Green Chemistry PAPER - IMDEA Materials

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H.-B. Yue,a,b Y.-D. Cui,*a,c P. S. Shuttleworthd and James H. Clark*d. Received ... bIMDEA Materials Institute, C/Profesor Aranguren s/n, 28040 Madrid,. Spain.
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Preparation and characterisation of bioplastics made from cottonseed protein† H.-B. Yue,a,b Y.-D. Cui,*a,c P. S. Shuttleworthd and James H. Clark*d Received 31st January 2012, Accepted 14th May 2012 DOI: 10.1039/c2gc35509d

Cottonseed protein bioplastics (CPBs) from cottonseed flour were successfully prepared by hot-press molding in the presence of urea, aldehydes and glycerol. The effect of cross-linking treatment on the thermal stability, water absorption resistance and mechanical strength was investigated, and found to improve all properties. Increasing glycerol concentration resulted in a decrease in denaturation and α-relaxation temperature of the cottonseed protein as well as storage modulus of the plasticised CPBs. Interestingly, the colour and odor of the CPBs before and after hot compression changed. The mechanism proposed involved urea induced protein denaturation and Maillard-driven generation of the cross-linked structure, both in thermal and alkaline processed conditions. According to Fickian diffusivity, liquid transport and liquid permeability, chemical interactions and physical transport processes are responsible for the different water transport behaviours in the CPBs and the cross-linked CPBs, respectively. These findings could provide valuable in-depth information for tailoring the properties of the environmentally sustainable CPBs, which are attractive for low-load bearing applications, such as agriculture, packing and garden amenities, etc.

1 Introduction Bioplastics made from both animal waste protein (e.g., horn meal, leather scraps and feather meal) and plant waste protein (e.g., soy dreg, corn dreg, wheat gluten and cottonseed) have emerged as a new class of green and eco-friendly materials, known as second generation bioplastics (SGBs).1 Abundance of natural resources, low cost, easily modifiable properties and ease of manufacture make these SGBs suitable alternatives to petroleum-based plastics. For these reasons, both industry and academia are considering them in many environmentally sensitive industries, including agriculture (e.g. mulch films, greenhouse films, flower pots, planting pots, etc.), packaging (one-time or short-term use before disposal) and biomedical industries.2–4 Santin and Ambrosio3 have ascertained the bioactivity of soybean-based materials on various biochemical and cellular components of regenerating tissues. In 2008, worldwide oil seeds production reached 397.21 million metric tons, of which about 10% were

a

School of Materials Science and Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi 710072, China IMDEA Materials Institute, C/Profesor Aranguren s/n, 28040 Madrid, Spain c Green Chemical Engineering Institute, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong 510225, China. E-mail: [email protected] d Green Chemistry Centre of Excellence, Department of Chemistry, University of York, York YO10 5DD, UK. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c2gc35509d b

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cottonseed, representing the next most important source of plant proteins after soybean (53%) and rapeseed (14%).5 Except for cottonseed oil, cottonseed meal is the second most valuable product of cottonseed, accounting for over one-third of the total product value. In addition, cottonseed protein (CP) extracted from cottonseed meal has amino acid components and nutrition value, thus it is being used mainly as dairy cattle feed, and is not extensively used in non-food industries. Cottonseed proteins have almost the same primary amino acid components as soy protein, containing 60% of globulins with gossypin (11S) and congossypin (7S) and 30% of albumins (2S).6 Moreover, the processability of CP with respect to plasticiser efficiency (PE) calculated from the relative contents of amino acids was compared to that of other proteins (Table S1, in the ESI†). A relatively high PE value (>5) makes cottonseed protein a good candidate as a renewable raw material for bioplastics production since it exceeds the value (PE = 2.5) required for protein-based thermoplastic processing. To date, little research has been carried out on cottonseed protein bioplastics (CPBs). Casting of cottonseed protein films has been achieved by Marquié,7 but this process is time-consuming and film formation is limited. By comparison, hot compression molding is a more efficient strategy in achieving protein films. Grevellec et al.8 succeeded in obtaining a biodegradable CP film within a set temperature range through which the protein could be extruded or thermo-molded. However, there have only been a few studies reported on hotpress molding of CP for bioplastic fabrication, while on soy protein there are numerous articles.1,9,10 Protein-based bioplastics have inferior mechanical properties to commonly used synthetic polymers, principally due to their Green Chem., 2012, 14, 2009–2016 | 2009

complex composition, hydrophilic nature, impurities and high environmental sensitivity. To help address these issues, modification and the processability of protein have been optimised, including protein denaturation, cross-linking, plasticisation, etc. Also the addition of an appropriate compatibiliser into soy protein-based composites has been reported recently by Zhang’s group11,12 as an efficient way to improve protein dispersion in a polymeric matrix, thus obtaining higher mechanical performances. Protein denaturation induced by chemical modification could disrupt its secondary, tertiary or quaternary structures, exposing the amino acid side chains, and thereby introducing new interactions by means of hydrogen or ionic bonding.13 Urea is commonly used as a chemical denaturant for the preparation of protein-based bioplastics as it can destabilise the globular protein by forming strong hydrogen bonds with water molecules that surround the protein. This can disrupt the protein hydrogen bonds, resulting in flexible peptide chains.14 In this case, the denatured protein would facilitate the formation of entanglements and cross-linked structures during the chemical crosslinking process, resulting in plastics with high tensile strength and water absorption resistance. Although aldehydes, the most commonly used cross-linking agent, can readily react with amine groups, there has been no consensus on their reaction with proteins. Sun et al. found that formaldehyde and glutaraldehyde are able to cross-link the amine group of lysine as well as the side chains of other amino acids within the protein.15 Studies on the mechanism involved in formaldehyde-, glutaraldehyde-, and glyoxal-induced cross-linking of CP found that lysine had a key role in protein cross-linking with dialdehydes.7 Glandless cottonseed flour purified from cottonseed meal is used in this study as the main source of CP. In addition to its high protein content (Table S2, in the ESI†) compared with other oil seeds except soybean, it contains a small amount (∼3% dry weight basis) of subsidiary cellulose from crude fibre that acts as a reinforcing component in the resultant composite materials. As an initial study, the processability of cottonseed flour into the CPBs by hot-press molding in the presence of aldehydes as a cross-linking agent and glycerol as a plasticiser was investigated.16 In this paper, we developed a protein denaturation process by the addition of urea before cross-linking and plasticising treatments, and optimised the bioplastic processing conditions. The effects of plasticising and cross-linking treatments on various properties (thermal, mechanical, and moisture sensitivity) were characterised. Different water transport behaviours in the bioplastic networks were evaluated, and finally the mechanism involved in urea induced protein denaturation and aldehyde induced cross-linking was proposed and interpreted.

2 Experimental 2.1 Materials

Glandless cottonseed flour was obtained from Cotton-unis Bio-tech Co. Ltd, China, with the protein content previously being reported as 57.2 wt% from nitrogen analysis.17 Urea, formaldehyde (400 g L−1), glyoxal (400 g L−1), glutaraldehyde (500 g L−1) and glycerol were supplied by Guangzhou Chemical Reagent Factory, and used without further treatment. 2010 | Green Chem., 2012, 14, 2009–2016

2.2 Protein denaturation and cross-linking treatment

Cottonseed flour was first dissolved in deionized water at a solid–liquid weight ratio of 1 : 6, followed by the addition of 1 M urea solution. Using a magnetic stirrer the mixture was then agitated for 4 h at ambient temperature to obtain the denatured cottonseed protein (DCP). The pH of the DCP solution was then adjusted to 11 ± 0.1 with 1 N NaOH solution and stirred for a further 10 min. The alkaline solution was then transferred to a water bath (70 °C), and a cross-linking agent (formaldehyde, glyoxal or glutaraldehyde) at 10 wt% of cottonseed flour was added. The resultant mixture was vacuum-dried for 10 h at 80 °C prior to further fabrication. 2.3 Preparation of cottonseed protein bioplastics

Glycerol was added to the dried denatured cross-linked protein described above, and then homogenised in a high-speed mixer (HR1704, PHILIPS Ltd) for 5 min. The mixture was then ground and processed using a triple roller mill (SG-65, Goldenblan Chemical Machine Cop. Ltd) and later conditioned in a desiccator (silica gel as the desiccant) for 24 h at room temperature before hot-press molding. The conditioned mixture was placed in a sample mold (stainless steel plates) covered with aluminum foil mounted over two sides before hot press molding at 20 MPa, 130 °C for 5 min. The mold was subsequently cooled to room temperature, and the formed CPBs were placed in a desiccator prior to characterisation. Both the aluminum foil covering and subsequent cooling process promote the ease of de-mould handling. The modified CPBs are coded as CP-0CL, CP-FA, CP-GX, and CP-GA representing the sample made from CP without cross-linking treatment, CP cross-linked with formaldehyde, glyoxal, and glutaraldehyde, respectively. The glycerol (0, 10, 20 and 30 wt% of cottonseed flour) plasticised CPBs are coded as CP-0GC, CP-10GC, CP-20GC and CP-30GC, respectively. 2.4 Thermal analysis

To investigate the effect of the plasticiser (glycerol) content on the denaturation temperature of the CPBs upon heating, differential scanning calorimetry (DSC) measurements were carried out with a DSC-Q200 apparatus under a nitrogen atmosphere. The samples (CP-10GC, CP-20GC, and CP-30GC) were first heated from 25 to 100 °C at a heating rate of 20 °C min−1, held for 5 min, and then cooled from 100 to −80 °C at a rate of 20 °C min−1, holding again for a further 5 min. The second heating step from −80 to 250 °C at a heating rate of 10 °C min−1 was recorded. In addition to DSC, thermogravimetry analysis (TGA) of the CPBs was conducted on a TG 209 under a nitrogen atmosphere ( protective gas flow was 15 ml min−1) at a heating rate of 30 °C min−1 from 25 to 500 °C. Dynamic mechanical properties of the plasticised bioplastics were investigated from ∼25 to 200 °C at a rate of 5 °C min−1 using a dynamic mechanical analyzer (DMA Q800, TA) in a tension film configuration with preload force 20 mN, amplitude 20 μm and frequency 5 Hz. The sample dimensions (Length, Width, and Thickness) were about 20 mm (L), 4.5 mm (W) and 0.7 mm (T). For the specimens after the water This journal is © The Royal Society of Chemistry 2012

absorption test, another working condition for DMA measurements was applied: preload force 10 mN, amplitude 10 μm and frequency 1 Hz. 2.5 Mechanical properties

The tensile strength measurements were tested on a universal testing machine (CMT6503) according to ISO6239-1986 (E). Dog-bone shaped samples were evaluated under standard conditions (25 °C and 50% relative humidity) to obtain stress and modulus values from the mean of five tests. Micro-indentation tests were carried out on a Vickers micro-hardness tester (HMV-2) at ambient temperature. The specimen surface (6 mm2), with a thickness of 0.6 mm, was polished prior to the test, and the load applied on the sample was maintained for 15 s. Fig. 1 DSC thermograms of the glycerol plasticised CP-10GC, CP-20GC and CP-30GC CPBs. The temperatures denoted in the dashed rectangle indicate the CP denaturation temperature (Td).

2.6 Structure and morphology

Fourier transform infrared (FTIR) spectra of the samples (CP, DCP, CP-GA and CP-10GC) were obtained using an FTIR 2000 from 4000 to 500 cm−1 wavenumbers. Before analysis, each specimen was ground with a pestle and mortar, vacuum-dried at 80 °C for 12 h, and then mixed with KBr. Pure glycerol was used as a reference chemical for comparison with the CP-10GC. The surface morphology and fracture microstructure after tensile testing of the CPBs (CP-0CL, CP-FA, CP-GX, and CP-GA) were recorded using a scanning electron microscope (SEM, EVO® MA15). The specimens were dried in a desiccator at room temperature for 3 days, and then powder coated with gold. They were then tested on the SEM using an accelerating voltage of 10 kV at 2000× magnification, and 18 kV at 800× magnification. 2.7 Water absorption and kinetics

Water absorption of the CPB (CP-0CL, CP-FA, CP-GX, and CP-GA) films with a thickness of 0.6 mm was carried out according to ASTM D570-98 standards. The CPB water absorption kinetics were also determined. The pre-dried and weighted CPB films with a thickness of 0.6 mm were immersed in distilled water at 25 °C. Samples were removed from the water at regular intervals, dabbed with filter paper to remove excess water, and the weight recorded. Water absorption (WA) of the samples is calculated using eqn (1): WAð%Þ ¼

Wt  W 0  100 W0

ð1Þ

where W0 and Wt are the initial weight of the bioplastics and the weight of the sample after being immersed in water for t min, respectively.

3 Results and discussion 3.1 Thermal analysis 3.1.1 Plasticiser effect on CP denaturation. The heat induced denaturation behaviour of cottonseed protein within the CPBs is shown in the DSC scans (Fig. 1). As the protein This journal is © The Royal Society of Chemistry 2012

denatures, its structure unfolds with the breaking and rearrangement of intra- or inter-molecular protein interactions. During this unfolding process, the protein absorbs energy and this is recorded as an endothermic peak in the DSC thermograms, denoted as the denaturation temperature (Td). Glycerol plays a positive role in accelerating this denaturation process for CP. For instance, the Td of CP within the CPBs decreases from 156.2 °C to 136.0 °C when the glycerol content increases from 10 to 30 wt%, respectively. The role of glycerol as a plasticiser in the bioplastics system could be well explained by the “free volume” theory.18 Creation of free volume within the polymer matrix occurs due to the plasticiser molecules helping to lower the Tg and hence increases the polymer chain flexibility, via lubrication or chemical interaction with the polymer chains, in most cases substituting the polymer–polymer interactions with polymer– plasticiser ones, and thereby reducing the polymer chain rigidity. As a result, the susceptibility of the CP chains to external environments, such as heat, will be greatly increased, resulting in a lower Td for the glycerol plasticised CPBs. 3.1.2 TGA analysis of thermal stability. The TGA curves in Fig. 2 indicate three distinct stages of mass loss for all the CPBs. In the first stage, the mass loss (less than 10 wt%) occurs between room temperature and 100 °C (inset graph A, Fig. 2), mainly arising from the evaporation of absorbed moisture. In the following stage from 160 to 230 °C, the CPBs decomposed quickly with a mass loss of 20 to 40 wt% (inset graph B, Fig. 2) that is attributed to the decomposition of small molecules (glycerol and urea residues), as confirmed by Zhang and coauthors.19,20 In the last stage, decomposition of the cottonseed protein occurs at temperatures higher than 260 °C. The volatiles released as a consequence of degradation are CO2, CO, NH3 and unsaturated compounds with carbonyl groups presence according to Schmidt et al.21 using FTIR spectra. At all temperature ranges, the TGA thermograms of the CPBs (CP-FA, CP-GX, and CP-GA) showed less mass loss compared with CP-0CL, indicating an improvement of thermal stability for the cross-linked CPBs. The enhanced thermal stability is probably attributed to the formation of strong imine (–CHvN–) Green Chem., 2012, 14, 2009–2016 | 2011

Fig. 3 DMA measurements show glycerol content dependence of storage modulus and tan delta of the plasticised CPBs as a function of temperature. Note that the sample without glycerol (CP-0GC) was too brittle to record its dynamic mechanical behaviour; therefore the information on its storage modulus was only present in a temperature range from room temperature to 80 °C.

Fig. 2 Weight loss and DTG curves of the CPBs as a function of temperature. Inset graphs denote the first stage (A) and second stage (B) of the CPB weight loss.

bonds formed by the cross-linking agent and CP during the hot press molding, which is similar to cross-linked soy protein bioplastics.22 It is interesting to note that the CP-FA sample exhibited the least mass loss below 190 °C, corresponding to the highest thermal stability among all the CPBs over this temperature range, whereas above 190 °C, CP-GA exhibited the highest thermal stability. This might indicate that the interactions between CP and GA with increasing temperature are greater than those of CP and FA, probably due to the higher cross-linking efficiency of GA compared to FA upon heating.23 In addition to the thermal stability, both colour and smell of the CPBs were monitored. The colour of the CPBs changed from yellow-brown before preparation, to golden-brown after hot compression molding (Fig. S1, in the ESI†). Interestingly, the odor of the different CPBs was not the same after hot press molding. For example, CP-GA smelled like persimmon while the smell of the CP-FA CPBs was a bit malodourous. 3.1.3 Dynamic mechanical properties. The glycerol content dependence of storage modulus of the plasticised CPBs can be found in DMA measurements (Fig. 3). Storage modulus of the CPBs decreased with increasing temperature. In addition, the CPBs containing lower amounts of glycerol showed a higher value of storage modulus, especially at low temperature. For example, with 30 wt% of glycerol, the plasticised CPBs exhibited a fourfold decrease in storage modulus at 60 °C, compared to the CP-0GC. 2012 | Green Chem., 2012, 14, 2009–2016

Fig. 4 Water absorption behaviour of the CPBs measured in distilled water at 25 °C.

The tan delta peaks in Fig. 3 are assigned to α-relaxation temperature (Tα) of CP. From the DMA curves the change in location of the relaxation peak is due to the degree of freedom or order of the chain segment motions. For instance, the Tα decreases from about 145 °C for CP-10GC to 115 °C for CP-30GC, although the CP-10GC and CP-20GC have almost the same Tα value. The results are ascribed to the elevated amount of the mobile glycerol component as a plasticiser participating in the cross-linked matrix relaxation process. The variation in Tα value is in agreement with that in Td value observed from DSC thermograms. 3.2 Water absorption 3.2.1 Water absorption kinetics. Fig. 4 shows the water absorption kinetics for the CPBs in accordance with the ASTM D570-98 standard. It is clearly seen that water absorption of all bioplastics increased markedly over the first 2 h of immersion, followed by a gradual decrease in absorption rate over the This journal is © The Royal Society of Chemistry 2012

Table 1

Values of water absorption, n, k, diffusion, sorption and permeability coefficient for the CP-0CL, CP-FA, CP-GX and CP-GA CPBs Fickian diffusivity

Samples

Water absorption (%)

Liquid transport

n

k

R2

D × 108 (cm2 s−1)

R2

D′ × 107 (cm2 min−1)

S (g g−1)

P × 108 (cm2 min−1)

CP-0CL CP-FA CP-GX CP-GA

40.90 37.23 33.27 32.43

0.3889 0.4399 0.4629 0.4980

0.0782 0.0583 0.0465 0.0382

0.9358 0.9467 0.9874 0.9990

1.063 1.303 1.516 1.658

0.9564 0.9872 0.9983 0.9997

0.638 0.986 1.367 1.665

15.131 13.773 12.308 11.998

0.966 1.359 1.683 1.998

following 8 h. After this point, water absorption equilibrated, not showing any further mass increase. The cross-linked bioplastics (CP-FA, CP-GX, and CP-GA) absorbed less water than the standard sample (CP-0CL) over the given test period, indicating that cross-linking helps reduce water absorption. The cross-linked bioplastics have a more compact, tightly bound structure, which helps retard the distance water can diffuse within, and hence lowers water absorption. Generally, due to the hydrophilic nature of the carboxyl and hydroxyl groups within the protein, water absorption is a considerable problem for protein-based plastics. For instance, soy protein plastics increase in mass by 78.66% when conditioned in distilled water at room temperature for 2 h.24 Surprisingly, the percentage mass increase of the CP-0CL, CP-FA, CP-GX, and CP-GA CPBs under the same experimental conditions was 19.39, 16.35, 14.15 and 13.51%, respectively, showing a marked improvement in protein bioplastic water resistance. 3.2.2 Water transportation evaluation. The CPB water absorption test results were analysed using Fickian diffusivity, liquid transport and liquid permeability theories to help evaluate the liquid transit properties of the polymer network. The fitting parameters corresponding to these three different theories are summarised in Table 1. The widely adopted Fickian diffusion theory is based on the relative rates of water diffusion and polymer chain relaxation.25 The water absorption of the CPBs is plotted as a function of time for 0 ≤ (Mt/M∞) ≤ 0.6. The CPB data are fitted to eqn (2):

M t =M 1 ¼ kt n

ð2Þ

where Mt and M∞ are the mass of the samples at water absorption time t and at the equilibrium state, respectively. k is a characteristic constant related to the structure of the polymer network, and can indicate an interaction between the polymer and water. n is the swelling exponent which is indicative of different Fickian diffusion models:25 (a) Fickian diffusion (n = 0.5), known as Case I diffusion, occurs when the rate of diffusion is significantly slower than the rate of relaxation of the polymer chains; (b) Case II transport (n = 1) arises when the rate of diffusion is greater than the rate of relaxation of the polymer chains; (c) non-Fickian diffusion (0.5 < n < 1) occurs when the rates of diffusion and polymer relaxation are comparable, and is connected with the transition region between Case I and Case II scenarios. The values of n and k for the water sorption tests of the CPBs are obtained from the slope and intercept of the plot (Fig. S2, in the ESI†) of ln(Mt/M∞) vs. ln t, and given in Table 1. It is shown that the values of n and k vary for different bioplastics, with the n value increasing, whereas the k value decreases after addition of the cross-linking agent (FA, GX or This journal is © The Royal Society of Chemistry 2012

Liquid permeability

GA). For example, the n value increases nearly to 0.5 (CP-GA) from 0.388 (CP-0CL), while the k value drops from 0.078 (CP-0CL) to 0.038 (CP-GA). Fickian diffusion behaviour is found in the CP-GA (n = 0.5), implying a higher polymer chain relaxation rate in comparison with the water diffusion rate. However, the low value of n for CP-0CL indicates that the mechanism of water transport deviates from the Fickian diffusion. The liquid transport model, based on the microstructure of the materials and the affinity of the polymer components and plasticisers to water,26 was also applied to the CPBs. The mass of the water absorbed (Mt − M0) by the samples at time t can be expressed as " # 1 X Mt  M0 8 Dð2n þ 1Þ2 π 2 t ð3Þ ¼1 exp 2 2 M1 ð2LÞ2 n¼0 ð2n þ 1Þ π where 2L and D are the thickness of the specimen and the diffusion coefficient, respectively. Over a short period of time, eqn (3) can be rewritten as   Mt  M0 2 D 1=2 1=2 ¼ t M1 L π

ð4Þ

Eqn (4) is useful to calculate the diffusion coefficient D with the error in the order of 0.1%, when (Mt − M0)/M∞ ≤ 0.5.26 The data obtained in the water absorption experiments are fitted to eqn (4) and the D values are calculated and presented in Table 1. The reduction in D value for different CPBs is in the order CP-0CL < CP-FA < CP-GX < CP-GA. Interestingly, a high D value for the cross-linked bioplastics was found, indicating that water diffuses more easily in the cross-linked networks based on liquid transport theory. The last theory, liquid permeability, is based on diffusivity as well as the sorption or solubility of a liquid in the polymer networks.27 Three parameters, diffusion coefficient (D′), sorption coefficient (S) and permeability coefficient (P), are introduced in this theory to characterise the ability of a liquid to permeate the polymer chains. The diffusion coefficient D′ can be measured using eqn (5): D′ ¼ πðhθ=4M 1 Þ2

ð5Þ

where θ is the slope of the linear portion of the water sorption curve shown in Fig. 4, and h is the initial sample thickness. The sorption coefficient S is calculated using eqn (6): S ¼ M 1 =M 0

ð6Þ

The permeability coefficient P is a net effect of diffusion and sorption, and gives an indication about the amount of water Green Chem., 2012, 14, 2009–2016 | 2013

Fig. 5 Micro-hardness (MH) as a function of load applied to the CPBs for 15 s at ambient temperature.

permeating through the uniform area of the sample per second, and is given by eqn (7): P ¼ D′S

ð7Þ

The values of three coefficients (D′, S and P) fitted to the water absorption data of the CPBs are listed in Table 1. It can be seen clearly that D′ and P values increase while the S value decreases for the cross-linked bioplastics (CP-FA, CP-GX and CP-GA) compared to the untreated sample (CP-0CL). This finding suggests that the cross-linked structures promote the physical transport process, including water diffusion and permeability, while in the absence of crosslinking, it is chemical interactions such as hydrogen bonding and van der Waals interactions that facilitate water transport within the protein chains.

Fig. 6 FTIR spectra of (a) the pristine, denatured and cross-linked CP and (b) pure glycerol and the CP-10GC.

3.3 Mechanical properties

3.4 Structure and mechanism

Mechanical strength of the CPBs was measured by means of tensile testing, micro-indentation and DMA measurements. Low tensile strength and Young’s modulus of the CPBs were found previously.16 Cross-linking of the CPBs showed a slight improvement in modulus, in accordance with their thermal stability and water absorption properties as discussed earlier. Fig. 5 shows the dependence of micro-hardness (MH) on the load applied for the CPBs. As expected, an improved MH was found for the cross-linked samples. For instance, at a load of 50 g, the MH of the CP-GA (45 MPa) was significantly higher than that of the CP-0CL (2 MPa). From Fig. 5 it can be seen that the MH of the CPBs displays a minimum value (25 g) on increasing the load, suggesting that the bioplastics fabricated in our study do not show ideal plastic behaviour.28 For the cross-linked CPBs (CP-FA, CP-GX, CP-GA), the preservation of their mechanical properties after conditioning the samples in distilled water for 2 h was evaluated through DMA testing. The results showed a significant approximate 12-fold decrease (from ∼120 MPa to ∼10 MPa) in storage modulus measured at room temperature. The decreases in storage modulus after water adsorption are due to both the soft nature of the CPBs, resulting from hydrophilic groups inside of the protein, and the protein chain relaxation, ascribed to the function of water serving as a plasticiser in the protein matrix.

3.4.1 FTIR analysis. Fourier transform infrared (FTIR) spectra of the cottonseed proteins (denatured and cross-linked CP) are shown in Fig. 6a. The broad absorption bands around 3500 cm−1 for the CP are attributed to amide N–H stretching vibration bands. However, for the denatured cottonseed protein (DCP) two bands at 3439 and 3335 cm−1 are observed and assigned as the amide N–H stretching vibration bands in CP and CO(NH2)2. The characteristic absorbing band ranging from 1630 to 1680 cm−1 is attributed to the CvO stretch vibrations of the peptide linkages of amide I. This region is the most sensitive spectral region to the protein secondary structure.29 The amide II band, in contrast, is derived mainly from in-plane N–H bending and C–N stretching vibrations at 1480–1575 cm−1. Since the locations of both amide I and amide II bands are sensitive to the secondary structure of the protein, the frequency of these bands depends on the nature of the hydrogen bonding in CvO and N–H moieties. The presence of urea in CP may also result in component bands, including α-helices, β-sheets, turns and random structures.29 It was observed from Fig. 6a that the characteristic absorbing band of amide I shifts to a lower frequency, 1618 cm−1 (DCP) from 1648 cm−1 (CP), and that the amide II band shifts from 1541 cm−1 (CP) to 1467 cm−1 (DCP). In addition to the change in vibrational position for the amide bonds in the DCP sample, a new peak centred at 1665 cm−1 for

2014 | Green Chem., 2012, 14, 2009–2016

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CP-GA (Fig. 6a) was observed, and is associated with the imine (–CHvN–) stretching vibration. This confirms that cross-linking

has occurred between CP and GA during hot compression molding, which is absent in the CP and DCP samples. From FTIR spectra of pure glycerol and the glycerol plasticised CPBs (CP-10GC) shown in Fig. 6b, it is clear that for the CP-10GC a marginal decrease in the spectrum intensity of C–O (1000–1260 cm−1) and C–H (2800–2950 cm−1) stretching bands can be found when comparing with pure glycerol. Besides, OH (3300–3400 cm−1) stretching vibration bands in the CP-10GC are broader than those in pure glycerol. The changes in characteristic absorption bands frequency and intensity might partially support the role of glycerol acting as a plasticiser in the CPB networks. 3.4.2 Morphology. SEM micrographs of the surface morphology and fracture of the CPBs (Fig. 7) show that the CPB specimens (CP-FA, CP-GX and CP-GA) have a fluctuating and continuous structure, whereas the CP-0CL structure is discontinuous. The height of the asperities at the fractured surface of the bioplastics also indicates ductile failure for the crosslinked structures. This is verified from their rough surface morphology compared to CP-0CL that appears smooth with cracks, indicating its brittle nature. The plastics treated with GA or FA have smaller cracks than the plastics treated with GX, which is in agreement with the differences in their mechanical properties.

Fig. 7 SEM images of the fractured microstructure (left) and the surface morphology (right) of the CPBs.

3.4.3 Mechanism. A schematic illustrating the CP denaturation and cross-linking process is proposed in Fig. 8. It can be seen that urea denatures the protein via both indirect and direct hydrogen bonding to water and the cottonseed protein as observed by Bennion and Daggett30 using atomic-resolution molecular dynamics simulation. Indirect urea denaturation with CP occurs by reduction in the water–water and/or water–protein

Fig. 8 Schematic illustration of the urea induced CP denaturation via indirect and direct interactions, and of the Maillard-driven cross-linking reaction.

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Green Chem., 2012, 14, 2009–2016 | 2015

interactions, weakening cohesion of the water molecules. This decreases water diffusion, and thereby facilitates side chain functional group exposure, resulting in increased water–urea hydrogen bonding. Direct hydrogen bonding consists of the interaction between urea and the polar moieties and peptide backbone of the protein, particularly after disruption of the secondary structure of CP. In this case, the number of hydrogen bonds involved with urea and the peptide backbone of CP increases. It has been proved that the cross-linking agents GA, FA, and GX react with the amino acid side chains of CP, particularly with the lysine ε-NH2 group, to form Schiff bases.31 The change in colour and odor of the CPBs occurs via the reaction of the carbonyl group with the amino group.32,33 This Maillard reaction is further accelerated by increased nucleophilicity as the amino groups are deprotonated. In this study, the carbonyl groups in the cross-linking agents react with the amino groups in CP under an alkaline and heated environment, resulting in the formation of an imine group (–CHvN–), and thus improve thermal stability, mechanical strength, and moisture resistance of the CPBs (CP-FA, CP-GX, and CP-GA). Moreover, Maillard-driven formation of the complex mixture is expected to be responsible for the variation in colour and odor observed.

4 Conclusions Cottonseed protein bioplastics (CPBs) using cottonseed flour were successfully prepared by hot-press molding. Cottonseed protein (CP) was modified by denaturing and cross-linking it with urea and aldehydes, respectively. The cross-linked CPBs showed improved thermal stability, water absorption resistance and mechanical strength, mainly due to the Maillard-driven formation of the cross-linked structure (imine bonding). This was confirmed by FTIR and SEM characterisation as well as the thermal, mechanical and water absorption tests. Increased plasticiser content (glycerol) in the bioplastics led to a decrease in denaturation and α-relaxation temperature of CP due to reduced structural integrity and increased free volume. Variation in color and odor, before and after hot compression molding, was also found to be due to Maillard type reactions. By evaluating water transport in the bioplastic networks, it was found that the physical transport process is predominant in the cross-linked CPBs, while chemical interactions play a more important role in noncrosslinked CPBs. The improvements in mechanical and moisture resistant properties and their biodegradable nature may make these ‘green’ CPBs have potential in low-load bearing applications and environmentally sensitive industries.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Number: 20776164). J.P. FernándezBlázquez and J.C. Rubalcaba from IMDEA Materials Institute are gratefully acknowledged for their technical assistance in

2016 | Green Chem., 2012, 14, 2009–2016

DMA and SEM measurements, respectively. The authors would like to thank China Cotton-unis Bio-tech Co., Ltd for supplying glandless cottonseed flour.

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