International Journal of Biological Macromolecules 109 (2018) 863–871

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International Journal of Biological Macromolecules

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

Research Paper

New starch ester/gelatin based films: Developed and physicochemical

characterization
Chengmei Shi, Furong Tao ∗ , Yuezhi Cui
Shandong Provincial Key Laboratory of Fine Chemicals, Qilu University of Technology, Jinan 250353, PR China

a r t i c l e i n f o a b s t r a c t

Article history: A new starch-based ester Starch-EDTAD-NHS (SEN) was successful prepared firstly and used to mod-
Received 8 August 2016 ify gelatin films by amidation. 1 H NMR, FTIR spectra, XRD curves, elemental analysis and Van Slyke
Received in revised form method verified the synthesis and confirmed the crosslinking procedure. The properties of the com-
18 September 2017
posite SEN-Gel film, such as mechanical test, surface hydrophobicity, light barrier performance, water
Accepted 10 November 2017
uptake studies, morphology and solubility behavior were studied. The results of water contact angle
Available online 11 November 2017
showed the hydrophobicity of the films, and the obtained transparency values indicated that the films
were opaque. Finally, the modified films with better surface hydrophobicity, more flexible and pliable,
Keywords:
Composite film
improved light barrier performances and low solubility were obtained. All the results above illustrated
Gelatin that the composite films obtained in this work maybe have potential applications in many fields.
Starch © 2017 Elsevier B.V. All rights reserved.
Crosslinking

1. Introduction the purpose of substituting the synthetic petroleum-based poly-

In recent years, along with the increase in population and the
deterioration of the environment, green chemistry has attracted
people’s attention [1]. Biomass-derived carbohydrates represent
a promising carbon-based alternative as an energy source and a
sustainable chemical feedstock [2,3]. Gelatin, as an environmental-
friendly biological material, is produced by partial hydrolysis or
thermal degradation of collagen, and is mainly extracted from
bones, and connective tissues of mammalian sources [4]. On
account of the low cost, biodegradability, nontoxicity, sustainabil-
ity and abundance, gelatin is considered to be used in various fields
of the most promising candidates, such as biology, medicine, chem-
istry and other fields, particularly in terms of packaging materials
[5–8]. However, the main disadvantages of gelatin are connected
with its poor mechanical properties and thermal stability, easily
soluble in water and readily biodegradable, which limit its appli-
cations [9,10]. Therefore, improve the characteristics of gelatin to
enhance its value is very important.
Starch is readily available from renewable sources and has a
great ability to form transparent, tasteless, odorless films with very
good oxygen barrier properties, which is very useful for food preser-
vation [11–13]. In the last few years, the aim of many studies has
been to obtain starch-based biodegradable membrane materials for
∗ corresponding author.
E-mail address: frtao2015@126.com (F. Tao).
mers [14–17]. Fakhouri [18] prepared an edible film and coatings
based on starch/gelatin, developed and evaluated the physico-
chemical properties of composite films, and the result showed that
the addition of gelatin significantly increased mechanical strength,
solubility in water, permeability to water vapor, and thickness of
the biofilms, while also decreasing the opacity. Jiménez et al. [19]
studied the effect on the physical properties and stability of starch-
gelatin based films by the addition of esters of fatty acids, which
demonstrated that the lipids decreased the hardness and resis-
tance but enhanced the stretchability, and also improved water
barrier properties of the films. Martínez-Bustos [20] investigated
the possibility of using hydrolyzed corn starch–gelatin as a base
matrix and cellulose as reinforcement, to produce containers by
extrusion blow molding, the results showed that the addition of cel-
lulose decreased the viscosity of the starch–gelatin polymer matrix
and improved the dimensional stability. Dubruel [21] developed
hydrogel films consisting of methacrylamide-modified gelatin and
starch-pentenoate building blocks, and the results indicated that
both the mechanical properties and the swelling extent of the films
can be controlled by the reaction variation. Norziah [22] studied
the physical and mechanical properties of edible films based on
blends of sago starch and fish gelatin plasticized with glycerol or
sorbitol (25%, w/w), and verified the compatibility of the sago starch
and fish gelatin polymers to form films at the concentration lev-
els used. González-Martínez [23] prepared the starch-gelatin blend
films containing essential oils, which exhibited antifungal activity
against Fusarium oxysporum f.sp. gladiolo and Colletotrichum gloes-

https://doi.org/10.1016/j.ijbiomac.2017.11.073
0141-8130/© 2017 Elsevier B.V. All rights reserved.
864 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
poroides. Also, the addition of dispersed essential oils in the films
improved their barrier properties and transparency. Furthermore,
Fakhoury [24] investigated influence of different types of plasti-
cizer and different levels of macromolecules on the properties of
edible films, which prepared from blends of manioc starch and
gelatin. All the results showed that the performances of the films
were improved as the gelatin content increased in the formula-
tions. Therefore, the gelatin films in the above researches were
almost modified with blending methods, which limited their prac-
tical applications.
Composite film is commonly used in retaining water, inhibit-
ing fat oxidation, reducing the loss of volatile aromatic substances
and improving the treating performance. Based on these, in this
work, a composite film based on ester SEN and gelatin (Gel)
was successful prepared, and the possible formation process was
shown in Scheme 1. 1 H NMR, FTIR spectra, XRD curves, elemen-
tal analysis and Van Slyke method verified the accurate synthesis
and confirmed the crosslinking procedure. The properties of the
composites, such as mechanical test, surface hydrophobicity, light
barrier performance, water uptake studies and solubility behavior
in PBS were studied. On the basis of these results, the film material
obtained in this work was stable eco-friendly and excellent perfor-
mance. And all the properties of the SEN-Gel films were an evidence
to broaden its application areas.
2. Experimental
2.1. Materials
Type A Gelatin (obtained from pigskin, with an approx-
imate molecular weight of 50,000 and isoelectric point at
pH = 8 determined by fluorescence measurements) was obtained
from Sinopharm Chemical Reagent Co., Ltd. Starch (starch
from corn, pharmaceutical-grade) and EDTA were from Aladdin
Chemistry Co., Ltd. N-hydroxysuccinimide (NHS, AR, 98%) and 1-(3-
dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC,
AR, 99%) were bought from Energy Chemical Technology Co., Ltd
(Shanghai). Glycerol (AR, 99%), DMF (AR, 99.5%), acetic anhydride
(AR, 98.5%) and other agents were obtained from Tianjin Fu Yu Fine
Chemical Co., Ltd. All chemicals and reagents were used as received
without further purification.
2.2. Analytical methods
FTIR spectra were recorded on a Nicolet NEXUS 470 FT-IR spec-
trometer. KBr pellets were concisely prepared by mixing 1 mg of
the sample with 200 mg of KBr. Fourier Transform Infrared spec-
troscopy was performed in a range of wavenumber from 4000 to
400 cm−1 with a resolution of 2 cm−1 .
XRD analysis of samples were carried out by an X-ray diffrac-
tometer (D8-ADVANCER, Bruker AXE, Germany) with a thin film
attachment using Cu-K␣ radiation (␭ = 0.1541 nm) at a current of
40 mA and an accelerating voltage of 40 kV. All the samples were
recorded from 5 ◦ to 60 ◦ .
Scanning electron microscopy (SEM) analysis was examined the
microstructures of the obtained films by Quanta 200 environmental
scanning electron microscope (SEM, FEI Company, Holland). The
film surfaces and sections were coated with Au using SEM coating
device before observation.
1 H NMR data were recorded on a Bruker Advance 400 spec-
trometer, operating at 400 MHz for protons, equipped with pulse
gradient units capable of producing magnetic field pulsed gra-
dients. Chemical shifts (␦) for 1H spectrum are expressed inH
NMR data were recorded on a Bruker Advance 400 spectrometer,
operating at 400 MHz for protons, equipped with pulse gradi-
ent units capable of producing magnetic field pulsed gradients.
Chemical shifts (␦) for 1H spectrum are expressed in ppm and
calibrated according to the residual solvent signal (DMSO; ␦ = 3.3
ppm ␦ = 2.5 ppm).
The contents of elements in the samples were determined by
Elementary analysis. Specific data is recorded on the Vario EL III,
Elementar Analysensysteme, Germany. The amount of sample mea-
sured is more than 10 mg.
2.3. Preparation of raw materials
2.3.1. Synthesis of ethylenediamine tetraacetic dianhydride
(EDTAD)
EDTAD is an important small molecular chelating agent and
modification reagent. In this paper, EDTA dianhydride was obtained
according to the method depicted by Golikand [25] with minor
modifications. 18 g EDTA, 50 mL pyridine and 24 mL acetic anhy-
dride were placed in a 150 mL round-bottom flask. The mixture
was reacted under reflux and kept stirring at 65 ◦ C for 24 h. In
order to obtain the pure product, the mixture was filtered off
and washed in water at least three times, and then the yellowish
solids obtained were dried under vacuum at 40 ◦ C. The prepared
EDTAD was characterized by 1 H NMR spectrum (Bruker Advance
400 spectrometer) and FTIR spectrum (Nicolet NEXUS 470 FT-IR
spectrometer). 1 H NMR spectra (400 MHz, DMSO): ı 3.687 (s, 8H),
ı 2.652 (t, 4H), ı 3.317 (s, DMSO), ı 2.490 (m, DMSO), correspond-
ing to the characteristic peaks of H in the ideal product. The FTIR
spectra peak of EDTAD exhibited characteristic peak of the main
functional groups. C O and C N absorption peaks were observed at
about 1249 cm−1 and 1421 cm−1 , respectively. The wavenumbers
of 1807 cm−1 and 1763 cm−1 were associated to C O stretching
vibration. Besides, the absorption peaks appeared at 1132 cm−1 ,
1070 cm−1 and 996 cm−1 with the similar interval further proved
that EDTAD was successfully synthesized.
2.3.2. Preparation of starch-EDTAD (SE)
The functionalization of starch with EDTAD was prepared based
on Chen [26] with some modifications. 3 g starch and 9 g EDTAD
were dispersed into 150 mL DMF, the mixture was stirred and
heated under reflux at 60 ◦ C for 24 h. The functional materials were
then treated by vacuum filtration, washed in sequence with DMF,
distilled water, saturated NaHCO3 solution (in order to release car-
boxylate and amine functions), distilled water, and finally with
ethanol. The white powder (SE) obtained was dried under vacuum
at 40 ◦ C and stored in a desiccator before usage.
2.3.3. Preparation of ester starch-EDTAD-NHS (SEN)
The ester SEN was prepared via the method of Li [27,28] with
slight improvement. The specific steps are as shown below: 1 g
SE, 1 g NHS, 2 g EDC were dispersed/dissolved in 50 mL distilled
water, the mixture were slowly stirred at room temperature for
1.5 h. After reaction, the product was processed with centrifugal
separation, and then washed with distilled water at least three
times. At last, the sample was dried under vacuum at 40 ◦ C and
stored in a desiccator before usage. The resulting products SE and
SEN were characterized by FTIR spectrum (Nicolet NEXUS 470 FT-IR
spectrometer), Elemental Analyzer (Vario EL III, Elementar Analy-
sensysteme, Germany).
2.4. Preparation of composite films
2.4.1. Preparation of film-forming solution
1.2 g of gelatin particles were soaked in 20 mL distilled water,
0.18 g glycerol (15% dry gelatin weight) was added into the above
gelatin aqueous solution as plasticizer. The mixture was then
 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
Scheme 1. The formation process of modified gelatin with SEN.
heated and stirred at 45 ◦ C for 2 h until the solution became homo-
geneous. The content of SEN was based on the mass ratio of dry
gelatin, which meant WSEN /W gelatin = 0%, 5%, 15%, 25%, 35% and
45%. SEN powder with different weight was dispersed in 20 mL dis-
tilled water and stirred at room temperature for 12 h to produce a
homogeneous suspension liquid, and then the SEN suspension liq-
uid was added to gelatin solution above drop by drop, together
with 1.2 mL acetic acid to the whole system in order to make them
more uniform and promote the ester ammonolysis reaction. Finally,
the above SEN-gelatin reaction solution was gently stirred at 45 ◦ C
for 12 h to obtain the filmogen. And the modified gelatin samples
were named Gel, 5%SEN-Gel, 15%SEN-Gel, 25%SEN-Gel, 35%SEN-
Gel and 45%SEN-Gel when the ratio of SEN to gelatin was 0, 5%,
15%, 25%, 35% and 45%, respectively. The free NH2 content of
SEN-gelatin reaction solution was determined by the improved Van
Slyke method.
2.4.2. Formation of composite films
The films were prepared using a casting process. 30 g SEN-
gelatin reaction solution was transferred into a teflon mold and
placed at room temperature for 2 h, and then put it in an oven at
40 ◦ C until dried. The dried films were peeled off and stored in a des-
iccator with relative humidity ≤20% before using. Besides, part of
SEN-gelatin reaction solution was freeze dried at −55 ◦ C, 70 Pa with
vacuum freeze drier (FD-1A-50, Beijing, China) and the lyophilized
powder was characterized by FTIR spectrum (Nicolet NEXUS 470
FT-IR spectrometer).
865
2.5. Characterization of composite films
2.5.1. Determination of residual amino group
The residual −NH2 groups in modified gelatin solution was
determined by the improved Van Slyke method at 45 ◦ C [29]. Sam-
ple solutions were mixed with acetic acid, sodium nitrite and stirred
for 45 min. The residual primary amine (mol/g) was calculated
according to the volume of N2 . All samples were tested in three
times.
2.5.2. Mechanical properties testing
After dried to a constant weight, the tensile strength (TS),
elasticity modulus (EM) and elongation at break (EB) of the com-
posite films were measured on a Computerized Eletronic Universal
Testing Machine (WDL-005, Jinan, China) equipped with tensile
load cell of 500 N. Before the test, the cross-head speed was set
at 10 mm/min. The initial grip length, width and thickness of
each film were measured by Vernier Caliper (0.02 mm/150 mm,
Shanghai, China) and electronic digital micrometer (0–25 mm/0-1
“0.001 mm/0.0001”, SYTEK, Zhejiang, China), respectively.
2.5.3. Water contact angles measurement
The equipment of DSA100 contact angle measuring system from
Krüss was applied to measure the water contact angles (CAs) of
all films by the sessile drop method. The gelatin reaction solution
was coated on the surface of glass sheet to obtain films with thick-
ness about 0.1 mm, and then stored in a desiccator with relative
humidity 15 ± 5% before use. When the droplet was deposited to
866 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
the surface of the film, the contact angle was immediately tested,
all the measurements are carried out at least three times to ensure
minimum error.
2.5.4. Light barrier properties and transparency
The ultraviolet and visible light barrier properties of the films
(1 cm × 2 cm) were measured by an ultraviolet-visible spectropho-
tometer (UV-7504C, Shanghai, China) at selected wavelengths from
200 to 800 nm. The transparency of the films was calculated by the
following Eq. (1):
Transparency = −logT/x
In this equation, T is transmission (%) at each wavelength; x is film
thickness (mm). According to the equation, the higher transparency
indicates that more opaque.
2.5.5. Water uptake behavior
Water uptake behavior of the films was determined on the basis
of the methods reported by Ruseckaite [30] with a little modifica-
tion. The sample films were cut into rectangular and the specimens
sized was about 15 mm × 10 mm. Prior to investigate the water
uptake property, the samples were conditioned at 20 ◦ C in a des-
iccator containing silica gel (RH 20% ± 5%) three days to constant
weight (W0 ). Then, the film samples were transferred into des-
iccators at 98% relative humidity (supersaturated salt solution of
CuSO4 ·5H2 O) at 20 ◦ C for ten days to absorb water until the weight
reached to equilibrium. The weight of samples at the adsorption
time of t was noted as Wt . The amount of water adsorbed at differ-
ent intervals even equilibrium was calculated by Eq. (2). All tests
were the means of at least three measurements.
Wt − W 0
Water uptake (%) = × 100
W0
2.5.6. Solubility studies
The films’ solubility was determined according to the method
reported by Cerqueira [31] with minor modifications. The solubility
study of gelatin films was carried out by incubating in phosphate
buffer (pH 7.40) at 37 ◦ C for different intervals (2, 4, 6, 8, 10 and
12 h). The gelatin films were dried at 50 ◦ C to constant weight before
using and named as M0 . After different intervals, the samples were
washed with distilled water, filtrated with a vacuum pump and
dried at 50 ◦ C to constant weight (Mt ). The solubility of the films
was then determined according to Eq. (3).
M0 − M t
Solubility (%) = × 100
M0
2.6. Statistical analysis
The analysis of data was performed through the analysis of vari-
ance (ANOVA) using a SPSS computer program (SPSS 13.0, SPSS
INC., Chicago, Illinois, USA). The statistical significance of differ-
ences between mean values was established at p < 0.05 and the
least significant difference (LSD) test was applied for all statistical
analyses.
3. Results and discussion
3.1. Characterization of starch, SE and SEN
3.1.1. FTIR spectra
Fig. 1 shows the FTIR spectra of Starch, SE and SEN. Com-
pared with starch, bands for SE appeared at 1751 cm−1 was C O
stretching vibration of the ester group, the peaks at 1637 cm−1 and
1417 cm−1 were symmetrical and asymmetrical stretching vibra-
tion of COOH, respectively. The absorption peak for SEN located at
(1)
Fig 1. FTIR spectra of starch, SE and SEN.
Table 1
Elemental analysis of starch, SE and SEN.
Material C (%) H (%) N (%)
Starch 39.29 6.732 –
SE. 40.66 6.711 0.527
SEN 39.46 6.764 1.370
1744 cm−1 were related to C O stretching vibration of the ester
group, bands at 1646 cm−1 and 1363 cm−1 corresponded to ␥-
(2)
dicarbonyl and C N stretching of amide groups, respectively. These
characteristic peaks demonstrated the successful preparation of SE
and SEM.
3.1.2. Elemental analysis
The results for element analysis of SE and SEN are shown in
Table 1. From the table, it can be seen that the nitrogen content
increased obviously whether in SE or SEN. Compared with starch,
the N content of SE increased to 0.527%, indicating that the func-
tionalization of starch with EDTAD was successful. As for SEN, the
testing result of nitrogen content reached to 1.370%, 0.800% higher
than that of SE, which meant that the esterification occurred suc-
cessfully between SE and NHS, with linkage of a five-membered
(3)
nitrogenous ring. The results above were accordance with the FTIR
spectra.
3.2. Conformation of SEN crosslinking with gelatin
3.2.1. FTIR spectra of gelatin, SEN and SEN-Gel film
Fig. 2 shows the FTIR spectra of gelatin (curve a), SEN
(curve b) and SEN Gel film (curve c). As for gelatin, bands
appeared at 1665 cm−1 (the C O stretching vibration of the amide
I band), 1544 cm−1 (N H stretching vibration of the amide II
band), 1449 cm−1 (aliphatic C H bending vibrations), as well as
1337 cm−1 and 1206 cm−1 (the C N bond stretching vibrations).
In the case of SEN-Gel film, all the characteristic peaks of gelatin
and SEN were detected. Also, the absorption peak at 1742 cm−1
was attributed to C O stretching vibration of the ester group, the
intensity of this absorption peak is reduced, indicating the amida-
tion reaction between NH2 in gelatin and the ester base in SEN. In
addition, the new band at 1640 cm−1 was assigned to C O stretch-
ing vibration of amide groups. The bending vibration of N H and
C N of the amide group appeared at 1555 cm−1 , Meanwhile, the
peaks at 1404 cm−1 was assigned to the C N stretching vibration
 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
Fig. 2. FTIR spectra of gelatin, SEN and SEN-Gel film.
Fig. 3. XRD spectra of gelatin, SEN and SEN-Gel.
of amide group. The results above confirmed the successful reaction
between gelatin and crosslinker SEN.
3.2.2. XRD spectra of gelatin, SEN and SEN-Gel
XRD patterns can examine the effect of SEN on the crystal struc-
ture and crystallinity of gelatin, the spectra of gelatin (curve a), SEN
(curve b) and 25%SEN-Gel (curve c) are illustrated in Fig. 3. From
the figure, the diffraction pattern of gelatin exhibited an extensive
broadening peak in the 2␪ range of 15.0–25.0◦ , originated from a-
helix and triple-helical structure of gelatin [32]. As for SEN, the
diffraction pattern showed the typical peaks of the native starch
with the main diffraction signals of 2␪ at around 15 ◦ ,17 ◦ , 18 ◦ and
◦
23 , respectively. However, for 25%SEN-Gel, the diffraction pattern
displayed the wide peaks of gelatin, and also expressed the char-
acteristic peaks of SEN at about 23 ◦ . In addition, SEN-Gel patterns
also formed the new crystal planes (0 1), (2 0), (2 1 0) at approxi-
◦ ◦
mately 10 ◦ , 19 and 23 , respectively. All the results demonstrated
the success modification of gelatin by SEN, corresponding with the
FTIR results.
867
Fig. 4. Residual amino group content of gelatin solution modified with SEN at dif-
ferent dosages.
3.2.3. Free −NH2 in the film formation solution
Testing the concentration of residual amino group in film-
formation solution can trace the reaction of gelatin effectively. Fig. 4
illustrates the changing curve of residual primary amino groups in
the film-formation solution against the ratio n (n = WSEN /WGel ). It
could be seen that the content of free −NH2 was determined by the
dosage of SEN, that is, the content of residual primary amino group
decreased obviously with the increase of the SEN dosage from 0%
to 45%, correspondingly. The minimum free NH2 content reached
to 3.28 × 10−4 mol/g (p < 0.05) while n = 45%. The main reason was
that the ester group in SEN reacted with the amino in gelatin,
thereby consumed −NH2 in gelatin. All the results showed that the
whole system overcame the inhibition of the interfacial reaction,
the maximum −NH2 conversion rate was around 42% (p < 0.05),
which confirmed the happening of amidation between gelatin and
SEN again.
3.3. Performance of gel-SEN films
3.3.1. Mechanical properties
Table 2 exhibits tensile strength (TS), elastic modulus (EM) and
elongation at break (EB) of the modified films as a function of
SEN content. The results demonstrated that the addition of SEN
exhibited the lower TS and EM than that of control film (with-
out incorporated glycerin and SEN) with the concomitant higher
EB. It could be seen from the table that TS (p < 0.05) decreased
with the increase of SEN content, which indicated that the mod-
ified gelatin films produced lower stress than pure gelatin film.
Also, EB reached the maximum value 108% while SEN content was
25%, meaning that the elasticity of the 25%SEN-Gel film was the
largest. TS decreased by 55% with the addition of 5 wt% SEN. This
behavior could be assigned to the fact that the crosslinking effect is
counterbalanced by the plasticization exerted by the hydroxylated
polymeric backbone of SEN, including its ability to attract water
to the plasticized protein system [33]. In addition, water could be
also used as a plasticizer from protein films. Therefore, the more
hydrophilic the film the higher plasticization was expected. The
plasticizer exhibited a great influence on flexibility of the film, due
to the reduced hydrogen interaction between adjacent polymer
chains, thereby increasing the flexibility of the 5% SEN-Gel film by
about 90%, as well as leading to the decrease of tensile strength and
Elasticity Modulus of the films. The increase of SEN concentration
868 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
Table 2
Mechanical properties of different Gel-SEN films.
Films Thickness (mm) Tensile Strength (MPa)
Gel 0.135 ± 0.011 73.07 ± 1.1
5%SEN- Gel. 0.170 ± 0.009 32.98 ± 0.7
15%SEN- Gel 0.200 ± 0.013 26.83 ± 0.4
25%SEN- Gel 0.221 ± 0.007 23.66 ± 0.6
35%SEN- Gel 0.241 ± 0.003 22.32 ± 0.5
45%SEN- Gel 0.259 ± 0.001 12.96 ± 0.6
Fig. 5. Photos of water contact angles of different gelatin films. (a Gel, b 5%SEN- Gel, c 15%SEN- Gel, d 25%SEN- Gel, e 35%SEN- Gel, f 45%SEN- Gel.).
had a statistically significant influence (p < 0.05) on the values of
TS, EB and EM.
In addition, Table 2 also shows the thickness of the different
modified gelatin film. And the result showed that films with high
ester (SEN) concentration showed greater thickness. The increase
of thickness seems to be related with the higher amount of SEN
in the films formulation. The EM improved in modified films with
the increase of SEN content, which would contribute to increase
thickness and mechanical resistance of the films.
3.3.2. Surface hydrophobicity
Surface hydrophobicity is very important for food packag-
ing materials, the formation of amido bonds can improve the
hydrophobicity of the modified gelatin films, which was confirmed
by the results of the contact angle measurements. In Fig. 5, labels of
a, b, c, d, e and f represents the contact angle results of the pure
gelatin, 5%SEN-Gel, 15%SEN-Gel, 25% SEN-Gel, 35% SEN-Gel and
45% SEN-Gel, respectively. From the figure, the pure gelatin film
◦
with a typical contact angle of 77.8 was observed, which was due
to the hydrophilic group, such as amino, carboxyl and hydroxyl,
exposed in gelatin chains. After modified by SEN, the contact angle
◦
of the films presented an increase (p < 0.05) from 77.8 to 126.7
◦
. Also, the higher SEN content, the better surface hydrophobicity.
The phenomenon was due to the reaction between SEN and gelatin,
which consumed the exposed hydrophilic group in gelatin, and
produced the hydrophobic acidamide. In addition, hydrogen bond
formed between the hydrophilic groups in gelatin and starch-based
crosslinker was also a major factor to improve the hydrophobic.
The modified films with improved hydrophobicity overcame the
shortcoming of water-sensitive for gelatin.
3.3.3. SEM pictures
Fig. 6 shows the SEM photographs of gelatin composites
with/without SEN, labels a, b and c represents the surface, cross-
section and freeze drying of the film, respectively. From the figure,
almost all of the films displayed highly uniform, dense surface and
cross-section. Also, there was an obvious difference between the
Elongation at Break (%) Elasticity Modulus (MPa)
8.1 ± 3.7 1927.52 ± 1.7
101.2 ± 8.4 183.06 ± 1.2
104.7 ± 8.9 189.45 ± 0.9
108.0 ± 9.2 189.31 ± 1.3
83.3 ± 7.5 194.29 ± 1.2
42.0 ± 5.8 209.52 ± 1.5
native gelatin film and the 25%SEN-Gel film no matter from a, b
or c. As for the surface feature, control gelatin film (a1 ) exhibited
a homogeneous and smooth surface, while a relatively low con-
tent of particles with different sizes and shapes into the matrix
were observed when SEN was added [Fig. 6(a2 )]. Compared with
the cross-section of the native gelatin film (b1 ), the modified film
[Fig. 6(b2 )] showed obvious stratification, and there were obvious
melting phenomena in the stratified junction, proving the suc-
cess of the reaction between gelatin with SEN. In our study, phase
separation, seems far less likely to have occurred at lower SEN con-
centration (i.e. 5 wt%), but the size of the dispersed particles was
small enough to produce transparent films. For higher amounts
of SEN in the mixture (45 wt%), the inhomogeneity of the reac-
tant was evident, larger particles were observed. These results also
explained the followed opacity of the modified films. In addition,
for lyophilized films, the control gelatin film (c1 ) presented thinner
pore and wall, while the modified film [Fig. 6(c2 )] showed the oppo-
site effect, the pore canal almost disappeared, the wall of the hole
was thick, thus, hindering the permeability of water into molecules.
Through the way of vacuum freeze drying, the dehydration of the
film was more thorough, the freeze-dried samples were stable and
easy to store for a long time. The results above provided an evi-
dence for the hydrophobic property and the followed water-uptake
behavior of the modified films.
3.3.4. Light transmission and transparency of gel-SEN films
Transparency of films is greatly relevant to the films function-
ality due to their great impact on the appearance of the products.
Visually, SEN-modified films turned from pale yellow to light brown
as SEN content increased from 0 to 45 wt%, which may be attributed
to the formation of conjugated Schiff’s bases during the reaction.
Light transmission and transparency in the UV and visible light
range from 200 nm to 800 nm of the SEN-Gel films are shown in
Table 3. From the table, it could be seen that the transmission of
UV light was very low at 200 nm and 280 nm for all films, while
transparency value was high at 280 nm for Gel and 5%SEN-Gel.
Based on this, SEN-Gel films exhibited excellent barrier properties
 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871 869

Fig. 6. SEM images of surface (a, c) and cross-section (b) of gelatin composites. (a1 Surface of Gel, b1 Cross-section of Gel, c1 Surface of freeze-dried Gel, a2 Surface of
25%SEN-Gel, b2 Cross-section of 25%SEN-Gel, c2 Surface of freeze-dried 25%SEN-Gel.).

Table 3
Light barrier properties of modified Gel-SEM films.

Films Light transmission at different wavelength (%) Transparency Value

200 nm 280 nm 400 nm 500 nm 600 nm 700 nm 800 nm 280 nm 600 nm

Gel 0.0 1.1 45.2 54.4 58.6 61.4 63.7 9.60 1.14
5%SEN-Gel 0.0 0.4 25.2 32.2 35.2 37.6 39.7 11.75 2.22
15%SEN-Gel 0.0 0.0 13.4 19.5 22.8 25.4 27.6 – 3.15
25%SEN- Gel 0.0 0.0 8.7 13.4 16.2 18.5 20.5 – 3.87
35%SEN- Gel 0.0 0.0 7.5 11.7 14.3 16.3 17.9 – 4.14
45%SEN- Gel 0.0 0.0 4.6 7.3 9.1 10.6 11.9 – 5.10

against UV light in the range of 200–280 nm, which mainly due

to the high content of aromatic amino acids in gelatin to absorb
UV light. Table 3 also shows that the transmission of visible light
was found lower than 65% at 600 nm and 800 nm for all films,
while transparency value was over than 1 at 600 nm, indicating
that the modified SEN-Gel films were opaque. It was noteworthy
that the transparency value of SEN-Gel films at 600 nm boosted
obviously with the increase of SEN content, which represented
the opacity of the modified gelatin films. It was assumed that the
increase tendency in opacity with SEN content increased could be
a consequence of phase separation to a certain degree, which was
also confirmed by SEM. It is reported that protein-polysaccharide
systems are characterized by limited compatibility between their
components [34]. In short, all the results revealed that the addition
of SEN improved the UV barrier properties of gelatin films, result-
ing from the formation of amido bonds between the ester groups
in SEN and the amino groups of the lysine or hydroxylysine side
chains in gelatin, which exhibited high UV light absorbance. It was
interesting that this high level of absorption in the UV–vis range Fig. 7. Water uptake behavior of gelatin films incorporated with different dosage of
SEN.
was a desirable film characteristic in food packaging.

3.3.5. Water uptake behavior Fig. 7 shows the water uptake curves of SEN-Gel films versus
The humid environment was also a significant factor inducing time at the relative humidity of 98%. For all films, water uptake
food decomposition except for the UV and visible light referred was more rapid at the initial stages and then the absorption
above, any improvement in water resistance is greatly important. rate became gentle, even hardly changed, indicating that samples
870 C. Shi et al. / International Journal of Biological Macromolecules 109 (2018) 863–871
Fig. 8. Solubility properties of Gel-SEN composite films.
became equilibrated with the storage RH. And the obtained val-
ues for modified gelatin films are lower and statistically different
(p < 0.05) from the values for blank films. Compared with the native
gelatin film, the water uptake tendency declined (p < 0.05) with the
increase (p < 0.05) of SEN content. The reason was that the devel-
opment of SEN in the protein structure, rendered amino-side chain
groups were not available to bind water by hydrogen bonding, so
hydrophilic groups along protein chains became less exposed and,
therefore, less accessible to water molecules. Therefore, the adding
of SEN into reaction system was expected to enhance the water
resistance of modified gelatin/starch films. This phenomenon was
accordance with the results of the contact angle test.
3.3.6. Solubility test
The solubility profiles of modified gelatin films with different
SEN content in phosphate buffered saline (PBS, pH 7.4) was inves-
tigated, the results are illustrated in Fig. 8. It is found that the
solubility of the controlled film was very high, while the modified
films decreased (p < 0.05) continuously with the increase (p < 0.05)
of SEN content, no matter at what time stage. This is related with the
hydrophobic behavior of SEN and with the increase of ester bonds
on gelatin films matrix with the increase of SEN concentration, that
are more available to resist with the water molecules. There were
a lot of hydrophilic amino and carboxyl groups in gelatin molec-
ular chains, which facilitated the solubility of the films. However,
as for the modified gelatin films, partially hydrophilic groups in
gelatin reacted with SEN, thereby reducing the occurrence of water
molecules into the membrane matrix. In addition, it was indicated
that the solubility of all the films increased with the dissolution
time prolonged, the maximum solubility of controlled film reached
about 82% (p < 0.05) at 12 h, while the modified films with 45%SEN
content dissolved the slowest, and the value of solubility was only
42% (p < 0.05) at 12 h, which was a half of the controlled film. This
is mainly because of the reaction between gelatin and SEN, which
formed a new amide bond to resist adverse factor outside. Also,
the strong hydrogen bond and electrostatic interaction between
gelatin and SEN also restrained PBS buffer solution diffusion and
protected gelatin polypeptide chains from dissolution. In short, the
special properties of SEN endowed the composites with good low
solubility ability, improved the service life of the films and provided
a basis for its application.
4. Conclusion
In summary, a novel ester SEN was successfully synthesized for
the first time and introduced into gelatin. 1 H NMR, FTIR spectra
and element analysis proved the structure of SEN, also, FTIR, XRD
spectra, SEM images and free NH2 test confirmed the reaction
between SEN and gelatin. The higher EB and lower EM demon-
strated a more flexible and pliable material was obtained. The
highest water contact value 126.7◦ of 45%SEN-Gel film material
indicated the better surface hydrophobicity. The light barrier per-
formances were improved obviously and the opacity of composite
films was dosage dependent. In addition, the modified film also
exhibited better resistance to solubility, decreased the water sensi-
tivity and reduced the swelling. In a word, the incorporation of SEN
into gelatin-based composite films exhibited excellent properties
than the native gelatin film.
Acknowledgment
We gratefully acknowledge the National Natural Science Foun-
dation of China (NO. 31570570 and 21401114), and the Program for
Scientific Research Innovation Team in Colleges and Universities of
Shandong Province.
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