Preparation of bioplastic using soy protein

Journal Pre-proof Preparation of bioplastic using soy protein

Masanori Yamada, Sakura Morimitsu, Eiji Hosono, Tetsuya Yamada PII:

S0141-8130(19)39826-5

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.025

Reference:

BIOMAC 14641

To appear in:

International Journal of Biological Macromolecules

Received date:

30 November 2019

Revised date:

26 January 2020

Accepted date:

4 February 2020

Please cite this article as: M. Yamada, S. Morimitsu, E. Hosono, et al., Preparation of bioplastic using soy protein, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.02.025

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© 2018 Published by Elsevier.

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Preparation of bioplastic using soy protein

Department of Chemistry, Faculty of Science, Okayama University of Science, Ridaicho,

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Kita-ku, Okayama 700-0005, Japan

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Masanori Yamada1,*, Sakura Morimitsu1, Eiji Hosono2, and Tetsuya Yamada3

Research Institute for Energy Conservation, National Institute of Advanced Industrial

Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

[*]

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Science and Technology, 1-1-1 Umezono, Tsukuba 305-8568, Japan

Corresponding author

Department of Chemistry, Faculty of Science, Okayama University of Science, Ridaicho, Kita-ku, Okayama 700-0005, Japan Tel: +81 86 256 9550 Fax: +81 86 256 9757 E-mail: [email protected]

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Abstract Soybean, one of the most abundant plants, has been cultivated around the world as a familiar crop. Especially, most of the soybean is globally used as a crop to obtain the oil. The degreased soybean contains a lot of protein in it. The part of the degreased soybean is used for the food of human consumption and livestock feed, however most of this are discarded as industrial waste throughout the world. Therefore, we demonstrated the preparation of

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bioplastics consisting of soy protein. Although the soy protein without the cross-linking

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reaction by formaldehyde (HCHO) was collapsed in water, bioplastics were stable in water. Additionally, the bending strength of the bioplastic increased with the HCHO concentration

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and showed the maximum value of approximately 35 MPa at a 1% HCHO concentration.

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Surprisingly, this bending strength value was the same as that of polyethylene. In contrast, the

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infrared spectra indicated the formation of methylene cross-linking between the basic amino acids, such as lysine and arginine. Finally, we estimated the biodegradable property of the

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bioplastic by pronase, one of the proteolytic enzymes. As a result, this bioplastic showed the

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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.

Keywords Bioplastic, Soy protein, Biodegradable, Cross-linking, Amino acid

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1. Introduction Artificial plastics [1], made from petroleum, have various properties, such as light weight, low cost, high mechanical strength, and easy processing, and have been produced in large quantities around the world. These artificial plastics have been used in various products including industrial 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

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or toxic compounds during production are serious problems for the global environment.

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Additionally, since common plastics, such as polyethylene, polypropylene, and polyvinyl chloride, are not degraded in nature, these plastic materials, when discarded in the

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environment, are believed to remain for hundreds of years [2-4]. Especially, the plastic waste

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that pollutes the ocean is a major problem around the world [2,5]. Therefore, an alternative

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material 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

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renewable biomass sources that are related to natural biopolymers, such as carbohydrates,

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proteins, woodchips, natural rubber, recycled food waste, etc.[6-8] Generally, since biopolymers possess a biodegradable property, bioplastics consisting of biopolymers easily decompose in nature. Additionally, 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 environmentally benign material. Therefore, the bioplastics consisting of biopolymers, 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 3

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sauce, and natto. In contrast, soybean contains approximately 35% protein 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

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[19], and graphene [20]. However, the procedures, which are prepared the composite material

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with the degreased soybean, are complicated processes. Therefore, the preparation of bioplastics through simple process is reasonable in a green chemistry approach.

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Soy protein mainly consists of the acidic amino acids of aspartic acid (Asp) and

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glutamic acid (Glu), neutral amino acids of glycine (Gly), alanine (Ala), valine (Val), and

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leucine (Leu), and basic amino acids of lysine (Lys) and arginine (Arg) [21]. These amino acids possess modifiable functional groups, such as the carboxyl or amino groups, in these

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residues. Especially, the amino or imino group reacts with the formaldehyde (HCHO) in an

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aqueous solution under a moderate condition, 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 protein (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 solution. 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 4

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biodegradable activity when the bioplastic underwent biodegradation using a pronase, one of the proteolytic enzymes.

2. Experimental sections 2.1. Material Soy

protein,

37%

formaldehyde

(HCHO),

sodium

chloride,

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tris-(hydroxymethyl)aminomethane (Tris), and pronase (EC 3.4.24.4) [25] were purchased

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from Wako Pure Chemical Industries, Ltd., Osaka, Japan. The plastic plates of polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP),

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polycarbonate (PC) as commercial polymer materials were obtained from the AS ONE

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Corporation, Osaka, Japan. The thickness of these plastic plates was 1.0 mm. Analytical grade

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solvents were used in all of the experiments. Ultrapure water (Merck KGaA, Darmstadt,

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Germany) was used in this experiment.

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2.2. Preparation of bioplastic

The soy protein (200 mg) was compacted into pellets with a diameter of 13 mm using a hand press at a pressure of approximately 600 MPa for 15 minutes. These pellets were immersed in 0 – 37 % aqueous HCHO solutions for 24 hours at room temperature. These immersed pellets were removed from the HCHO solutions, rinsed with ultrapure water, dried at room temperature for 12 hours, and then heated for 2 hours 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 5

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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 environment of the bending strength was controlled by a laboratory air conditioner 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

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of five measurements. In this measurement, commercial polymer materials of PMMA, PVC,

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2.4. Structural analysis of bioplastic

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PE, PP, and PC were used for the control materials.

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The infrared (IR) absorption spectra of the RNA-SiNSi hybrid material were

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measured by the KBr method using an FT-IR 8400 Fourier transform infrared spectrometer (Shimadzu Corp., Kyoto, Japan). The IR samples was prepared as follows: the surface of the

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bioplastic was scraped by a knife. The obtained powder was mixed with KBr and pelleted by

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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 was prepared as follows: the surface of the bioplastics was scraped by a knife. The TG-DTA measurement was carried out at the heating rate of 10 °C min-1 from room temperature 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 6

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The swelling ratio (%) of the bioplastic was determined by the following 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 equation (1). 𝑊

Swelling ratio (%) = 𝑊s × 100 0

(1)

where W0 and Ws are the initial and swelling weights of bioplastic, respectively. The values of

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swelling ratio was expressed as an average of five measurements.

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2.7. Biodegradable property of bioplastic

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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

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mM NaCl and pronase [25]. The concentrations of pronase in aqueous solution were 1 or 10

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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

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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 acceleration 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 immersed in water, this material collapsed and could not be pinched 7

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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 solution for 24 hours, rinsed with water, and then dried at room temperature for 12 hours. The dried samples were heated for 2 hours at 80 °C. By this procedure, the concentrations of HCHO changed from 0% to 37%. Figure 1 shows a photograph of the bioplastic which was prepared in a 1%

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aqueous HCHO solution. Although the pelleted soy protein without the reaction of HCHO

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was white, the obtained bioplastic was a brown translucent material. Additionally, this bioplastic could not easily break by hand. In contrast, although the bioplastics, which were

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prepared at the HCHO concentration of < 1%, were soft in an aqueous HCHO solution, these

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materials did not collapse when pinched by tweezers. Additionally, the bioplastics, which

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were prepared under the HCHO concentration of ≥ 10%, were white. These results suggested

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of the bioplastic.

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that the concentration of HCHO affects the reaction with the soy protein and the preparation

3.2. Bending strength of the bioplastic The hardness of the obtained bioplastic varied with the HCHO concentration. Therefore, we measured the bending strength of the bioplastics which were prepared at various HCHO concentrations. The bending stress was loaded from above. Figure 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 demonstrated for the various bioplastics which were prepared under differential HCHO concentrations. Figure 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 8

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protein without the HCHO reaction was approximately 3 MPa and this material could easily break by hand. The bending strength of the bioplastic increased with the HCHO concentration and showed the maximum value at the 1% HCHO condition. This bending strength value was approximately 35 MPa and one order of magnitude higher than that of the non-reacted pelleted soy protein. In contrast, at the high HCHO concentrations of ≥ 5%, the bending strengths decreased. The bending strength of the bioplastic, which was prepared in a 37%

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aqueous HCHO solution, was approximately 10 MPa and this value was less than one-third of

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the maximum value of ca. 35 MPa. These results suggested that the aqueous HCHO solution at a high concentration is unsuitable to prepare the bioplastic consisting of soy protein. In

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bioplastics with a high mechanical strength.

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particular, the 1% aqueous HCHO solution was the appropriate concentration to prepare the

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We next measured the bending strength of commercial polymer materials, such as polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polyethylene (PE),

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polypropylene (PP), and polycarbonate (PC), compared to the bending strength of the

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bioplastics. The bending strengths at the broken point of the PMMA, PVC, PE, PP, and PC were approximately 76 MPa, 69 MPa, 35 MPa, 37 MPa, and 65 MPa, respectively, and the values of PE and PP were as same as that of the bioplastic of ca. 35 MPa. Figure 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 prepared 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 9

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of the bioplastic with a knife. Figure 4 shows the IR spectra of the bioplastics which were prepared in an aqueous 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 formation of the C–N bonding by the reaction with amino acid residues in the soy protein and HCHO.

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Additionally, the absorption band at approximately 3400 cm-1, attributed to the stretching

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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 Figure 4). These results suggested that

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the amino or imino groups in the amino acid residue form the C–N bonding by the reaction

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with HCHO. Similar phenomenon, such as the formation of the C–N bonding by the reaction

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with the HCHO, has been reported to be the interaction between the ribonucleic acid (RNA)-inorganic hybrid material and HCHO [29].

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Generally, the HCHO molecules react with the amino or imino groups, produce the

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methylol derivative of the amino or imino group, then form the methylene cross-linking, such as N–CH2–N [22-24]. Similar phenomena have occurred in the bioplastic consisting of soy protein. 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 Figure 3). The reason for this 10

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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 hour (see IR spectrum (b) in Figure 4). Therefore, at the high HCHO concentrations 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

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concentration of < 5%, the HCHO molecules could penetrate into the material. As a result, the

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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

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solution, effectively formed the three-dimensional network not only on the surface but also on

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the inside of pelleted soy protein, and this material showed the highest bending strength of the

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various materials. In a further experiment, the bioplastic, which was prepared in the 1%

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aqueous HCHO solution, was used.

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3.4. Thermal property of the bioplastic

Figures 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 < 100°C, related 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 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 endothermic peak 11

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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 decomposition, was also obtained for the non-reacted soy protein. At > 200°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 formation of a three-dimensional network with cross-linking of the peptide chain. Similar phenomena, such as the thermal stabilization by the cross-linking

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between polymer chains, have been reported for various organic-inorganic hybrid materials

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[27,31,32] and a cross-linked protein film [33]. These results suggested that the soy protein is thermally stabilized by the reaction with HCHO and the prepared bioplastic can be stably

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3.5. Biodegradation of the bioplastic

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used at < 200°C.

The swelling behavior of bioplastic is important for biodegradable properties. Before

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the biodegradable measurement of the bioplastic, we demonstrated the swelling test in water.

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The swelling ratio of the bioplastic was calculated from the weight of swelling materials. As a result, 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 biodegradable samples were immersed in water for 24 hours 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 more than 10 types of protease [25]. Therefore, the pronase has been used at the biodegradable experiments, such as the degradation of polyamino acids and polyesteramides, for one of general proteolytic enzymes [34-36]. Furthermore, there are various proteolytic enzymes in nature, such as soil, river, sea, 12

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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. Figure 6 () and () show the biodegradable amount of the bioplastic using the pronase concentrations of 1 unit ml-1 and 10 units ml-1, respectively. The incubation

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temperature was performed at 37 °C. The biodegradable amounts were calculated from the

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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 aqueous pronase solution. In

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addition, the biodegradable rate varied with the pronase concentration. When the bioplastics

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were incubated at the pronase concentrations of 1 unit ml-1 and 10 units ml-1, the

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biodegradable amounts of the bioplastics were 17% and 38%, respectively, after the incubation for 144 hours. The biodegradable amount in 10 units ml-1 was two or more than

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that in 1 unit ml-1. These results suggested that the bioplastic consisting of soy protein

such as pronase.

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possesses a biodegradable property in an aqueous solution containing a proteolytic enzyme,

Figure 7 shows photographs of the bioplastic which was incubated in the (a) absence and (b) presence of pronase for 144 hours. 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 Figure 7(b)). Therefore, we observed the bioplastic by scanning electron microscopy (SEM). Figures 8 (a) and (b) show the SEM images of the bioplastic which was incubated in the absence of pronase for 144 hours. 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 Figure 8 (b)). Figures 8 (c) 13

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and (d) show SEM images of the bioplastic which was incubated in the presence of pronase for 144 hours. The biodegraded bioplastic showed a large crack on the surface. The width of the crack on the surface was over 100 m. Therefore, we observed the inside of the crack. The magnified SEM image showed on unevenness over a wide range (see Figure 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

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the peptide chain on the surface of the bioplastic and hydrolyzed the peptide bonding. As a

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result, the crack was produced on the surface. In addition, since the a three-dimensional

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networks through the cross-linking between the peptide chains are formed at high density on the surface of bioplastic than inside, the hydrolysis of bioplastic by pronase occurs

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preferentially at the inside of the crack. Consequently, the inside of the crack showed a

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4. Conclusion

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significant unevenness.

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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 concentration. When the bioplastic was prepared in a 1% aqueous HCHO solution, the highest bending strength of ca. 35 MPa was obtained. This strength of the bioplastic was the same as that of commercial polyethylene. Additionally, the bioplastic showed a thermal stability at < 200°C by the formation of a three-dimensional network through the methylene cross-linking between the peptide chains. Furthermore, 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, industrial parts, and alternative substance of artificial plastics. In particular, since the soy protein is derived from discarded soybeans, the bioplastic 14

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consisting of soy protein may have the potential for application of agricultural fields, such as the agricultural mulch, seedling pots, and disposable poles.

Acknowledgements This work was supported by JSPS KAKENHI Grant Number JP19K12408,

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JP19K22298.

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John Wiley & Sons, New York, 1998.

biochemically-stable RNA hybrid material, Polym. Adv. Technol. 29 (2018) 2890-2898. A. Bozkurt, W.H. Meyer, Proton conducting blends of poly(4-vinylimidazole) with

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M. Yamada, S. Funaki, S. Miki, Formaldehyde interacts with RNA rather than DNA:

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phosphoric acid, Solid State Ionics 138 (2001) 259-265.

accumulation of formaldehyde by the RNA-inorganic hybrid material, Int. J. Biol.

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Macromol. 122 (2019) 168-173.

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M. Yamada, H. Aono, DNA-inorganic hybrid material as selective absorbent for harmful compounds, Polymer 49 (2008) 4658-4665.

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M. Yamada, S. Shiiba, Preparation of pectin-inorganic composite material as accumulative material of metal ions, J. Appl. Polym. Sci. 132 (2015) 42056-42063.

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biodegradation behavior of polyesters, Eur. Polym. J. 121 (2019) 109296-109326. 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

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Figure captions Figure 1.

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

Figure 2.

Bending stress-strain curves of (a) bioplastic, which was prepared in a 1%

of

aqueous HCHO solution, and (b) commercial polyethylene. The distance

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between the fulcrums was 5.0 mm. These measurements were done at 25 °C

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under a 50  10% relative humidity condition. Five experiments gave similar

Bending strength of the bioplastic. The concentrations of the aqueous HCHO

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Figure 3.

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results.

solution were varied from 0% to 37%. The bending strength was expressed by an

Figure 4.

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average of five measurements.

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.

Figure 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.

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Figure 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

Photographs of bioplastic which was incubated in the (a) absence and (b)

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Figure 7.

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were expressed by an average of three measurements.

presence of pronase for 144 hours. The concentrations of the aqueous pronase

-p

solution was 1 unit ml-1. The obtained samples were dried overnight at room

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SEM images of bioplastic which was incubated in the (a, b) absence and (c, d)

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presence of pronase for 144 hours. The (b) and (d) images show the SEM images of (a) and (c) at higher magnifications, respectively. The concentration of the

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Figure 8.

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temperature.

aqueous pronase solution was 1 unit ml-1. The obtained samples were dried overnight at room temperature.

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Author 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

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acquisition

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8