Food Hydrocolloids 108 (2020) 106016

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Food Hydrocolloids
journal homepage: http://www.elsevier.com/locate/foodhyd

Fabrication of films with tailored properties by regulating the swelling of

collagen fiber through pH adjustment
Jinlong Xu a, b, Fei Liu a, b, Tao Wang a, b, H. Douglas Goff c, Fang Zhong a, b, *
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
b
School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
c
Department of Food Science, University of Guelph, Guelph, ON, N1G 2W1, Canada

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

Keywords: Acid swelling collagen fibers have been commonly used to prepare casing or film, but how the swelling degree
Collagen fiber regulates the properties of collagen fiber dispersions (CFD) and their related films are still unclear. Herein,
Dispersions variant swelling degrees of collagen fiber were obtained by controlling the pH of CFD. The swelling ratio of
pH
collagen fiber increased and then decreased with the decrease of pH from 4.0 to 1.5. The maximum viscosity,
Acidic swelling
Films
modulus and denaturation temperature appeared at pH 3, where the fibers interconnected to form a dense
network structure. This sufficient swelling enabled excellent mechanical and thermodynamic properties of films,
whereas insufficient swelling of CFD led to inferior properties. Films from collagen fiber swelled at pH 3 further
showed a more ordered layer structure, but cross-sectional microstructures with disintegrated tiny fibers along
with the reduction of triple helix content for those at low pH were observed. This study provided a better un­
derstanding and guidance of pH regulation in actual production of collagen films using bovine dermis.

1. Introduction
Collagen constitutes a class of proteins that represent the main
component of connective tissues of all vertebrates, accounting for about
25%–30% of the total protein content in the body (Pati, Adhikari, &
Dhara, 2010). Type I collagen, the most common and abundant type,
features a triple helix chain with a length of 300 nm and a diameter of
1.5 nm. The molecules assemble into thin (20–500 nm) and long (>1
mm) fibrils by lateral aggregation and axial dislocations (Gautieri,
Vesentini, Redaelli, & Buehler, 2011; Park et al., 2012; Svensson,
Mulder, Kovanen, & Magnusson, 2013). The coaxial overlapping stipu­
lates the radial fibril growth, affording the formation of type I collagen.
Due to the outstanding molecular architecture, collagen features
intriguing properties such as high strength, flexibility, biocompatibility,
and biodegradability, contributing to a wide variety of uses in tissue
engineering, cosmetics and the food industry (Deng, Kang, Liu, Feng, &
Zhang, 2018; Long et al., 2017; Oechsle, Bugbee, Gibis, Kohlus, & Weiss,
2017).
Collagen is also an excellent film-forming material with many
bioengineering applications (Liu, Ren, & Wang, 2013; Veeruraj, Liu,
Zheng, Wu, & Arumugam, 2019). Currently, collagen from bovine
dermis is widely applied in the food industry as edible films, exclusive to
protein films that are commercially available in the meat industry (Wolf,
Sobral, & Telis, 2009; Wu, Liu, Wang, & Ye, 2018). Collagen casing, a
commercial film merchandise, is capable of obstructing the transmission
of oxygen from the environment and reducing the exudation of meat
juice. At the same time, collagen casing has uniform size, suitable
strength, sanitary, easy-stuffing property, and other advantages over
natural casing obtained from cattle, sheep or pig intestines (Shi et al.,
2019).
Collagen film is generally produced from a formulated colloidal
aqueous dispersion (Deiber, Peirotti, & Ottone, 2011; Shi et al., 2019;
Wang et al., 2017) containing around 0.5–4% w/w of total suspended
collagen fibers or clusters (Deiber et al., 2011). Formation of casing/­
films is enabled by extrusion or casting after swelling of collagen fibers
in acidic or alkaline conditions. The swelling of collagen fiber is gov­
erned by the Donnan membrane effect characteristic of osmotic pressure
contrast between the protein phase and the external solution (Bowes &
Kenten, 1950; Lloyd, Marriott, & Pleass, 1933). Another perspective
ascribes the swelling of the collagen fiber to electrostatic repulsions
(Shan & Wang, 2001; Tang, 2012). pH is an important factor for the
swelling of collagen fibers. Kaye and Lloyd (1924) reported that the
swelling maxima of collagen fiber appeared at pH ¼ 2.2 and pH ¼ 11.8.
Others reported similar findings (Marriott, 1932; Shan et al., 2001). Acid

* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
E-mail address: fzhong@jiangnan.edu.cn (F. Zhong).

https://doi.org/10.1016/j.foodhyd.2020.106016
Received 5 December 2019; Received in revised form 22 April 2020; Accepted 11 May 2020
Available online 12 May 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
J. Xu et al.
swelling of collagen fiber is the most common approach for the pro­
duction of collagen casing and collagen films using bovine dermis (Wolf
et al., 2009; Wu et al., 2018). However, the effect of acidic pH on the
physicochemical properties of collagen fiber dispersions (CFD) is still
unclear, which is important for the actual production of collagen
casing/films.
In the present study, dispersions and films with varied collagen fiber
swelling degrees were prepared by controlling the pH from 1.5 to 4.0.
The rheological behavior and microstructure by fluorescence staining of
dispersions were studied. Furthermore, the mechanical and thermody­
namic properties of films were studied using texture analysis and dif­
ferential scanning calorimetry. The micromorphology and structure of
collagen fiber in films were investigated by scanning electron micro­
scopy, Fourier-transform infrared spectra, and X-ray diffraction. This
study will provide a better understanding of the effects of pH on in­
dustrial production of collagen casing/film when using the animal skin.
2. Materials and methods
2.1. Materials
Bovine skin (dermis) was offered by China Beijing Qiushi Agriculture
Development Co., LTD (Beijing, China). Fluorescein isothiocyanate
(FITC) was purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO,
USA). All other reagents of analytical grade were purchased from China
Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai, China).
2.2. Swelling test of collagen fiber
The swelling ratios of collagen fiber at different pH were tested ac­
cording to previous methods with some modifications (Marriott, 1932;
Shan et al., 2001). Bovine skins were repeatedly washed with distilled
water and the pH of skins was adjusted to 4.8 with citric acid and sodium
citrate solution (4.35 and 5.89 g/L, respectively). The washed skins were
cut into 1 cm � 1 cm strips and ground with ice at the ratio of 1/2 (w/w).
Then the skin slurries were lyophilized to get the collagen fiber. Collagen
fiber was weighed (0.5 g) and placed in 150 mL acid solution. The pH of
the acid solutions was adjusted with HCl to 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0,
respectively. After being held at 4 � C for 24 h, the collagen fibers were
taken out and the surface moisture was removed by filter papers. The
final weights were recorded and the swelling ratio was determined using
equation (1):
wf wi
SRð%Þ ¼ � 100 (1)
wi
where wi and wf are the initial and final mass of each sample. The
experiment was carried out in triplicate.
2.3. Preparation of CFD
Bovine skins were repeatedly washed with distilled water and the pH
of skins was adjusted to 4.8 with citric acid and sodium citrate solution
(4.35 and 5.89 g/L, respectively). The washed skins were cut into 1 cm
� 1 cm strips and ground with ice at the ratio of 1/2 (w/w). The skin
slurries were mixed with different concentrations (0.38, 0.26, 0.18,
0.13, 0.09 and 0.05 mol/L) of HCl solution and stirred using a digital
cantilever stirrer (IKA Works Inc., Wilmington, USA) to form homoge­
nous collagen fiber pastes (pH ¼ 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0). After
being held at 4 � C for 24 h, the collagen fiber pastes were dispersed in
deionized water and homogenized at 30 MPa by a homogenizer (PANDA
PLUS 2000; GEA Niro Soavi Co., Parma, Italy) to obtain the homoge­
neous CFD (0.5 wt%, pH ¼ 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0).
2
Food Hydrocolloids 108 (2020) 106016
2.4. Rheological measurements
A rotational rheometer (DHR-3, TA, USA) equipped with a stainless-
steel parallel plate (40 mm diameter, 1 mm gap) was used to study the
rheological properties of the CFD. About 1.5 mL of the dispersion was
placed in the equipment and compressed to 1 mm gap between the
plates. All the experiments were conducted at 25 � C and the temperature
control was established by a Peltier plate.
2.4.1. Viscosity test
The apparent viscosity of the dispersions with different pH was
measured as the shear rate (_γ ) was increased from 0.1 to 1000 1/s.
2.4.2. Dynamic viscoelastic behavior
Dynamic viscoelastic interval of the dispersion was determined by
strain sweep with a strain range from 0.01 to 1000%. Dynamic visco­
elastic behavior of the dispersions was tested in the angular frequency
range from 0.1 to 100 rad/s and the strain was fixed at 1%.
2.4.3. Dynamic temperature sweep
Dynamic temperature sweep measurements of tan δ were performed
in the temperature range from 20 to 50 � C at a heating rate of 1.0 � C/
min. The strain was controlled at 1% and the angular frequency was
fixed at 6.28 rad/s.
2.5. Microstructure of CFD
The microstructure of CFD was observed using fluorescent staining
method. Samples were stained by fluorescein isothiocyanate (FITC) ac­
cording to the method of Sow, Chong, Liao, and Yang (2018) with some
modifications. The dye solution was dissolved in DMSO, and 10 μL was
added into 100 mL samples. Labelling was carried out at 4 � C for 2 h.
Fluorescence images were observed with a fluorescence microscope
(Nikon 80i, Nikon Corporation, Japan) at a magnification of 400�.
2.6. Preparation of films
40 g of CFD were poured into Petri dishes (10 cm � 10 cm) and air-
dried at room temperature. The peeled films were placed in a constant
temperature and humidity chamber (Boxun Instrument HC BXS150S,
China) at 25 � C and 52% relative humidity (RH) for 48 h to serve as test
samples.
2.7. Mechanical properties
Tensile strength (TS) and elongation at break (EB) were analyzed
according to the ASTM standard method D882 with some modifications
using a texture analyzer (TA. XTplus100, Lloyd Instruments, U.K.)
equipped with a tension grip system A/TG. Samples were cut into
rectangular strips (10 mm � 50 mm) and then clamped and stretched to
breaking point. The initial grip separation and crosshead speed were 30
mm and 0.5 mm/s, respectively. A micrometer caliper was used to
measure the film thickness. TS (MPa) and EB (%) were calculated from
the following equations (2) and (3):
F
TS ¼ (2)
A
ðL L0 Þ � 100
EB% ¼ (3)
L0
where F is the maximum load (N) to stretch film at the breaking point, A
is the cross-sectional area (mm2) of films, L0 (mm) is the initial length of
films and L (mm) is the length of films when it breaks. Eight replicates
were tested for each film.
J. Xu et al.
2.8. Differential scanning calorimetry (DSC)
Thermodynamic properties of films were studied using a DSC (204
F1, NETZSCH Scientific Instruments, Germany). Specimens were sliced
into several flakes and 5–6 mg of samples were hermetically sealed into
aluminum pans. An empty sealed aluminum pan served as a reference.
The sample was heated from 20 � C to 150 � C at a heating rate of 10 � C/
min. The sample chamber was purged with nitrogen gas at a flow rate of
70 mL/h.
2.9. Scanning electron microscopy (SEM)
The morphology of the films was determined by scanning electron
microscopy (SEM, SU8220, Hitachi, Japan) under high vacuum mode at
an accelerating voltage of 5.0 kV. The film strips were cryo-fractured in
liquid nitrogen and then broken off in order to observe the cross-
sections. Samples were dehydrated in a desiccator for 5 d. Cross-
sections were cut from the dehydrated samples and fixed on a bronze
stub, and then sputter-coated with gold.
2.10. Fourier transform infrared (FTIR) spectroscopy
According to a previous method (Shi et al., 2019), FT-IR spectra of
films were recorded on an FT-IR spectrometer (Nicolet IS 10, Thermo
Electron, USA) using attenuated total reflection (ATR) mode at room
temperature. A total of 64 scans were made for each specimen at 4 cm 1
resolution, all between 4000 and 400 cm 1. An OMNIC 8.2 data
collection software program (Thermo Fisher Scientific Inc., USA) was
used to analyze the spectra.
2.11. X-ray diffraction (XRD)
XRD analysis of the films was performed on an X-ray diffractometer
(D2 Phaser, Bruker AXS Germany). A copper target at 40 mA and 40 kV
was used as the radiation source (0.154 nm), and data were collected
from 5� to 60� of 2θ incidence angle at a speed of 0.5� and the scanning
rate was 12� min 1.
2.12. Statistical analysis
Data was presented as mean value � standard deviation of at least
three replications. SPSS 24.0 package (IBM, New York) was used to
analyze the data by one-way analysis of variance (ANOVA). Duncan’s-
multiple range test was applied to determine the significant differences
of the mean values (P < 0.05).
3. Results and discussion
3.1. Swelling ratio
The swelling curve of collagen fiber was determined and shown in
Fig. 1. The water could enter the collagen fiber and cause the swelling of
the fiber under acidic conditions. With the decrease of pH from 4.0 to
1.5, the swelling ratio of collagen fiber increased and then decreased
with a maximum appearing at pH 3.0. The result was similar to Marriott
(1932) but different from the results of Kaye et al. (1924) and Shan et al.
(2001), which showed a maximum swelling ratio at around pH 2.0. This
may be due to the different materials used as the former used bovine skin
and the latter used goat skin. The swelling ratio of collagen fiber
decreased at low pH, indicating that too low pH was not conducive to
the swelling of collagen fiber.
3.2. Rheological properties of CFD
The variation of shear viscosity (η) relative to shear rate (_γ) is shown
in Fig. 2A. Like other polymer systems, CFD exhibited shear-thinning
3
Food Hydrocolloids 108 (2020) 106016
Fig. 1. Swelling ratio of collagen fiber at different pH.
behavior at all pH conditions, featuring a pseudoplastic fluid. With the
reduction of pH, the viscosity of dispersions increased and maximized at
pH 3.0, followed by a sharp decrease. It is well known that collagen fiber
swells under acidic condition (Marriott, 1932), the maximum viscosity
of fiber bundles at around pH 3.0 demonstrated the maximum swelling
ratio. Collagen molecules curled at isoelectric point and extended and
entangled beyond the isoelectric point, over which the viscosity was
increased. For materials exposed to a low pH, increased ionic strength
enabled the curling of the molecules again, and, therefore, decreased
viscosity (Fu, 2002). Viscosity of CFD affects its flow in the pipeline, and
is pivotal to production.
To further understand the shear-thinning behavior of the CFD at
different pH, the Power law constitutive equation η ¼ k_γn 1 was used to
fit the viscosity curves, where η is the apparent viscosity, k the viscosity
coefficient, γ_ the shear rate, and n the power law exponent. Pseudo­
plastic fluids have n value between 0 and 1 while Newtonian fluid has an
n value equal to 1 (Xu, Zhang, Liu, Sun, & Wang, 2016). The values of k
and n of the dispersions are shown in Table 1. All the R2 values were
higher than 0.99, so the shear-thinning behavior of the dispersion can be
well fitted. The pseudoplastic property was indicated by n value that was
between 0 and 1 for all the tested samples, with the dispersion at pH 3.0
obtaining the lowest n value representative of the strongest
pseudoplasticity.
The dependence of storage modulus (G0 ) and loss modulus (G00 ) on
angular frequency (ω) for CFD is shown in Fig. 2B and C. All of the G0 and
G00 increased with increasing ω. Initially, the G0 and G00 were higher at pH
3.0 than at pH 3.5 and they were reversed with the increase of ω. Spe­
cifically, decrease occurred at pH 4.0 and pH 2.5, and the modulus
decreased further when the pH reached 2.0 and 1.5. For the sample at
pH 4.0, there was insufficient swelling with less water inside the
collagen fiber. By reducing the pH below 3.0, collagen molecules curled
again together with sharp decreases of modulus and swelling ratios (Fu,
2002). The relational expression of G’ ~ ωn’ was used to fit the linear
correlation between log G0 and log ω, where the value of the exponent n’
can be obtained. The system exhibits a n’-dependent gel behavior and
demonstrates strengthening elasticities when n’ is from 1 to 0. As shown
in Fig. 2D, the CFD behaved like an elastic gel as all the n’ values were
close to 0, and the strongest gels were obtained for those formulated at
pH 3.0. The counterparts, especially those at lower pH, presented as
weak gels.
There was a concave peak on the curve of tan δ, which represented
the denaturation temperature of the CFD. It was observed that the
denaturation temperature of collagen moleculea generally decreased
with greater acidity. As shown in Fig. 2E, a slight increase from 41.7 to
J. Xu et al. Food Hydrocolloids 108 (2020) 106016

Fig. 2. Rheological properties of CFD (A) Dependence of the steady shear viscosity (η) on the shear rate for CFD at different pH (B) Storage modulus G0 and (C) Loss
modulus G00 as a function of angular frequency ω for CFD at different pH (D) Exponent n’ of G’ ~ ωn’ as a function of pH for CFD (E) Loss tangent (tan δ) as a function
of temperature for CFD at different pH.

decreases of the denaturation temperature took place when the pH was

Table 1
at 2.0, indicating that acid had greater impact on the denaturation
Power law model-fitted parameters of collagen fiber dispersion at different pH.
temperature of collagen fibers.
pH K n-1 n R2

1.5 7.510 0.787 0.213 0.995 3.3. Microstructure analysis of CFD

2.0 12.732 0.828 0.172 0.998
2.5 20.076 0.851 0.149 0.998
In this study, fluorescein isothiocyanate (FITC) was used to label the
3.0 32.034 0.869 0.131 0.999
3.5 29.829 0.861 0.139 0.998 collagen fiber and the microscopic images were observed by a fluores­
4.0 20.968 0.839 0.161 0.991 cence microscope equipped with a high-resolution camera. The CFD
presented different microstructures at different pH and the collagen
fiber showed various morphologies (Fig. 3). Collagen fibers tend to
43.2 � C and a prominent decrease of denaturation temperature from aggregate and form interior voids when the pH was 4.0, probably due to
43.2 to 35.0 � C occurred when the pH reduced from 4.0 to 3.0 and 3.0 to exudation of moisture from the collagen fibril intervals. The fibers
1.5, respectively. The results were in agreement with previous studies interconnected with each other to form a dense network structure at pH
(Yang, Xu, Shen, Liu, & Li, 2016). The swelling ratio may have exerted 3.5 and pH 3.0. When the pH reached 2.5, the morphology of collagen
influences on the denaturation of CFD. However, much more apparent fibers was ill-defined and no visible structures could be observed at pH

Fig. 3. Morphological characterization by fluorescence microscopy of collagen fiber in the dispersions at different pH.

4
J. Xu et al. Food Hydrocolloids 108 (2020) 106016

2.0 and pH 1.5. Different swelling ratios of collagen fibers resulted in the Table 2
differences in fiber morphology. FITC was apt to bond to bigger fiber Effect of pH on the mechanical properties of collagen fiber films.
bundles, whose fluorescence intensity was different under different pH pH TS (MPa) EM (Mpa) EB (%) Thickness (μm)
conditions. The change of fluorescence intensity was related to the d c b
1.5 77.62 � 2.37 23.09 � 1.62 5.71 � 1.35 17.61 � 1.10b
change of collagen fibers aggregation. The fibers may disintegrate into 2.0 84.61 � 4.43c 24.75 � 1.67bc 5.10 � 0.64b 17.74 � 0.83b
smaller individuals, which were difficult to observe at pH 2.0 and pH 2.5 87.78 � 4.72bc 26.81 � 1.34ab 4.60 � 0.64b 18.40 � 1.02b
1.5. Despite that the change of fluorescence intensity may also be 3.0 95.70 � 7.59a 27.32 � 1.52a 5.26 � 0.66b 18.50 � 0.44b
affected by the change of pH, the morphological changes of collagen 3.5 94.14 � 4.13ab 27.13 � 2.31ab 5.63 � 0.73b 18.65 � 0.52b
4.0 93.81 � 6.70ab 23.55 � 0.89c 8.33 � 1.34a 20.03 � 1.01a
fibers seemed to be the key factor.
a
Values are expressed as mean � standard deviation. Different lowercase
3.4. Tensile properties of films letters in the same column indicate a statistically significant difference (P <
0.05). TS, EM and EB represent tensile strength, elastic modulus and elongation
at break respectively.
The effects of pH on the mechanical properties of collagen films were
studied. Stress-strain curves are shown in Fig. 4, the tensile strength
(TS), elastic modulus (EM) and elongation at break (EB %) are listed in
Table 2. The highest TS of the film appeared at pH 3 and significant
reduction happened when the pH further decreased. When the pH was
3.5 and 4.0, the TS had a slight decrease, although with no statistical
significance. Sufficient swelling could make the collagen fibers to be
more intertwined with each other, being beneficial to the final TS of
films. Too low pH (pH 1.5, pH 2.0 and pH 2.5) of the dispersion was not
conducive to the TS of the final films, which was related to the swelling
ratio of collagen fibers in the dispersions. Collagen fibers had smaller
swelling ratios under low pH conditions. The EM was related to the
stiffness of films. It varied with pH and showed a similar trend to the
swelling ratio of collagen fiber. There was no significant difference in the
elongation at break (EB %) of films except for the films at pH 4.0. The
exudation of moisture from the collagen fibers led to the increase in fiber
bundles, which resulted in the increase of film flexibility.

3.5. Differential scanning calorimetry (DSC) of films

The thermodynamic properties of collagen fiber films were detected
by DSC and the results are shown in Fig. 5. It could be seen that the films
had denaturation peaks between 90 and 100 � C and their variations
were consistent with the swelling ratio of collagen fiber. The collagen
molecules and fibers were readily bonded and connected to each other at
the maximum swelling degree (pH 3.0), thus forming a stable film
structure. The acid seemed to have more significant impact at lower pH
(pH 1.5, 2.0 and 2.5) where the films were denatured at lower tem­
perature. The denaturation temperature displayed in DSC was different
from that in rheological measurement. Rheological measurements
showed the denaturation temperature of the collagen fibers in the
dispersion, and DSC showed the denaturation temperature of the
collagen fibers in the films. Different moisture content would affect the
Fig. 5. DSC curves of films formed by CFD at different pH.
denaturation temperature of the protein, and the denaturation temper­
ature of the sample would decrease with the increase of moisture con­
tent (Atuonwu, Ray, & Stapley, 2017; Rao & Labuza, 2012).
3.6. SEM analysis of films
The cross-sectional microstructures of the collagen fiber films at
different pH were observed by SEM (Fig. 6). It could be seen that the
collagen fibers formed a layered network in the films. The internal
structure at pH 3.0 was ordered and similar to the film at pH 3.5. The
fiber structure of the film at pH 4.0 was disordered and rough, attributed
to the aggregation of fibers in the dispersions. With the decrease of pH,
the internal structure of films, especially those at pH 1.5, presented
dense fiber network consisting of very tiny collagen fibers, which
confirmed the previous speculation in Sect. 3.3 that the collagen fibers
disassembled into smaller forms at low pH. The results of SEM coincided
with the observation of fluorescence studies. Compared with the results
of tensile test, it was suggested that larger fiber structures and higher
ordered networks were more beneficial to the tensile properties of
collagen fiber films.

3.7. FT-IR analysis

The secondary structures of collagen were investigated by a FT-IR

Fig. 4. Stress-strain curves of films formed by CFD at different pH.
spectrometer and the results were shown in Fig. 7. All the films had
the typical characteristic bands of amide A, B, I, II and III, associated
with the peptide bonds in collagen. The amide A band represented a free
N–H stretching vibration and the peak positions of the films formed at
pH 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 were 3291, 3295, 3296, 3304, 3305,
3305 cm 1, respectively. When the N–H group of a peptide was involved
in hydrogen bonding, the band would be shifted to a lower wavenumber
(Li et al., 2013; Liu, Dan, & Dan, 2016), which coincided well with the

5
J. Xu et al.
Fig. 6. SEM micrographs of cross sections (magnification 2.4 K) of films formed by CFD at different pH.
Fig. 7. FT-IR spectra of films formed by CFD at different pH.
samples under decreasing pH in this study, indicating that the acidifi­
cation promoted the disintegration and degradation of collagen fibers.
The amide B bands at about 3077 cm 1 were mainly attributed to the
asymmetrical stretching vibrations of CH2. The amide I bands at about
1629 cm 1 and amide II bands at approximately 1540 cm 1 were
ascribed to proteinic amide C– – O stretching vibrations and the N–H
bending vibrations coupled to C–N stretching vibrations, respectively.
The amide III at around 1235 cm 1 arose from the C–N stretching and
N–H bending vibrations from amide linkages, as well as the absorptions
arising from wagging vibrations from CH2 groups in the glycine back­
bone and proline side chains (He et al., 2011). Additionally, the value of
the absorbance of amide III relative to that at 1450 cm 1 (AIII/A1450) was
calculated to quantify the relative amounts of integrated triple helical
structures (Liu et al., 2017; Veeruraj, Arumugam, Ajithkumar, & Bala­
subramanian, 2015). With the decrease of pH, the value of AIII/A1450
increased and then reduced (Table 3). The swelling of collagen fiber
promoted the exposure of the triple helix structure, which resulted in the
value of AIII/A1450 increasing from 1.2152 (pH 4.0) to 1.2996 (pH 3.0).
Upon further acidification, the value began to decrease as a result of the
Table 3
Absorption ratio of AIII/A1450 of films with different pH.
pH 1.5 2.0 2.5 3.0 3.5 4.0
Absorption ratio 1.1054 1.2853 1.2881 1.2996 1.2532 1.2152
6
Food Hydrocolloids 108 (2020) 106016
adverse effect of excessive acids on the collagen fibers. The AIII/A1450 for
the film at pH 1.5 was significantly smaller than its counterparts, indi­
cating that excessive acid was detrimental to the integrity of the triple
helix structures.
3.8. X-ray diffraction (XRD) analysis of films
The crystal diffraction patterns of the films at different pH were
recorded by an X-ray diffractometer. A major sharp peak appeared at 2θ
of ~8� (Fig. 8A). This is a crystal diffraction peak of triple helix, which
indicated an intermolecular lateral packing between collagen molecular
chains that were assembled into fibrils by the Schmitt model, by which
the formation of the collagen fibers were enabled (Hu et al., 2017). A
secondary broad peak at 15� to 25� (2θ) was assigned to the amorphous
fractions. The intensity of the peak at 2θ of ~8� , varying within the
selected pH, could represent the content of triple helix (Chen, Dan,
Wang, Liu, & Dan, 2016; Liu, Antoniou, Li, Ma, & Zhong, 2015). The
changes of crystal diffraction peak of triple helix were clearly shown in
Fig. 8B, showing that the intensity increased and then decreased when
the pH varied from 4.0 to 1.5. The swelling of collagen fiber promoted
the exposure of triple helix in the film structures.
4. Conclusion
The pH significantly affected the swelling degree of collagen fibers in
J. Xu et al.
Fig. 8. X-ray diffraction patterns of films formed by CFD at different pH.
the dispersions. The viscosity and modulus increased and then decreased
with the decrease of pH, which would affect the flow of dispersions in
practical production. The pH of CFD should be controlled at appropriate
value as high pH caused the collagen fibers to aggregate and low pH led
to insufficient swelling of collagen fibers. Sufficient swelling could
enable extensive swollen and intertwined collagen fibers with excellent
mechanical and thermodynamic properties. However, the reduction of
swelling degree at low pH would not be desired. Films formed at high pH
had a rough internal structure whereas those at low pH showed dis­
integrated tiny fibers along with the reduction of the triple helix. This
study provides significant knowledge in understanding the role of pH in
the production of collagen films using bovine dermis.
Declaration of competing interest
The authors declare no competing financial interest.
CRediT authorship contribution statement
Jinlong Xu: Investigation, Methodology, Software, Writing - orig­
inal draft. Fei Liu: Conceptualization, Data curation, Writing - review &
editing. Tao Wang: Visualization, Writing - review & editing. H.
Douglas Goff: Methodology, Writing - review & editing. Fang Zhong:
Resources, Supervision, Project administration.
Acknowledgement
This research was supported by National Natural Science Foundation
of China (31871846, 31801589), Natural Science Foundation of Jiangsu
Province (BK20180615). The research is also supported by 111 project-
B07029, national first-class discipline program of Food Science and
Technology (JUFSTR20180204) and the program of “Collaborative
Innovation Center of Food Safety and Quality Control in Jiangsu Prov­
ince”, China.
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