S/G-CB film than in the control, indicating that CB inhibited the
gelatinization of starch. The more disordered and discontinuous film
network structure caused by the formation of larger CB particles pro­
moted the evaporation of water, which was important for starch gela­
tinization (Fabra, Pérez-Masiá, Talens, & Chiralt, 2011; Xiao et al.,
2016). In addition, some cracks inside the films were noticed in the
cross-section images, indicating structural disruption and phase sepa­
ration caused by the CB molecules. No wax particles were observed in
the S/G-BW and S/G-CL films, indicating the good compatibility and
dispersibility of waxes in the film matrix. In contrast, the presence of CB
in large particles was clearly observed in the cross-sectional morphology
of the S/G-CB film, indicating its poor compatibility with the S/G
matrix.
As expected, the presence of ungelatinized starch granules resulted
in the rough surface of S/G films with “steamed bread”-like bulges. The
addition of natural waxes resulted in greater roughness on the film
surface, manifesting as many small wax crystal particles, which were
different from the S/G film. Similar observations were reported by
Muscat, Adhikari, Mcknight, Guo, & Adhikari (2013) in starch films
incorporated with waxes. The small particles of wax crystals on the film
surface significantly increased the surface roughness of the films.
Compared with the S/G-BW and S/G-CL films, less small particles were
observed on the surface of the S/G-CB film, which may indicate that the
CB was mainly distributed inside the film matrix rather than on the
surface.
3.3. Atr-FTIR
Physicochemical interactions between multiple components in
composites are reflected based on spectral changes, such as peak shifting
and broadening (Wu, Zhong, Li, Shoemaker, & Xia, 2013; Zhang et al.,
2021). Fig. 3a shows the ATR-FTIR spectra of all prepared films. S/G is a
phase separation system in films, and each polymer shows its own
characteristic band (Zhang et al., 2013). The band at 1020 cm− 1 is
attributed to the stretching vibration of C–O in C–O–C bonds mainly
present in the glycosidic linkages. There was no significant change in
this peak with the addition of waxes. Generally, the addition of lipids
caused the shift of the C–O stretching vibration peak in polysaccharide
films due to the molecular interaction between polysaccharides and
lipids (Gao et al., 2020; Zhang et al., 2019). This result implied that the
waxes and starch had weaker interactions in the S/G systems because of
Y. Cheng et al. Food Chemistry 401 (2023) 134081

Fig. 3. ATR-FTIR spectra (a) and XRD patterns (b) of S/G, S/G-BW, S/G-CL and S/G-CB films.

the hydrophobic group in starch. The broad band at approximately
3288 cm− 1 corresponds to the O–H or –NH stretching vibration mainly
from starch and gelatin (Kowalczyk, Kordowska-Wiater, Nowak, &
Baraniak, 2015). The characteristic peak at 3288 cm− 1 shifted to a high
wavenumber (3290 cm− 1) with the addition of BW, indicating weak­
ened hydrogen bonding interactions (Zhang et al., 2020). This outcome
was related to the fact that the addition of BW destroyed the hydrogen
bonding interactions that maintained the structural stability of the
starch molecule, which could be proved by the SEM results that BW
promoted the starch gelatinization. In addition, the formation of hy­
drophobic interactions between wax and gelatin molecules might reduce
the interactions between the –NH groups of peptides via hydrogen bonds
(Hoque, Benjakul, & Prodpran, 2010; Nilsuwan et al., 2016).
The appreciable band at approximately 1640 cm− 1 is mainly
attributed to the stretching vibration of C–– O in the amide present in
gelatin (Noorbakhsh-Soltani, Zerafat, & Sabbaghi, 2018). This band
shifted from 1640 cm− 1 to 1644 cm− 1 after adding BW to films, which
might be due to the interference of the BW molecules on the intermo­
lecular interaction between gelatin polypeptides at the C– – O groups
(NúñEz-Flores, Giménez, Fernández-Martín, López-Caballero, Montero,
& Gómez-Guillén, 2013;). A similar phenomenon was also observed in
the S/G-CL film. The shift of this peak indicated that the waxes first
affected the film network structure by weakening the interaction be­
tween gelatin molecules, which supported the above implication of a
weaker interaction between the waxes and starch in the S/G system. The
shift of these bands indicated that BW and CL were able to cause
conformational changes and reduce the interactions of the S/G matrix.
CB showed no visible effect on the infrared absorption peak, indicating
negligible interaction between CB and the S/G matrix. In addition to the
above-mentioned peak shifts, the S/G film with waxes also showed new
bands related to the various functional groups derived from wax. The
presence of fatty acid chains in waxes formed sharp peaks at 2940 and
2848 cm− 1, which were due to the methylene asymmetric and sym­
metric stretching vibrations of aliphatic C–H groups (Muscat, Adhikari,
Mcknight, Guo, & Adhikari, 2013).
3.4. Xrd
bonds. The decrease in peak intensity of the S/G-BW and S/G-CL films
implied that BW and CL were uniformly distributed inside the film
matrix (Omar-Aziz et al., 2021).
According to the Scherrer equation, the half-width of the charac­
teristic diffraction peak is positively correlated with the crystalline
domain size. The three natural waxes showed two similar diffraction
peaks at 2θ = 21.4◦ and 23.6◦ (Muscat et al., 2013), indicating their
similar crystalline domain sizes. York et al. (2019) also reported similar
crystalline structures of BW and CB in filter paper samples coated with
waxes. However, great differences appeared after adding them to S/G
films, indicating their different particle size distributions in the film
matrixes (Zhang, et al., 2020). Among the three natural waxes, CB
exhibited the highest |η*| value, as shown in Fig. 1a, which made it
difficult to break into small droplets during processing, resulting in the
presence of larger wax particles. Thus, the S/G-CB film showed the
strongest intensity of the diffraction peak associated with waxes here,
indicating the presence of a stronger crystalline structure. In contrast,
BW and CL with lower |η*| values tended to be turned into smaller
droplets. Xiao et al. (2016) also reported that liquids with lower |η*|
values were beneficial to emulsification and the formation of smaller
droplets in film matrixes. Moreover, the melting point of natural wax
also affected the crystallization of wax during film formation. When the
cooling temperature of the films was below the melting point of natural
waxes during film formation, the natural waxes tended to crystallize
rapidly and randomly, resulting in a random distribution inside the film
(Zhang et al., 2020). The waxes with higher melting points reached their
crystallization temperature faster and aggregated more during film
cooling, thereby increasing crystallinity of materials (Matsumoto,
Kimura, Iwao, & Itai, 2017). Moreover, the addition of waxes increased
Table 1
Crystallinity, water vapor permeability (WVP), water solubility, swelling de­
gree, and moisture content of S/G, S/G-BW, S/G-CL and S/G-CB films.
Sample Crystallinity WVP (10-12⋅g Water Swelling Moisture
(%) m− 1⋅s− 1⋅Pa− 1) solubility degree content
(%) (%) (%)

S/G 14.87 2.75 ± 0.13b 34.24 ± 478.41 18.84 ±

The XRD patterns of the prepared films are shown in Fig. 3b. Gelatin 0.23 a ± 12.23 a 0.37 a
S/G- 16.36 1.26 ± 0.09 d 28.94 ± 317.23 17.42 ±
exhibited the presence of helix and triple-helix structures that generated
BW 0.79b ± 8.27c 0.46b
broad peaks in the 2θ range of 15◦ –25◦ (Ki et al., 2005). Starch produced S/G-CL 15.21 1.78 ± 0.12c 26.21 ± 280.67 18.65 ±
relatively weak diffraction peaks bewteen 17◦ and 23◦ , which over­ 0.13c ± 10.28 d 0.18 a
lapped with the gelatin peaks (Kang et al., 2020). As shown in Fig. 3b, S/G- 17.81 3.37 ± 0.29 a 30.95 ± 387.19 16.23 ±
the addition of BW and CL caused a decrease in the intensity of the CB 0.54b ± 8.74b 0.24c

overlapping peak, indicating a weakened crystallization. This effect was Different lowercase letters within the same column indicate significant differ­
attributed to the possible reduction in the ability to form hydrogen ences among the samples (p < 0.05).

5
Y. Cheng et al. Food Chemistry 401 (2023) 134081

the crystallinity of S/G-natural wax films, as shown in Table 1, which

was mainly attributed to the wax crystals formed in S/G-natural wax
films. Among them, the S/G-CB film exhibited the greatest crystallinity
because of the most crystalline structure of CB in the film matrix.

3.5. TgA

TGA and DTG curves of all film samples are shown in Fig. 4a and b,
respectively. The thermal decomposition of S/G films displayed three
main stages observed from the DTG curves in the range from 25 ℃ to
600 ℃. The first weight loss occurred in the temperature range of
50–150 ℃, which was mainly attributed to the evaporation of water in
the film. The second stage, which appeared at approximately 270 ℃,
might be related to the volatile substances in carbohydrates, proteins,
and waxes, the loss of bound water, and the degradation of glycerol
(Oluwasina et al., 2019). The third stage of thermal decomposition
occurred at approximately 305 ℃, which was attributed to the thermal
decomposition of starch and gelatin (Wang, Wang, Xiao, Liu, Zhao, &
Liu, 2017; Zhang, Dutilleul, Li, & Simpson, 2019).
The temperature corresponding to the maximum mass loss rate of the Fig. 5. Dynamic water contact angles of S/G, S/G-BW, S/G-CL and S/G-CB
film increases from 297 ◦ C for the S/G film to 317 ◦ C for the S/G-BW films within 150 s. The inset shows images of a water droplet taken soon
film, which was mainly attributed to the interactions between the wax after dropping water on the films.
component and the polar side groups in gelatin, such as hydroxyl,
carbonyl and amino groups (Halal et al., 2016). However, the thermal that of pure starch film reported by Zhai et al. (2020). This finding was
stability of the S/G-CB film in this study was not improved at this stage, attributed to the orientation of the hydrophobic groups of gelatin mol­
which may be due to the weak interaction between CB and the film ecules on the film surface (Wiącek, 2015). Liu, Antoniou, Li, Ma, &
matrix as previously discussed. In addition, all the S/G-natural wax films Zhong (2015) also reported similar findings and indicated that the
presented a new thermal decomposition stage in the range of 450–500 exposure of hydrophobic groups in gelatin significantly enhanced the
℃, which was attributed to the thermal degradation of the incorporated surface hydrophobicity of films. Besides, increased initial contact angles
natural wax (Cheng, Wang, Zhang, Zhai, & Hou, 2020; Silva Freitas, and reduced drop within 150 s occurred after the addition of waxes. This
Pinto Vieira, Machado Sousa, Muniz, Leonia, & Florindo Guedes, 2016). phenomenon indicated a noticeable improvement in the hydrophobicity
The stage of wax thermal decomposition was almost consistent with the of films, which was attributed to the presence of wax particles on the
study of gelatin films reported by Oliveira Filho et al. (2020). In this film surface.
decomposition stage, the weight loss varied between the three natural The chemical composition of waxes significantly affected their hy­
waxes (4.42 % for S/G-BW, 6.16 % for S/G-CL, and 6.94 % for S/G-CB), drophobicity. Generally, these natural waxes contain similar constitu­
which supported the XRD analysis that three natural waxes existed in the ents (i.e., hydrocarbons, wax esters, alcohols, and fatty acids), among
composite film with different aggregation states. which the hydrocarbons showed the strongest hydrophobicity (Hollo­
way, 1969). The hydrocarbon content of CL is 50 %, higher than that of
BW and CB (Muscat et al., 2013). However, BW was more effective than
3.6. Surface hydrophobicity
CL and CB in terms of increasing the surface hydrophobicity of S/G films
in the present study. In addition, the contact angle of the S/G-BW film
Surface hydrophobicity is an important properties of food packaging,
was comparable to those of filter papers coated with BW
which could be quantified by water contact angle. As shown in Fig. 5,
(102.7◦ –116.3◦ ) (York, Collins, & Rantape, 2019). This might be caused
the water contact angles of all films decreased with prolonged time,
by the high concentration of BW particles on the film surface, which was
which was attributed to the reorientation of polar groups on the film
supported by the maximum surface roughness of the S/G-BW film in the
surface (Wirbel et al., 2015). The initial angle of the S/G film was 50◦
SEM images. In contrast, CB and CL were unable to develop their full
and decreased by 8◦ within 150 s, which was significantly higher than

Fig. 4. TGA (a) and DTG (b) curves of S/G, S/G-BW, S/G-CL and S/G-CB films.

6
Y. Cheng et al.
hydrophobic potential, as more of them were buried within the S/G
matrix. It was worthwhile to mention that the S/G-BW film exhibited a
higher contact angle (102.6◦ ) than that of the S/G films previously re­
ported, achieving a hydrophobic surface (Wang et al., 2022). The above
results showed that the addition of natural waxes significantly improved
the surface hydrophobicity of S/G films, which was significantly affected
by the wax crystal particles formed on the film surface.
3.7. Water vapor barrier properties
A lower WVP value indicates a stronger water vapor barrier prop­
erty. The addition of natural waxes to S/G films has a significant effect
on their WVP values (p < 0.05) as shown in Table 1. Natural waxes are
solid lipids with high contents of nonpolar long-chain fatty alcohols and
alkanes that can prevent water vapor from permeating through films.
Therefore, their addition usually reduces the WVP of edible films
(Kowalczyk & Baraniak, 2014; Zhang, Dutilleul, Li, & Simpson, 2019).
In this study, the addition of BW and CB significantly reduced the WVP
values, while a significant increase in the WVP value appeared in the S/
G-CB film. The permeation of water vapor through the film is the result
of a combination of adsorption, diffusion, and desorption (Kowalczyk
et al., 2015). Barrier properties of biopolymer films are affected by not
only the composition of the film but also their microstructure, such as
the presence of pinholes or cracks (Baümler et al., 2014; Kowalczyk &
Baraniak, 2014). In the present study, pores and cracks were observed in
SEM images of the S/G-CB film. The immiscibility between CB and
polymer might also lead to phase separation (Núñez-Flores et al., 2013).
These allowed water vapor to easily permeate through the S/G-CB film.
In addition, the distribution of lipids in film matrixes also played a vital
role in the water vapor barrier property (Perez-Gago, Krochta, &
Chemistry, 2000; Pérez-Gago & Rhim, 2014). More homogeneously
distributed lipids generally produce films with lower WVP values (Galus,
Mathieu, Lenart, & Debeaufort, 2012). SEM results (Fig. 2) showed that
the larger CB crystalline particles in the S/G-CB film were difficult to
disperse homogeneously in the matrix, resulting in higher WVP values.
3.8. Water solubility, swelling degree, and moisture content
The WS and SD of edible films are often used to measure water
resistance (Wu et al., 2019). The high water resistance of edible films is
essential to maintain the integrity of the package in practical applica­
tions. Table 1 lists the WS and SD values obtained for all films. The S/G-
natural wax films exhibited significantly lower WS and SD values than
the control, which indicated the addition of natural waxes effectively
improved the water resistance of S/G films. Zhang et al. (2019) also
found that the addition of beeswax improved the water resistance of the
agar/maltodextrin edible films.
Generally, starch and gelatin could combine with water molecules to
form hydrogen bonds due to their hydrophilic nature, which promoted
their dissolution in water (Ciannamea, Castillo, Barbosa, & De Angelis,
2018). In addition, the porous nature of the gelatin network might also
be responsible for the strong water absorption of the films (Kavoosi,
Rahmatollahi, Seyed, & Mohammadi Purfard, 2014). In this study, due
to the high-content long-chain fatty alcohols and alkanes, waxes might
interact with the hydrophobic groups of polymer chains when incor­
porated into the S/G matrix, which would reduce the void spaces and
cover the water binding sites. This hypothesis was supported by the shift
of the amide A peak to a higher wavenumber in the ATR-FTIR spectra,
indicating a reduction in the hydrogen bonds between the –NH groups of
polypeptides (Syahida et al., 2020). Moreover, the addition of BW and
CL promoted the gelatinization of starch to form a denser film network
structure, which could reduce water absorption, thereby weakening film
swelling (Cheng et al., 2022). This might be the main reason that the S/
G-BW and S/G-CL films showed lower WS and SD values than the S/G-
CB film. S/G-CL film exhibited the minimum WS value due to the strong
hydrophobicity of CL. The WS values of the S/G films obtained by
7
Food Chemistry 401 (2023) 134081
extrusion blowing were higher than those obtained by solution casting
method (Najwa et al., 2020), which might be attributed to the lower
degree of hydration of the polymer as the result of the three cycles inside
the extruder mixture (Fakhouri et al., 2013).
Table 1 also showed the highest MC value of the S/G film due to the
strong hydrophilicity of starch and gelatin, while reduced MC values
were found in S/G-natural wax films. These results were due to the fact
natural waxes are equivalent to a fraction of solids with almost no water
absorption capacity. The lowest MC value of the S/G-CB film was mainly
attributed to the presence of pores and cracks in the film matrix, which
promoted the evaporation of water.
3.9. Mechanical properties
Appropriate mechanical properties are required to withstand various
stresses during food packaging, storage and transportation, but too high
strength also possibly leads to poor edibility of edible films for the
elderly (Gallego, Barat & Talens, 2021). The mechanical properties (TS,
EAB, and YM) of all films are illustrated in Fig. 6. The TS value of the S/G
film was 4.3 MPa, meeting the requirement proposed by Hosseini et al.
(2015) that the TS values of films must exceed 3.5 MPa to satisfy food
packaging. Films prepared by the solution casting technique showed
higher TS and EAB than those produced by extrusion blown (Najwa
et al., 2020), which was possibly due to the denaturation of the protein
by high temperature, making it contribute less to the films mechanical
strength. Although the addition of BW and CL reduced the TS values of
the films, the S/G-BW and S/G-CL films still met packaging re­
quirements. The significant decrease in TS values indicated that the
presence of BW and CL could destroy the network structure of the film
matrix.
This decline in the TS of the S/G films could be attributed to various
causes. The mechanical properties of the films were mainly driven by the
gelatin phase because gelatin was usually the continuous phase
responsible for absorbing stress under loading (Schwach, & Averous,
2004; Zhang et al., 2013). As discussed in ATR-FTIR, the partially
stronger gelatin-gelatin interactions were replaced by weaker gelatin-
wax interactions in the film network after the addition of BW and CL,
which would cause a decline in TS (Haq, Hasnain, Jafri, Akbar, & Khan,
2016; Hromiš et al, 2015). Besides, the formation of gelatin triple-helix
structures played an important role in improving the mechanical prop­
erties of the films (Chen et al., 2017). The addition of BW or CL might
decrease the number of compact and ordered triple-helix structures in
the film, which caused the deterioration of the film mechanical prop­
erties (Cheng, 2021). Generally, the incorporation of hydrophobic sub­
stances into the starch-based films matrix induced structural disruptions
and then preferential breaking zones. The presence of these three nat­
ural wax in the films matrix exhibited the same effect in this study. Thus,
the addition of BW and CL produced a significant decrease in EAB values
as shown in Fig. 6b. Although CB did not promote starch gelatinization,
the S/G-CB film showed a lower EAB than the control film. The greater
hindrance and discontinuity caused by the wax particles to the film
structure might be another reason for the decline in the EAB. As shown
in Fig. 6c, the addition of CL and CB led to a significant increase in YM
values, indicating increased rigidity. These results were attributed to the
higher hardness of CL and CB than BW (Muscat et al., 2013), which gave
the composite films higher resistance to pressure.
4. Conclusions
Highly hydrophobic S/G edible blown films were successfully pre­
pared by adding natural waxes. The addition of natural waxes decreased
the |η*| values of the S/G blends, which was beneficial for film extrusion
blowing. The presence of BW weakened the intermolecular hydrogen
bonds, causing a decrease in the TS value. The surface micromorphology
of the S/G films became more irregular and rougher after the incorpo­
ration of BW and CL, which was conducive to the improvement of the
Y. Cheng et al.
Fig. 6. Mechanical properties of S/G and S/G-BW, S/G-CL and S/G-CB films: (a) tensile strength, (b) elongation at break, and (c) Young’s modulus. Different
lowercase letters within the same indicator indicate significant differences among the samples (p < 0.05).
surface hydrophobicity of the film. All S/G-natural wax films exhibited
significantly decreased EAB, WS, and MC values compared with those of
the S/G film. Due to the strong hydrophobicity of CL, S/G-CL film
showed the lowest WS value. CB exhibited poor dispersion and
compatibility in the S/G film matrix, which increased the WVP value.
The S/G-BW film showed the strongest hydrophobicity, water vapor
barrier properties, and thermal stability. In conclusion, BW was the
optimal wax for constructing highly hydrophobic edible films. In the
future, the hydrophobic properties of bio-based edible films incorpo­
rated with natural waxes can be further improved by adjusting the
distribution and crystal structure of the wax.
CRediT authorship contribution statement
Yue Cheng: Methodology, Writing – original draft. Xiaosong Zhai:
Investigation, Data curation. Yuhao Wu: Validation, Visualization.
Cheng Li: Software. Rui Zhang: Formal analysis. Chanchan Sun:
Formal analysis, Visualization. Wentao Wang: Validation, Formal
analysis, Visualization. Hanxue Hou: Supervision, Funding acquisition,
Project administration, Resources, Writing – review & editing.
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.
Acknowledgments
This work was supported by National Natural Science Foundation of
China (grant number 32102015), Great Innovation Program of Agri­
cultural Application Technology in Shandong Province, Natural Science
Foundation of Shandong Province (ZR2020QC222), Project funded by
China Postdoctoral Science Foundation (2022 M711965), Young and
Innovative Talents Introduction and Education Program of Universities
in Shandong Province, the Key Technology Research and Development
Program of Shandong province (2019GNC106035), Natural Science
Foundation of Tianjin City (18JCQNJC84200).
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