International Journal of Biological Macromolecules 149 (2020) 1077–1083

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International Journal of Biological Macromolecules

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

Preparation of bioplastic using soy protein

Masanori Yamada a,⁎, Sakura Morimitsu a, Eiji Hosono b, Tetsuya Yamada c
a
Department of Chemistry, Faculty of Science, Okayama University of Science, Ridaicho, Kita-ku, Okayama 700-0005, Japan
b
Research Institute for Energy Conservation, National Institute of Advanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba 305-8568, Japan
c
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

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

Article history: Soybean, one of the most abundant plants, has been cultivated around the world as a familiar crop. Especially,
Received 30 November 2019 most of the soybean is globally used as a crop to obtain the oil. The degreased soybean contains a lot of protein
Received in revised form 26 January 2020 in it. The part of the degreased soybean is used for the food of human consumption and livestock feed, however
Accepted 4 February 2020
most of this are discarded as industrial waste throughout the world. Therefore, we demonstrated the preparation
Available online 5 February 2020
of bioplastics consisting of soy protein. Although the soy protein without the cross-linking reaction by formalde-
Keywords:
hyde (HCHO) was collapsed in water, bioplastics were stable in water. Additionally, the bending strength of the
Bioplastic bioplastic increased with the HCHO concentration and showed the maximum value of approximately 35 MPa at a
Soy protein 1% HCHO concentration. Surprisingly, this bending strength value was the same as that of polyethylene. In con-
Biodegradable trast, the infrared spectra indicated the formation of methylene cross-linking between the basic amino acids, such
Cross-linking as lysine and arginine. Finally, we estimated the biodegradable property of the bioplastic by pronase, one of the
Amino acid proteolytic enzymes. As a result, this bioplastic showed the weight loss of approximately 30% after the incubation
time of 6 days. These results suggested that the bioplastic consisting of soy protein possesses a biodegradable
property. Therefore, the bioplastic consisting of soybean may have the potential to be used as a biodegradable
material, such as agricultural materials, industrial parts, and disposable items.
© 2020 Published by Elsevier B.V.

1. Introduction consisting of biopolymers easily decompose in nature. Additionally,

Artificial plastics [1], made from petroleum, have various properties,
such as light weight, low cost, high mechanical strength, and easy pro-
cessing, and have been produced in large quantities around the world.
These artificial plastics have been used in various products including in-
dustrial parts and disposable items. On the other hand, since artificial
plastics are mainly made from petroleum, the depletion of this resource
and the release of carbon dioxide or toxic compounds during produc-
tion are serious problems for the global environment. Additionally,
since common plastics, such as polyethylene, polypropylene, and poly-
vinyl chloride, are not degraded in nature, these plastic materials, when
discarded in the environment, are believed to remain for hundreds of
years [2–4]. Especially, the plastic waste that pollutes the ocean is a
major problem around the world [2,5]. Therefore, an alternative mate-
rial is an artificial plastic that has been investigated, and one of these
materials is bioplastics consisting of natural biopolymers [2,3].
Bioplastics are a material produced from renewable biomass sources
that are related to natural biopolymers, such as carbohydrates, proteins,
woodchips, natural rubber, recycled food waste, etc. [6–8] Generally,
since biopolymers possess a biodegradable property, bioplastics
⁎ Corresponding author.
E-mail address: myamada@chem.ous.ac.jp (M. Yamada).
the biopolymers are obtained from various living things and there is
no cost to synthesize the polymers. Furthermore, biopolymers can be
considered to be extremely non-hazardous to humans and an environ-
mentally benign material. Therefore, the bioplastics consisting of bio-
polymers, such as starch [9], agarose [10], casein [11], lignin [8,12],
and keratin [13], have been reported. In this study, we focused on the
bioplastics employing soy protein.
Soybean, one of the most abundant plants, has been cultivated
around the world as a familiar crop. Especially, in East Asia, soybeans
are eaten as not only an unfermented food, such as boiled soybean,
tofu, and tofu skin, but also as fermented food, such as miso, soy
sauce, and natto. In contrast, soybean contains approximately 35% pro-
tein and 20% fat by weight of the dry soybean [14–16]. Therefore, most
of the soybean is globally used as a crop to obtain the oil. The degreased
soybean contains a lot of protein in it. Although part of the degreased
soybean is used for the food of human consumption and livestock
feed, most of this are discarded as industrial waste throughout the
world [14]. These degreasing soybeans have been reported for use in
composites with the cellulose [17], glycerol [18], polyacrylamide [19],
and graphene [20]. However, the procedures, which are prepared the
composite material with the degreased soybean, are complicated pro-
cesses. Therefore, the preparation of bioplastics through simple process
is reasonable in a green chemistry approach.

https://doi.org/10.1016/j.ijbiomac.2020.02.025
0141-8130/© 2020 Published by Elsevier B.V.
1078 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083
Soy protein mainly consists of the acidic amino acids of aspartic acid
(Asp) and glutamic acid (Glu), neutral amino acids of glycine (Gly), ala-
nine (Ala), valine (Val), and leucine (Leu), and basic amino acids of ly-
sine (Lys) and arginine (Arg) [21]. These amino acids possess
modifiable functional groups, such as the carboxyl or amino groups, in
these residues. Especially, the amino or imino group reacts with the
formaldehyde (HCHO) in an aqueous solution under a moderate condi-
tion, produces the methylol derivative of the amino or imino group,
then forms the methylene cross-linking, such as N–CH2–N [22–24].
Therefore, the degreased soybean, which possesses the Lys and Arg, is
expected to react with HCHO in an aqueous solution and produce the
cross-linked soybean. Furthermore, the bioplastic, which derived from
crop, with the biodegradable property might be used in an agricultural
material, such as agricultural mulch and seedling pots.
In this study, we prepared the bioplastics by the reaction of soy pro-
tein (degreasing soybean) and formaldehyde (HCHO). These bioplastics
formed a three-dimensional network through the methylene cross-
linking between the peptide chains and were stable in an aqueous solu-
tion. Additionally, this bioplastic showed a high mechanical strength
and the bending strength value was the same as that of polyethylene.
Furthermore, this bioplastic showed a biodegradable activity when the
bioplastic underwent biodegradation using a pronase, one of the proteo-
lytic enzymes.
2. Experimental sections
2.1. Material
Soy protein, 37% formaldehyde (HCHO), sodium chloride, tris-(hy-
droxymethyl)aminomethane (Tris), and pronase (EC 3.4.24.4) [25]
were purchased from Wako Pure Chemical Industries, Ltd., Osaka,
Japan. The plastic plates of polymethyl methacrylate (PMMA), polyvinyl
chloride (PVC), polyethylene (PE), polypropylene (PP), polycarbonate
(PC) as commercial polymer materials were obtained from the AS
ONE Corporation, Osaka, Japan. The thickness of these plastic plates
was 1.0 mm. Analytical grade solvents were used in all of the experi-
ments. Ultrapure water (Merck KGaA, Darmstadt, Germany) was used
in this experiment.
2.2. Preparation of bioplastic
The soy protein (200 mg) was compacted into pellets with a diame-
ter of 13 mm using a hand press at a pressure of approximately 600 MPa
for 15 min. These pellets were immersed in 0–37% aqueous HCHO solu-
tions for 24 h at room temperature. These immersed pellets were re-
moved from the HCHO solutions, rinsed with ultrapure water, dried at
room temperature for 12 h, and then heated for 2 h at 80 °C.
2.3. Bending strength of bioplastic
The bioplastic were cut into 10 × 5 mm2. The thickness of the
bioplastic was measured by the ID-C X series thickness gauge (Mitutoyo
Corporation, Kanagawa, Japan). The thickness of the material was 1 mm.
The bending strength was measured using the ZTA-50N digital force
gauge (Imada Co., Ltd., Aichi, Japan) and MX2-500N test stand (Imada
Co., Ltd.). The distance between the fulcrums was 5.0 mm. The bending
stress was loaded from above and the bending strength was estimated
from the loaded stress at the break point. The measurement environ-
ment of the bending strength was controlled by a laboratory air condi-
tioner and the temperature and humidity were 25 °C and 50 ± 10%,
respectively. The crosshead speed was 50 mm min−1. The bending
strength value was expressed as an average of five measurements. In
this measurement, commercial polymer materials of PMMA, PVC, PE,
PP, and PC were used for the control materials.
2.4. Structural analysis of bioplastic
The infrared (IR) absorption spectra of the bioplastic were measured
by the KBr method using an FT-IR 8400 Fourier transform infrared spec-
trometer (Shimadzu Corp., Kyoto, Japan). The IR samples were prepared
as follows: the surface of the bioplastic was scraped by a knife. The ob-
tained powder was mixed with KBr and pelleted by a hand press. The IR
spectrum was measured with the resolution of 4 cm−1.
2.5. Thermal analysis of bioplastic
The thermal stabilities of the bioplastics were analyzed using a DTG-
60 thermogravimetric (TG) - differential thermal analyzer (DTA)
(Shimadzu Corp.). The TG-DTA samples were prepared as follows: the
surface of the bioplastics was scraped by a knife. The TG-DTA measure-
ment was carried out at the heating rate of 10 °C min−1 from room tem-
perature to 300 °C under dry flowing nitrogen. The sample weights of
the TG-DTA measurements were normalized at 1 mg.
2.6. Swelling ratio of the bioplastic
The swelling ratio (%) of the bioplastic was determined by the fol-
lowing procedure: the dried bioplastics were immersed in ultrapure
water for 1 min or 10 min, and the weight of these swelling materials
was measured. The swelling ratio of bioplastic was estimated from
Eq. (1).
Ws
Swelling ratio ð%Þ ¼  100 ð1Þ
W0
where W0 and Ws are the initial and swelling weights of bioplastic, re-
spectively. The values of swelling ratio were expressed as an average
of five measurements.
2.7. Biodegradable property of bioplastic
The biodegradable property of the bioplastic was estimated by the
following method: the bioplastic was added to 10 ml of a 20 mM Tris-
HCl buffer (pH 7.4) in the presence of 5 mM NaCl and pronase [25].
The concentrations of pronase in aqueous solution were 1 or
10 units ml−1. These samples were incubated for various times at
37 °C. The biodegradable amounts of the bioplastics were calculated
from the weight difference of the bioplastic in the absence and presence
of pronase. The biodegradable amounts of the bioplastics were
expressed as an average of three measurements.
The biodegraded bioplastic was measured by optical and scanning
electron microscopies (SEM). The SEM images were obtained by an
LEO EM-922 (Carl Zeiss Co., Oberkochen, Germany) with an accelera-
tion voltage of 5 kV.
3. Results and discussion
3.1. Preparation of the bioplastic
The white soy protein was compacted into a pellet by a hand press.
This pelleted soy protein could easily break by hand and possessed a
brittle property. Additionally, when the pelleted soy protein was im-
mersed in water, this material collapsed and could not be pinched by
tweezers. Therefore, the soy protein without the chemical treatments
could not be used as a material, such as a bioplastic.
The pelleted soy proteins were immersed into an aqueous HCHO so-
lution for 24 h, rinsed with water, and then dried at room temperature
for 12 h. The dried samples were heated for 2 h at 80 °C. By this proce-
dure, the concentrations of HCHO changed from 0% to 37%. Fig. 1 shows
a photograph of the bioplastic, which was prepared in a 1% aqueous
HCHO solution. Although the pelleted soy protein without the reaction
 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083 1079

-20

-10

Bending stress / MPa

0

10

20

30

40 (a)
Fig. 1. Photograph of bioplastic which was prepared in a 1% aqueous HCHO solution. The (b)
thickness of this bioplastic is approximately 1 mm. The scale bar in the figure is 5 mm.

50
of HCHO was white, the obtained bioplastic was a brown translucent 0 200 400 600
material. Additionally, this bioplastic could not easily break by hand. Strain / %
In contrast, although the bioplastics, which were prepared at the
HCHO concentration of b1%, were soft in an aqueous HCHO solution, Fig. 2. Bending stress-strain curves of (a) bioplastic, which was prepared in a 1% aqueous
these materials did not collapse when pinched by tweezers. Addition- HCHO solution, and (b) commercial polyethylene. The distance between the fulcrums was
ally, the bioplastics, which were prepared under the HCHO concentra- 5.0 mm. These measurements were done at 25 °C under a 50 ± 10% relative humidity
tion of ≥10%, were white. These results suggested that the condition. Five experiments gave similar results.

concentration of HCHO affects the reaction with the soy protein and
the preparation of the bioplastic.
3.2. Bending strength of the bioplastic
The hardness of the obtained bioplastic varied with the HCHO con-
centration. Therefore, we measured the bending strength of the
bioplastics which were prepared at various HCHO concentrations. The
bending stress was loaded from above. Fig. 2(a) shows the bending
stress-strain curve of a bioplastic, which was prepared in a 1% aqueous
HCHO solution. When the stress was applied to the bioplastic, the
bioplastic broke at approximately 35 MPa. The strain at the broken
point was approximately 70%. Similar measurements were demon-
strated for the various bioplastics which were prepared under differen-
tial HCHO concentrations. Fig. 3 shows the bending strength at the
broken point of the bioplastics which were prepared under different
concentrations. The bending strength of the pelleted soy protein with-
out the HCHO reaction was approximately 3 MPa and this material
approximately 76 MPa, 69 MPa, 35 MPa, 37 MPa, and 65 MPa, respec-
tively, and the values of PE and PP were as same as that of the bioplastic
of ca. 35 MPa. Fig. 2(b) shows the bending stress-strain curves of PE. The
strain at the broken point of the PE was approximately 350%. This value
of PE was five times higher than that of the bioplastic consisting of the
soy protein. These results suggested that the bioplastic, which was pre-
pared using the 1% aqueous HCHO solution, possesses hard and brittle
properties.
3.3. Molecular structure of the bioplastic
The molecular structure of the bioplastic consisting of soy protein
was confirmed by IR spectrometry using the KBr method. The IR sample
was prepared by scraping the surface of the bioplastic with a knife. Fig. 4
40
Bending strength / MPa

could easily break by hand. The bending strength of the bioplastic in-
creased with the HCHO concentration and showed the maximum
value at the 1% HCHO condition. This bending strength value was ap- 30
proximately 35 MPa and one order of magnitude higher than that of
the non-reacted pelleted soy protein. In contrast, at the high HCHO con-
centrations of ≥5%, the bending strengths decreased. The bending 20
strength of the bioplastic, which was prepared in a 37% aqueous
HCHO solution, was approximately 10 MPa and this value was less
than one-third of the maximum value of ca. 35 MPa. These results sug- 10
gested that the aqueous HCHO solution at a high concentration is un-
suitable to prepare the bioplastic consisting of soy protein. In
particular, the 1% aqueous HCHO solution was the appropriate concen- 0
tration to prepare the bioplastics with a high mechanical strength. 0 0.2 0.5 1 5 10 20 37
We next measured the bending strength of commercial polymer Concentration of HCHO / %
materials, such as polymethyl methacrylate (PMMA), polyvinyl chloride
(PVC), polyethylene (PE), polypropylene (PP), and polycarbonate (PC), Fig. 3. Bending strength of the bioplastic. The concentrations of the aqueous HCHO
compared to the bending strength of the bioplastics. The bending solution were varied from 0% to 37%. The bending strength was expressed by an average
strengths at the broken point of the PMMA, PVC, PE, PP, and PC were of five measurements.
1080 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083

shows the IR spectra of the bioplastics which were prepared in an aque- 100

ous HCHO solution at the incubation times of (a) 0 h, (b) 1 h, (c) 3 h,
(d) 6 h, (e) 12 h, and (f) 24 h. The bioplastic indicated an absorption
band at ca. 1000 cm−1 that is related to the stretching vibration of
C\\N [26,27]. This absorption band increased with the incubation time
in the aqueous HCHO solution. These phenomena are due to the forma-
tion of the C\\N bonding by the reaction with amino acid residues in the
soy protein and HCHO. Additionally, the absorption band at approxi-
mately 3400 cm−1, attributed to the stretching vibration of N\\H in
the amino acid residue [26,28], relatively decreased by comparison
with the non-reacted soy protein (see spectra (a) and (f) in Fig. 4).
These results suggested that the amino or imino groups in the amino
acid residue form the C\\N bonding by the reaction with HCHO. Similar
phenomenon, such as the formation of the C\\N bonding by the reac-
tion with the HCHO, has been reported to be the interaction between
the ribonucleic acid (RNA)-inorganic hybrid material and HCHO [29].
Generally, the HCHO molecules react with the amino or imino
groups, produce the methylol derivative of the amino or imino group,
then form the methylene cross-linking, such as N–CH2–N [22–24]. Sim-
ilar phenomena have occurred in the bioplastic consisting of soy pro-
tein. The soy protein contains basic amino acids lysine (Lys) and
arginine (Arg) which possesses the amino and imino groups in its side
chain, respectively [21,30]. Therefore, on the surface of the pelleted
soy protein, the amino or imino groups react with the HCHO molecule
and form the three-dimensional network through the methylene
cross-linking between peptide chains in a pellet. As a result, the
bioplastic with the three-dimensional network was stabilized against
the aqueous solution and showed a high mechanical property when
compared to the non-reacted soy protein.
On the other hand, the bending strength of bioplastics significantly
varied depending on the concentration of the aqueous HCHO solution
(see Fig. 3). The reason for this phenomenon is as follows: the formation
of methylene cross-linking quickly occurs in the peptide chain. In fact,
the methylene cross-linking was produced at the incubation time of
1 h (see IR spectrum (b) in Fig. 4). Therefore, at the high HCHO concen-
trations of ≥5%, the cross-linking reaction occurred on the surface of a
pelleted soy protein and the HCHO molecules could not penetrate into
the pelleted material. Thus, the methylene cross-linking was produced
only on the surface of the pelleted material. In contrast, at the low
HCHO concentration of b5%, the HCHO molecules could penetrate into
(a)
TG weight / %
80
60
(a) (1)
40
(2)
20
0
Exo
(b)
Endo
10 µV
0 50 100 150 200 250 300 350
Temperature / ºC
Fig. 5. (a) TG and (b) DTA curves of (1) non-reacted soy protein and (2) bioplastic at the
heating rate of 10 °C min−1 to 300 °C under dry flowing nitrogen. Sample weights of the
TG-DTA measurements were normalized at 1 mg. Triplicate experiments gave similar
results.
the material. As a result, the methylene cross-linking reaction occurred
not only on the surface but also on the inside of the material. Especially,
since the bioplastic, which was prepared in the 1% aqueous HCHO solu-
tion, effectively formed the three-dimensional network not only on the
surface but also on the inside of pelleted soy protein, and this material
showed the highest bending strength of the various materials. In a fur-
ther experiment, the bioplastic, which was prepared in the 1% aqueous
HCHO solution, was used.
3.4. Thermal property of the bioplastic
Fig. 5 (a) and (b), respectively, show the thermogravimetric analysis
(TG) and differential thermal analysis (DTA) curves of the (1) non-
reacted soy protein and (2) bioplastic, which was prepared in the 1%
aqueous HCHO solution, at the heating rate of 10 °C min−1 up to
300 °C under dry flowing nitrogen. The non-reacted soy protein showed
the TG weight of approximately 10% at 100 °C. In addition, the non-
reacted soy protein indicated a large endothermic peak at b100 °C, re-
lated to the evaporation of water. Therefore, the TG weight loss of
non-reacted soy protein at 100 °C is due to the evaporation of water.
In contrast, the bioplastic, which was prepared in the 1% aqueous

(b)
Amount of biodegradation / %

100
Transmittance / %

(c)
50
(d) 40
30
(e)
20
(f)
10
0
0 50 100 150

4000 3500 3000 2500 2000 1500 1000

Wavenumber / cm-1
Fig. 4. IR spectra of bioplastics which were prepared in an aqueous HCHO solution with
incubation times of (a) 0 h, (b) 1 h, (c) 3 h, (d) 6 h, (e) 12 h, and (f) 24 h. Scale bar in
the figure shows the transmittance of 20%. Triplicate experiments gave similar results.
Fig. 6. Biodegradation of bioplastic which was prepared in a 1% aqueous HCHO solution.
The bioplastic was incubated in an aqueous pronase solution at 37 °C. The
concentrations of the pronase are (▲) 1 unit ml−1 and (●) 10 units ml−1. The
biodegradable amounts were calculated from the weight difference of the bioplastic in
the absence and presence of pronase. The biodegradable values were expressed by an
average of three measurements.
 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083
Fig. 7. Photographs of bioplastic which was incubated in the (a) absence and (b) presence
of pronase for 144 h. The concentrations of the aqueous pronase solution was 1 unit ml−1.
The obtained samples were dried overnight at room temperature.
HCHO solution, showed the TG weight loss of ≤5% at 150 °C, related to
the evaporation of water and non-reacted HCHO, which was remained
in the pelleted soy protein. Additionally, the bioplastic showed an endo-
thermic peak at 205.06 °C, attributed to the decomposition of the
bioplastic. As a result, the bioplastic indicated the TG weight loss at
≥200 °C. Similar phenomenon, such as the TG weight loss due to decom-
position, was also obtained for the non-reacted soy protein. At N200 °C,
the TG weight loss of the bioplastic was lower than that of the non-
reacted soy protein. This is due to the thermal stabilization by the for-
mation of a three-dimensional network with cross-linking of the pep-
tide chain. Similar phenomena, such as the thermal stabilization by
the cross-linking between polymer chains, have been reported for vari-
ous organic-inorganic hybrid materials [27,31,32] and a cross-linked
protein film [33]. These results suggested that the soy protein is ther-
mally stabilized by the reaction with HCHO and the prepared bioplastic
can be stably used at b200 °C.
Fig. 8. SEM images of bioplastic which was incubated in the (a, b) absence and (c, d) presence of pronase for 144 h. The (b) and (d) images show the SEM images of (a) and (c) at higher
magnifications, respectively. The concentration of the aqueous pronase solution was 1 unit ml−1. The obtained samples were dried overnight at room temperature.
1081
3.5. Biodegradation of the bioplastic
The swelling behavior of bioplastic is important for biodegradable
properties. Before the biodegradable measurement of the bioplastic,
we demonstrated the swelling test in water. The swelling ratio of the
bioplastic was calculated from the weight of swelling materials. As a re-
sult, the swelling ratios of the bioplastic which was immersed in water
for 1 min or 10 min, were 103% or 106%, respectively and the bioplastic
showed the slightly swelling in an aqueous solution. Therefore, the bio-
degradable samples were immersed in water for 24 h to be fully swollen
and used.
Finally, we estimated the biodegradable property of the bioplastic in
an aqueous solution. In this experiment, we used a pronase (EC 3.4.24.4)
[25] as the proteolytic enzyme. The pronase is a mixture of proteolytic
enzyme and include N10 types of protease [25]. Therefore, the pronase
has been used at the biodegradable experiments, such as the degrada-
tion of polyamino acids and polyesteramides, for one of general proteo-
lytic enzymes [34–36]. Furthermore, there are various proteolytic
enzymes in nature, such as soil, river, sea, etc., and the biodegradable
materials are hydrolyzed by various enzymes in natural condition.
Thus, in our research, the biodegradable properties of the bioplastic in
an aqueous solution were estimated by the pronase. In contrast, the
bioplastic, which was prepared in a 1% aqueous HCHO solution and
fully swollen in water, was used in this experiment.
Fig. 6 (▲) and (●) show the biodegradable amount of the bioplastic
using the pronase concentrations of 1 unit ml−1 and 10 units ml−1, re-
spectively. The incubation temperature was performed at 37 °C. The
biodegradable amounts were calculated from the weight difference of
the bioplastic in the absence and presence of pronase. The biodegradable
amount of the bioplastic increased with the incubation time in an
1082 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083
aqueous pronase solution. In addition, the biodegradable rate varied
with the pronase concentration. When the bioplastics were incubated
at the pronase concentrations of 1 unit ml−1 and 10 units ml−1, the bio-
degradable amounts of the bioplastics were 17% and 38%, respectively,
after the incubation for 144 h. The biodegradable amount in
10 units ml−1 was two or more than that in 1 unit ml−1. These results
suggested that the bioplastic consisting of soy protein possesses a bio-
degradable property in an aqueous solution containing a proteolytic en-
zyme, such as pronase.
Fig. 7 shows photographs of the bioplastic which was incubated in
the (a) absence and (b) presence of pronase for 144 h. When the
bioplastic was incubated in the absence of pronase, the bioplastic did
not show any structural change, such as a crack and transformation. In
contrast, the biodegraded bioplastic showed many cracks on the surface
(see Fig. 7(b)). Therefore, we observed the bioplastic by scanning elec-
tron microscopy (SEM). Fig. 8 (a) and (b) show the SEM images of the
bioplastic which was incubated in the absence of pronase for 144 h.
The surface of the bioplastic without any biodegradation was flat and
no unevenness could be observed over a wide range. Additionally, the
magnified SEM image also show almost no unevenness (see Fig. 8
(b)). Fig. 8 (c) and (d) show SEM images of the bioplastic which was in-
cubated in the presence of pronase for 144 h. The biodegraded bioplastic
showed a large crack on the surface. The width of the crack on the sur-
face was over 100 μm. Therefore, we observed the inside of the crack.
The magnified SEM image showed on unevenness over a wide range
(see Fig. 8(d)). Additionally, the unevenness of the inside at the crack
was larger than that on the surface. The biodegradable mechanism of
the bioplastic by the pronase is as follows: the pronase attacked the pep-
tide chain on the surface of the bioplastic and hydrolyzed the peptide
bonding. As a result, the crack was produced on the surface. In addition,
since the a three-dimensional networks through the cross-linking be-
tween the peptide chains are formed at high density on the surface of
bioplastic than inside, the hydrolysis of bioplastic by pronase occurs
preferentially at the inside of the crack. Consequently, the inside of the
crack showed a significant unevenness.
4. Conclusion
A bioplastic consisting of soy protein was prepared by the cross-
linking reaction with formaldehyde (HCHO). The mechanical
strength of the bioplastic varied depending on the HCHO concentra-
tion. When the bioplastic was prepared in a 1% aqueous HCHO solu-
tion, the highest bending strength of ca. 35 MPa was obtained. This
strength of the bioplastic was the same as that of commercial poly-
ethylene. Additionally, the bioplastic showed a thermal stability at
b200 °C by the formation of a three-dimensional network through
the methylene cross-linking between the peptide chains. Further-
more, when these bioplastics were incubated in an aqueous pronase
solution, these materials showed a biodegradable property. The soy
protein with the reaction of HCHO might play an important role in
the use of biodegradable materials, such as disposable items, indus-
trial parts, and alternative substance of artificial plastics. In particu-
lar, since the soy protein is derived from discarded soybeans, the
bioplastic consisting of soy protein may have the potential for appli-
cation of agricultural fields, such as the agricultural mulch, seedling
pots, and disposable poles.
CRediT authorship contribution statement
Masanori Yamada:Conceptualization, Validation, Formal analysis,
Visualization, Writing - original draft, Funding acquisition.Sakura
Morimitsu:Formal analysis, Visualization.Eiji Hosono:Formal analysis,
Visualization.Tetsuya Yamada:Formal analysis, Visualization, Writing -
review & editing, Funding acquisition.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
JP19K12408, JP19K22298.
References
[1] R.C. Thompson, C.J. Moore, F.S. vom Saal, S.H. Swan, Plastics, the environment and
human health: current consensus and future trends, Phil. Trans. R. Soc. B 364
(2009) 2153–2166.
[2] R.E. Hester, R.M. Harrison, Plastics and the Environment, The royal society of chem-
istry, London, 2018.
[3] G.E. Luckachan, C.K.S. Pillai, Biodegradable polymers - a review on recent trends and
emerging perspectives, J. Polym. Environ. 19 (2011) 637–676.
[4] S.M. Emadian, T.T. Onay, B. Demirel, Biodegradation of bioplastics in natural envi-
ronments, Waste Manag. 59 (2017) 526–536.
[5] Y. Picó, D. Barceló, Analysis and prevention of microplastics pollution in water: cur-
rent perspectives and future directions, ACS Omega 4 (2019) 6709–6719.
[6] C. Xu, M. Nasrollahzadeh, M. Selva, Z. Issaabadi, R. Luque, Waste-to-wealth:
biowaste valorization into valuable bio(nano)materials, Chem. Soc. Rev. 48 (2019)
4791–4822.
[7] S. Lambert, M. Wagner, Environmental performance of bio-based and biodegradable
plastics: the road ahead, Chem. Soc. Rev. 46 (2017) 6855–6871.
[8] M. Brodin, M. Vallejos, M.T. Opedal, M.C. Area, G. Chinga-Carrasco, Lignocellulosics
as sustainable resources for production of bioplastics - a review, J. Clean. Prod. 162
(2017) 646–664.
[9] D. Sagnelli, K.H. Hebelstrup, E. Leroy, A. Rolland-Sabaté, S. Guilois, J.J.K. Kirkensgaard,
K. Mortensen, D. Lourdin, A. Blennow, Plant-crafted starches for bioplastics produc-
tion, Carbohydr. Polym. 152 (2016) 398–408.
[10] A. Awadhiya, D. Kumar, V. Verma, Crosslinking of agarose bioplastic using citric acid,
Carbohydr. Polym. 151 (2016) 60–67.
[11] E. Sutermeister, F.L. Browne, Casein and Its Industrial Applications, Reinhold pub-
lishing corp, New York, 1939.
[12] D. Kai, M.J. Tan, P.L. Chee, Y.K. Chua, Y.L. Yap, X.J. Loh, Towards lignin-based
functional materials in a sustainable world, Green Chem. 18 (2016)
1175–1200.
[13] N. Ramakrishnan, S. Sharma, A. Gupta, B.Y. Alashwal, Keratin based bioplastic film
from chicken feathers and its characterization, Int. J. Biol. Macromol. 111 (2018)
352–358.
[14] H. El-Shemy, Soybean Bio-Active Compounds, IntechOpen Limited, London, 2013.
[15] P.M. Visakh, O. Nazarenko, Soy Protein-Based Blends, Composites and Nanocompos-
ites, John Wiley & Sons, Hoboken, 1998.
[16] FoodData Central, https://fdc.nal.usda.gov/index.html, Accessed date: 1 November
2019.
[17] I. Paetau, C.Z. Chen, J. Jane, Biodegradable plastic made from soybean products. II. Ef-
fects of cross-linking and cellulose incorporation on mechanical properties and
water absorption, J. Environ. Polym. Degrad. 2 (1994) 211–217.
[18] H. Tian, W. Wu, G. Guo, B. Gaolun, Q. Jia, A. Xiang, Microstructure and properties of
glycerol plasticized soy protein plastics containing castor oil, J. Food Eng. 109 (2012)
496–500.
[19] F. Xu, Y. Dong, W. Zhang, S. Zhang, L. Li, J. Li, Preparation of cross-linked soy protein
isolate-based environmentally-friendly films enhanced by PTGE and PAM, Ind. Crop.
Prod. 67 (2015) 373–380.
[20] X. Jiang, Z. Li, J. Yao, Z. Shao, X. Chen, One-step synthesis of soy protein/graphene
nanocomposites and their application in photothermal therapy, Mater. Sci. Eng. C
68 (2016) 798–804.
[21] H. Taira, Heat destruction of amino acids in soybean products, Jpn. Agr. Res. Q. 7
(1973) 267–273.
[22] C. Klockenbusch, J.E. O’Hara, J. Kast, Advancing formaldehyde cross-linking to-
wards quantitative proteomic applications, Anal. Bioanal. Chem. 404 (2012)
1057–1067.
[23] D. Hopwood, Fixatives and fixation: a review, Histochem. J. 1 (1969) 323–360.
[24] J.D. McGhee, P.H. von Hippel, Formaldehyde as a probe of DNA structure. I. Re-
action with exocyclic amino groups of DNA bases, Biochemistry 14 (1975)
1281–1296.
[25] M.M. Burrell, Enzymes of Molecular Biology, Humana press, New Jersey, 1993.
[26] R.M. Silverstein, F.X. Webster, Spectrometric Identification of Organic Compounds,
John Wiley & Sons, New York, 1998.
[27] M. Yamada, C. Tohyama, T. Yamada, Preparation of water-insoluble and
biochemically-stable RNA hybrid material, Polym. Adv. Technol. 29 (2018)
2890–2898.
[28] A. Bozkurt, W.H. Meyer, Proton conducting blends of poly(4-vinylimidazole) with
phosphoric acid, Solid State Ionics 138 (2001) 259–265.
[29] M. Yamada, S. Funaki, S. Miki, Formaldehyde interacts with RNA rather than DNA:
accumulation of formaldehyde by the RNA-inorganic hybrid material, Int. J. Biol.
Macromol. 122 (2019) 168–173.
[30] J. Crowe, T. Bradshaw, P. Monk, Chemistry for the Biosciences, Oxford University
Press, New York, 2006.
[31] M. Yamada, H. Aono, DNA-inorganic hybrid material as selective absorbent for
harmful compounds, Polymer 49 (2008) 4658–4665.
[32] M. Yamada, S. Shiiba, Preparation of pectin-inorganic composite material as ac-
cumulative material of metal ions, J. Appl. Polym. Sci. 132 (2015)
42056–42063.
 M. Yamada et al. / International Journal of Biological Macromolecules 149 (2020) 1077–1083 1083

[33] S. Li, E. Donner, M. Thompson, Y. Zhang, C. Rempel, Q. Liu, Preparation and charac-
terization of cross-linked canola protein isolate films, Eur. Polym. J. 89 (2017)
419–430.
[34] D.F. Williams, Biodegradation of surgical polymers, J. Mater. Sci. 17 (1982)
1233–1246.
[35] A. Larrañaga, E. Lizundia, A review on the thermomechanical properties and biodeg-
radation behavior of polyesters, Eur. Polym. J. 121 (2019) 109296–109326.
[36] B. Romberg, J.M. Metselaar, T. deVringer, K. Motonaga, J.J. Kettenes-van den Bosch, C.
Oussoren, G. Storm, W.E. Hennink, Enzymatic degradation of liposome-grafted poly
(hydroxyethyl L-glutamine), Bioconjug. Chem. 16 (2005) 767–774.