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Development of Soy Protein Isolate/Montmorillonite Biocomposites Obtained by Injection Moulding

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The development of biocomposites requires a deep knowledge of physical, chemical and functional properties of biopolymers and fillers used. Soy protein, in combination with glycerol and montmorillonite (MMT‑Na+), was used to obtain suitable biocomposites materials. On the one hand, soy protein is one of the cheapest proteins in global markets, since it is a co‑product from soybean oil industry. On the other hand, natural MMT-Na+ was used since it is widely available in nature as micron-size tactoids. In addition, the processing technique used for the development of polymeric matrixes plays a key role. In this sense, injection-molding technique was used since it is one of the most versatile and used polymer processing techniques. However, the dispersion of such particles within a polymer structure is complex, and protein-nanoclay interactions have a high impact on techno-functional properties of biocomposites. The structure of blends was characterized by scanning electron microscopy (SEM) and X-Ray diffraction. Mechanical properties of biocomposites were evaluated by dynamic mechanical analysis (DMTA) and tensile tests as well as water uptake capacity was also determined. Results suggest that the addition of nanoclay does not improve tensile parameters since nanoclay particles are not exfoliated. However techno-functional properties i.e. water uptake capacity was highly improved. In this sense, these results suggest that the biocomposites obtained would be suitable for developing new bio‑based superabsorbent materials.

Keywords: Biocomposites; Nanoclay; Soy protein; Tensile strength test; Water uptake capacity (WUC)

1. Introduction

Plastic materials are the most commonly used materials due to their extraordinary properties [1], increasing the number and the new applications each day [2]. Indeed, combining different polymers or materials with different specifications allows us to modulate their properties and make materials without limits. The problem is that a big majority of plastics materials are petroleum-based and it is known that oil resources are not eternal, moreover, a large part of this plastics are non recyclable and they cause an accumulation of wastes. In this context, the substitution of petroleum-based plastics by bio-based plastics is seen as a promising alternative [3,4]. However, nowadays, the challenge is divided in two parts. On the one hand, scientists have to develop technologies which allow us to processing those new materials and on the other hand, they have to adapt it for existing applications. One alternative has been the development of biodegradable materials from renewable resources (mainly proteins and polysaccharides) [5]. New researches are devoted to develop new protein-based materials, which were able to replace fossil-based polymer for high quality applications such as superabsorbent. Nowadays, bioplastic applications are limited mostly to food, medical or agriculture industry. However, it appears that more and more bioplastics replace conventional plastics. For example, biopolymers were used in order to replace PVC pipes or make cell-phone coating [6].

The European bioplastics production had double in 2013 compared to 2010 for reach 509,000 tons and increases each year [7]. However, bioplastics production remains low compared to the 240 million tons of conventional plastic [1]. Bioplastics are composed by a polymer matrix, such as polysaccharides or proteins, a plasticizer, in order to reduce intermolecular forces among polymer chains, increasing mobility and reducing the glass transition, and some additives to improve the processability or properties of the final product. In this study, soy protein (polymer matrix), glycerol (plasticizer) and nanoclay (additive) were used. The combination of them builds up a product called biocomposite.

Soy protein isolates (SPI) are a co-product with soybean oil and they are one of the cheapest proteins in global markets, they show superabsorbent properties due to the presence of hydrophilic amino acids [8]. Soy protein concentrates are suitable raw materials for the production of bioplastics, and they have been suitable for performing bioplastics exhibiting a high water uptake [9,10]. In addition, lamellar nanofillers have been postulated to improve mechanical and barrier properties [11–13]. Natural Montmorillonite (MMT-Na+) is one clay mineral widely used in polymer science as filler [14]. It is widely available in the nature as micron-size tactoids. This special disposition consists on several hundred of individual platy particles that are hold together by electrostatic and Van der Waals forces. The gap between each layer is about 1 nm, the sum of several hundred of these plates forms the primary particles of the material (tactoids) [15]. The introduction of this materials leads to increase the water uptake capacity, while the mechanical properties of the hydrogel increase at the same time (especially strength and stiffness) [16]. However, the efficient dispersion of nanoclays in biopolymer matrices is a key problem in biocomposites, where exfoliation is the desirable arrangement for improving the properties of biocomposites [17]. The dispersion of these particles within the polymer structure is complex.

Most of the protein-based bioplastic properties can be easily controlled by adjusting different parameters such as the soy/plasticizer ratio, the quantity of filler or the moulding time and temperature [18]. However, the strength of the polymer is too low and these properties are really influenced by moisture absorption [9].

The overall objective of this work is to develop SPI/MMT‑Na+ biocomposite materials, plasticized with glycerol by using injection-moulding process. Nanoclay was incorporated with the intention of improving the water absorption. Rheological and tensile strength measurements have been carried out in order to evaluate the structure of bioplastics. Moreover, X-Rays diffraction and microscopy have been assessed to analyse the nanoclay incorporation into the material and evaluate its influence on the structure.

2. Material and methods

2.1. Materials

Soy Protein Isolated (SPI) Supro500E with a protein content of ca. 91 wt.% was supplied by Protein Technologies International (Leper, Belgium). Glycerol (GL), purchased from Sigma-Aldrich (St. Louis, Missouri, USA), was used as plasticizer. The nanoparticle introduced was Cloisite®‑Na+ (MMT-Na+). It was obtained from montmorillonites, and manufactured by Southern Clay Products, Inc. (USA).

2.2. Preparation of blends

Biocomposite materials were processed by a thermo-mechanical two-stage process: a mixing step, where blends were obtained, followed by the injection of blends. Hence, the first stage consists of a mixing at 25 ºC, 50 rpm and 60 min in a two-blade counter-rotating batch mixer (HaakePolylab QC; ThermoScientific, Germany). Samples were produced by adding different nanoclay (montmorillonite) concentration to blends with a protein/glycerol ratio of 50/50: 0 wt.% (0%), 3 wt.% (3%), 6 wt.% (6%) and 9 wt.% (9%).

2.3. Characterization of blends

2.3.1. Dynamic Mechanical Thermal Analysis (DMTA)

An oscillating compression was applied to the sample while being subjected to a thermal cycle. It allows to measure viscoelastic properties: E’, E’’ and tan  (E’/E’’) of the sample in a temperature range (from 20 to 150 ⁰C at 3°C/min). Those experiments were carried out by using a RSA3 (TA Instruments, New Castle, USA) connected to a Chiller in order to regulate the temperature.

2.3.2. X-Rays diffraction (XRD)

XRD studies of the composites probes were carried out using a D8 Discover (BRUKE, Massachusetts, USA) (40 kV, 30 mA) equipped with Cu-Kα radiation (λ=0.1516 nm). The scanning range (2θ) was from 2 to 40°, and the step size was 0.05°. Different crystalline phases were visualized, which may indicate systems whose microstructure could be different. X-Rays were carried out on the dough-like material after the mixing step.

2.4. Preparation of biocompoistes

Once the blends are prepared, the next step consists of the injection of the blend into a mould by means of MiniJet Piston Injection Molding System (ThermoHaake, Germany), obtaining biocomposite probes. Suitable processing conditions were selected on the basis of rheological properties found in previous studies. Thus, the mould temperature was 70 ºC and the injection pressure 500 bar over 20 s (200 bar as post-injection pressure was selected over 200 s) [19,20]. Two different shapes were produced: a 60×10×1 mm rectangular-shaped specimen for dynamic mechanical analysis (DMA) experiments as well as water absorption, and a dumb-bell-type specimen for tensile properties of plastics.

2.5. Characterization of biocomposites

2.5.1. Rheological Measurements

RSA3 rheometer (TA Instruments, USA) was used to carry out two different tests within the linear viscoelastic range, studying the viscoelastic properties of the composites:

(i) Frequency sweep test: from 0.02 to 20 Hz at room temperature, obtaining the mechanical spectra.

(ii) Temperature ramp: from 20 to 140 °C and at 3°C/min and 1Hz.

The critical stress was calculated in previous strain sweep tests. A stress was selected in order to perform frequency sweep and temperature ramps into the linear viscoelastic range.

2.5.2. Tensile strength measurements

Tensile tests were performed by using the Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA), according to by ISO 527 [21] for Tensile Properties of Plastics. Young’s Modulus, strain at break and maximum stress were evaluated from at least five replicates for each system using type IV probes and an extensional rate of 10 mm/min at room temperature.

2.5.3. Scanning Electron Microscopy (SEM)

After water immersion over 24 h, biocomposites were lyophilized and then observed by SEM. Microscopy examination has been assessed with a JEOL JSM 6460 LV (Tokyo, Japan) scanning electron microscope with secondary electron detector at an acceleration voltage of 20 kV.

2.5.4. Water uptake capacity (WUC)

Water uptake capacity of composites was measured according to the standard method for determining water absorption in plastics ASTM D570 [22]. Rectangular specimens of 60×10×1 mm were used. The specimens are subjected to drying (conditioning) in an oven at 50 ± 2 ºC for 5-6 h to determine dry weight, then introduced into distilled water and weighed after 24 h immersion. Finally, it is subjected to drying (reconditioning) again and weighed to determine the soluble material loss. All the experiments are performed in triplicate at room temperature. According to the methodology used, water uptake is determined by the following equation (Eq. 1):

% Water uptake= Wet Weight-Final Dry WeightFinal Dry Weight                 1

2.6. Statistical analysis

At least three replicates were carried out for each measurement. Statistical analyses were performed with t tests and one-way analysis of variance (p < 0.05) using PASW Statistics for Windows (Version 18: SPSS, Chicago, IL). Standard deviations were calculated for selected parameters.

3. Results and discussion

3.1. Preparation and characterization of blends

3.1.1 Preparation of blends

Samples with a 50/50 SPI/GL ratio were mixed with different nanoclay (MMT‑Na+) contents to form SPI/GL/MMT-Na+ blends. The MMT-Na+ concentration used were, 3 wt.% (3%), 6 wt.% (6%) and 9 wt.% (9%). Both torque and temperature were recorded along mixing time (Figure 1). Two different behaviours could be found: The samples without nanoclay and the lowest nanoclay concentration (3%) showed torque and temperature profiles which were almost constant during the whole mixing process (although the 3% system shows a soft increase from 50 min). On the other hand, it is worth pointing out that the most concentrated systems (6% and 9%), after an initially constant period, displayed a remarkable increase in both temperature and torque until they reach a maximum value, from which both parameters remain constant again. This is a relevant torque and thermal event that seems to be associated to some crosslinking effects which might be induced by the combination of shear forces and the presence of MMT-Na+. Moreover, these systems also anticipate the increase in torque and temperature. In fact, the maximum was found at ca. 31 and 18 min for the 6% and 9% systems, respectively. This information allows selecting optimal mixing times for each system. The time selected should be long enough to have a fairly homogenous product, but short enough to avoid shear-induced cross-linking. The chosen times are given in Table 1. In addition, the Specific Mechanical Energy (SME) for the mixing process of each blend was also obtained, showing a remarkable rise when nanoclay is added to the formulation (Table 1). Obviously, this rising in SME is truncated by the selection of such short mixing time periods.

3.1.2. X-Rays diffraction

Results of X-Rays diffraction of blends with different nanoclay content (0, 3, 6 and 9%) are plotted in Figure 2. The profiles obtained are similar in all cases, showing a good integration between the protein and the nanoclay present in the structure. It may be noticed that the height of the first characteristics peak increases according to the nanoclay content, what makes sense. This first peak corresponds to the first reflection of the dhkl‑spacing characteristic for MMT-Na+. Thus, as may be observed after the mixing process carried out there are still coherent specular reflections, which indicates the existence of parallel layers. Interesting, this peak appears at lower 2θ angle than the one for the pure MMT-Na+, which suggests a variation in dhkl spacing towards higher sizes [23]. Polymer/MMT-Na+ interactions may yield three different scenarios [24]:

– If the interactions are very weak, tactoids remain unaltered in the polymer matrix, i. e. no true biocomposite is formed.

– When there are moderate interactions, the clay interlayer expands, and under these conditions, polymer chains penetrate into the gaps between plates, leading to an intercalated structure.

– If there are strong interactions, the original structure of the nanoclay is lost and nanoclay particles are exfoliated. This last assumption has been related to the greatest improvements in polymer performance [24].

According to X-Ray measurements, the crystallinity of the MMT-Na+ has not been completely lost, and SPI/MMT- Na+ blends could be set in the second case. The dhkl spacing has increased as a consequence of protein intercalation between nanoclay layers.

Moreover, the two main peaks present in the SPI protein system have been previously related to the structure of the protein. The first peak has been associated to the 7S fraction, whereas the second peak has been related to the 11S fraction (the pattern of proteins belonging to the α-helix and β-sheet present in its tertiary structure) [25]. These peaks appeared after the mixing stage were carried out, what suggests the tertiary structure of the SPI protein system remains after this first stage.

3.1.3. Dynamic Mechanical Thermal Analysis (DMTA)

Figure 3 shows the values of elastic and viscous moduli (E’ and E’’, respectively) (A) and the loss tangent (tan δ) (B) from DMTA for the blends with different nanoclay content (0%, 3%, 6% and 9%) as a function of temperature (from 20ºC to 150ºC). The objective of the measurement is to select a suitable temperature to achieve a moderate viscosity in the mould in order to facilitate the injection from the injection chamber to the mould.

As can be seen, both viscoelastic moduli (E’ and E’’) decrease with temperature, although being less appreciable for 9%. For all blends, the thermal profile for E’ passes through an inflection point, which may be related to a glass transition. This transition yields a maximum value of tan (δ) at c.a. 70 °C for all samples. This glass transition temperature (Tg) is consistent with previous values determined by DSD analysis [18]. Furthermore, the profile for tan (δ) shows only one peak, regardless of the nanoclay content used, which reveals a good compatibility between the components of the blend.

3.2. Preparation and characterization of biocomposites

The influence of the addition of nanoclay in soy-based biocomposites was evaluated by performing a mechanical characterization by rheological measurements as well as microstructural characterization by SEM and water uptake measurements.

3.2.1. Rheological Measurements

The evolution of E’ and E’’ with frequency and temperature for composites without nanoclay and different nanoclay concentrations (3, 6 and 9%) is shown in Figure 4 (Figure 4A and 4B, respectively). In both studies, the value of E’ is always higher than E” value over the entire range, revealing the elastic behavior of all the samples.

Initially, two opposite effects can be seen. Both viscoelastic moduli, E’ and E’’, increase with frequency (Figure 4A), but a decrease in both moduli is observed when the temperature increase (Figure 4B). In any case, it is interesting to observe that the addition of nanoclay (from 0 to 3%) yields a remarkable increase in both moduli in the entire range of frequencies and temperatures studied. A further but moderate increase takes place with increasing nanoclay content from 3% to higher nanoclay concentration. However, there is a limit of concentration from which the viscoelastic properties are very similar (which is 6% for both tests). Thus, this fact confirms that there is a limit concentration of nanoclay to improve the mechanical properties of soy-based biocompoistes.

On the other hand, the critical strain of the biocomposites obtained was also measured at 20ºC and 80ºC (Table 2). All the systems exhibit an increase in the critical strain when the temperature is higher, so the biocomposites become more elastomeric with temperature. Moreover, the critical strain decreases when the amount of nanoclay present is higher (from 0.37% to 0.05% for the system with 0 and 9 % of MMT, respectively), leading the presence of nanoclay to more rigid but less deformable composites.

3.2.2. Tensile strength measurements

Figure 5 shows the results obtained from uniaxial tensile strength measurements up to rupture for all studied SPI-based Biocomposites containing 0, 3, 6 or 9 % MMT-Na+. On one hand, results from stress–strain curves are plotted in Fig. 5A. These curves are characterized by an initial linear elastic behaviour of high constant stress–strain slope (Young’s Modulus), followed by a plastic deformation stage with a continuous decrease in the slope after reaching the elastic limit, which is called maximum stress. Subsequently, a second constant slope is reached at the end of this plastic deformation stage, and the probe breaks down, reaching the maximum strain.

As may be observed, the addition of MMT-Na+ nanoparticles yields a general decrease in the strain at break values, as well as an increase in the initial sloped associated to elastic deformation (E). This opposite effect has been previously found in other polymeric materials [26]. In order to elucidate significant changes, Fig. 5B shows parameters from stress‑strain curves for systems containing 0, 3, 6 or 9 % MMT-Na+. As may be observed, the addition of nanoclay does not yield any significant change in maximum stress, only the system containing 9 % MMT‑Naexhibits significant lower maximum stress. However, this plot confirm the increase of the Young’s modulus after adding MMT-Na+. In fact, this increase reaches its maximum value at 6 % MMT‑Na+, andthe excess of MMT‑Na+ (9 % MMT‑Na+) yields lower elastic modulus. Eventually, this figure corroborates the decrease in strain at break value found in stress‑strain curves. Note that the SPI/MMT‑Nastructure previously assumed (intercalated) agree with these results. If nanoparticles were completely exfoliated, mechanical properties would have suffer significant improvements, as it was found previously by other authors [24,27]. The remaining structure of nanoclays does not help to improve mechanical properties, but it could play a key role in other technofunctional properties of these biocomposites i.e. water uptake capacity.

3.2.3. Water uptake capacity

Figure 6 shows results for water uptake capacity (WUC) after immersion biocomposite‑based probes over 24 hours. As may be observe, except for the system containing 3 % MMT-Na+, water uptake capacity of SPI-based biocomposites increases according to the nanoclay content. In fact, when the nanoclay content reaches up to 9 %, the WUC is twice the one found for reference system (0% MMT-Na+). These WUC values correspond to 12, 22 and 25 times their own dry weight (for 3, 6 and 9% MMT-Na+, respectively). According to these values, these systems could be considered as superabsorbent materials [28,29].

Moreover, these values are even higher than other achieved by protein functionalization [30,31], whereas the simple method carried out to obtain protein-based biocomposites in the framework of this study is cheaper and easily scalable. Despite the fact that the nanoclay is not exfoliated, the hydrophilicity of intercalated nanaclays remains after the processing of blends, which allows obtaining biocomposites materials highly hygroscopic.

3.2.4. Scanning Electron Miscroscopy (SEM)

Figure 7 shows pictures of all probes studied containing 0, 3, 6 and 9% MMT‑Na+ (Fig. A, B, C and D, respectively). Significant changes in the microstructure can be observed when the nanoclay is added. Thus, in absence of nanoclay particles a good three-dimensional network structure can be observed, where pores are distributed uniformly and whose pore diameter is around 100 μm. This porous region has been previously related to water permeation binding [29,30]. However, this porous structure is lost when the nanoclay content increases. SEM microscopy reveals laminar structures, which is clearer when the concentration of nanoclay is the highest. In fact, the structural differences between the samples may explain some of the measured properties. Indeed, it is observed for the nanoclay-free system that the pores are able to deform easily without breaking when elongation test are carried out. This is not the case for the samples containing nanoclay since presence of nanoclay seems to promote the formation of a laminar structure.

4. Concluding remarks

Soy protein based biocomposites have been performed by means of thermomechanical processing, which consists of a mixing process of proteins and nanoclay with plasticizer and a further injection moulding stage. Mixing stage conditions are important to get appropriate and homogenous dough. Indeed, self-heating need to be avoided in order to prevent cross linking reactions and structuring of the material inside the equipment. X‑Ray measurements reveals that protein chains are probably intercalated within nanoclay tactoids, being tactoids well integrated into the polymer matrix. Consequently, the addition of MMT‑NA+ does not result in biocomposites exhibiting greater mechanical properties, since they show higher elastic modulus, but lower strain at break. However, the handicap of these MMT‑Na+ composite materials is the high water uptake capacity. The intercalate structure of the SPI/nanoclay leads to biocomposites exhibiting a very high water absorption capacity (more than 2500% for some of them). Values obtained of water uptake yield biocomposites which could be used as potential sources of absorbent material, opening the possibility to manufacture cost-competitive bio‑based superabsorbent materials.


The authors gratefully acknowledge “Ministerio de Economía y Competitividad” because this work is part of a research project sponsored by MINECO, from the Spanish Government (Ref. CTQ2015-71164-P, MINECO/FEDER, UE) for the financial support and University of Seville for the grant of the VPPI-US. The authors also acknowledge the Microscopy Service (CITIUS – Universidad de Sevilla) for providing full access and assistance to the JEOL 6460LV equipment.


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Figure Captions

Fig. 1. Evolution of mixing torque and temperature over the mixing process for blends without nanoclay and with 3, 6 and 9% nanoclay content.

Fig. 2. X-Ray diffraction measurements for the different blends (without nanoclay and with 3, 6 and 9% nanoclay content), as well as for MMT‑Na+..

Fig. 3. Temperature ramps performed at constant frequency (1 Hz) and heating rate (3ºC·min-1) for all blends without nanoclay and with 3, 6 and 9% nanoclay content): Evolution of (A) elastic modulus (E’) and (B) loss tangent (tan δ).

Fig. 4. (A) Dynamic frequency sweep test and (B) temperature ramps for biocomposites without MMT‑Na+ and containing 3%, 6% and 9% MMT‑Na+.

Fig. 5. (A) Profile and (B) Parameters of tensile strength measurements: Maximum stress, Strain at break and Young’s modulus for biocomposites without MMT‑Na+ and containing 3%, 6% and 9% MMT‑Na+.

Fig. 6. SEM images for biocomposites without MMT‑Na+ (A) and three different nanoclay concentration: 3% (B), 6% (C) and 9% (D).

Fig. 7. Water uptake capacity after 24h for biocomposites without MMT‑Na+ and containing 3%, 6% and 9% MMT‑Na+.

Table Captions

Table 1. Mixing time and Specific Mechanical Energy (SME) for the blends processed without MMT‑Na+ and containing 3%, 6% and 9% MMT‑Na+.

Table 2. Critical Strain at 20ºC and 80ºC for the different systems processed without MMT‑Na+ and containing 3%, 6% and 9% MMT‑Na+.

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