Effective and efficient extrusion technology has been widely used to process polymers. An extruder consists of a heated, fixed metal barrel that contains either one or two screws which convey the raw material from the feed end of the barrel to the die.
The screws act by conveying the material through the heated barrel, inducing shear forces and increasing pressure along the barrel. Process variables include the feed material's composition, screw speed, barrel temperature profile, feed rates, and die size and shape. The degree of screw fill, specific mechanical energy (SME) input, torque, pressure at the die, residence time, and product temperature are influenced by these process variables.
Both single- and twin-screw extruders have been used successfully in producing protein-based materials.20, 22, 38, 48, 61 However, twin-screw extruders convey the material through positive displacement rather than friction forces, offering more efficient mixing and conveying, which is often a requirement during bioplastic compounding.
Extrusion requires the formation of a protein melt, implying processing above the protein's softening point. Proteins contain a vast range of inter-molecular interactions that reduce molecular mobility and increase viscosity, resulting in a high softening temperature, often above the decomposition temperature. To avoid degradation, additives that can alter the softening point are required for successful thermoplastic extrusion of proteins.
The formation of covalent cross-linking during extrusion will inhibit the formation of a thermoplastic material. Instead, extensive cross-linking (>10%), can result in the formation of a thermoset material, which cannot be remolded or reshaped. A thermoset will cause the extruder to fail, rising the torque and pressure above operative maximums.
To produce a thermoplastic material from proteins, cross-linking and non-covalent interactions have to be controlled. Barone et al.22 extruded poultry feathers (which contain keratin), with a combination of glycerol, water, and sodium sulfite as processing aids. Keratins are characterized by a large amount of cysteine. Cystine-cystine cross-links (disulfide bridges) provide strength and stiffness to keratin in the solid-state. However, they are an impediment to processing in the melt-state.22 With the use of a reducing agent, such as sodium sulfite, these cross-links can be broken, greatly improving processability. It was shown that the apparent viscosity during extrusion, decreased with increasing sodium sulfite up to 3 wt.-%, after which it increased with the addition of more sodium sulfite. It was hypothesized that the increase in viscosity after 3 wt.-% was due to increased chain mobility and entanglements after the complete reduction of disulfide bonds. It was found that protein chain mobility was sufficient above 4 wt.-% sodium sulfite, to allow chains to be orientated into crystalline structures, as revealed by NMR spectroscopy.22
Orliac et al., investigated the rheological behavior of sunflower protein isolate (SFPI) processed with water, glycerol, and in some cases sodium sulfite. It was shown that sodium sulfite can be used to reduce the required amount of plasticizer for thermoplastic extrusion. An initial decrease in viscosity was observed, followed by an increase upon further addition of sodium sulfite. It was concluded that greater protein unfolding at higher sodium sulfite concentrations, led to a more extended structure after processing.30
Ralston and Osswald31 used a screw-driven capillary rheometer to measure the viscosities of soy protein isolates extruded with cornstarch, glycerol, sodium sulfite, de-ionized water, and soy oil. In the absence of sodium sulfite, disulfide bonds reduced the effective chain length and led to a more globular protein conformation.31 Soy protein does not have extensive cross-links, and processing is therefore not hugely influenced by the addition of sodium sulfite. However, it was found that the use of sodium sulfite increased consolidation by enabling chain movement. This will lead to exposure of functional groups capable of forming new interactions.
The viscosity of a polymer melt depends on protein composition. Covalent cross-links are not always the inhibiting factor. Water insoluble and hydrophobic proteins, such as zein, require the addition of surfactants to enable thermoplastic processing. Sodium dodecyl sulfate (SDS) is an amphiphilic molecule, capable of electrostatic and hydrophobic interactions, causing dissociation of protein chains. Many studies of protein-based plastics formed by cast and compression molding have used SDS for added denaturation and dissociation, thereby improving processing.32–38
Sessa et al.38 studied the viscosity of zein during torque rheometry. Known amounts of water, triethylene glycol (TEG), and SDS were added in order to control viscosity. The addition of water resulted in a rapid torque rise after 1 min. Further addition of TEG and SDS delayed the rise by up to 12 min. They postulated that at a certain temperature and shear, zein denatures and forms entanglements and aggregates leading to a rapid increase in torque. It was concluded that the reduction in intermolecular forces, brought about by the presence of SDS, slowed the rate of aggregation induced by processing.38
Urea can be used to denature a protein molecule, and is extensively used in proteomics.39 It forms hydrogen bonds with amino acids, thereby preventing protein-protein interactions. The majority of bioplastic research using urea, has used compression molding as a processing technique. Mo and Sun40 investigated the thermal and mechanical properties of plastics molded from urea-modified soy protein isolates. Urea increased the degree of denaturation of soy protein and the glass transition temperature (Tg) decreased with increasing urea concentration, but mechanical properties reached a maximum value at 8 mol · L−1. At low concentration (X < 2 mol · L−1), urea functioned as a plasticizer, while at high concentrations (X > 4 mol · L−1) it acted as plasticizer, cross-linking agent, and filler.
At high concentrations, urea can form a surface residue after long-term storage. Pommet et al.19 used 30 wt.-% urea in wheat gluten and Mo and Sun41 investigated the effect of storage time on urea-modified soy protein, both observing the formation of urea on the surface of the material overtime. This shows that protein-protein interactions are stronger than urea-protein interactions, forcing the urea out of the material overtime.41 Compared to polyol plasticizers, urea is not as flexible and the plasticized materials do not show the same extensibility. Therefore urea is good as a denaturant, to increase chain mobility during processing. The use of a stronger plasticizer may further reduce strong protein-protein interactions which could enhance urea diffusion to the surface.
Denaturation and therefore chain mobility, is essential for successful extrusion. Proteins are semi-crystalline polymers and undergo several thermal transitions, similar to synthetic polymers. The most common techniques used to measure the glass transition temperature of polymers include differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). The glass transition appears as a step in the DSC baseline, resulting from a change in heat capacity (enthalpy). DMTA measures the change in viscoelastic properties of the polymer with changing temperature.43 The glass transition temperature is interpreted from the storage and loss moduli data obtained from a DMTA run.
Thermal transitions are related to chain mobility, most important of which is the glass transition (Tg) which is associated with the onset of main chain motions (Figure 1).9 It is the primary structure of a protein that determines its tendency toward cross-linking, as well as its sensitivity toward plasticization, or chain mobility.33, 72 Secondary transitions (short range motions) have also known to occur at lower temperatures, but do not influence the main rheological responses of proteinous biopolymers.9
Processing temperature is a critical parameter for protein extrusion. Mobility of the polymer increase with increase in temperature, but hydrophobic interactions and aggregation which follows denaturation will restrict chain movement.29 The glass transition temperature of proteins varies based on protein source (primary structure), thermal history and additives. Increasing the molecular mass, chain stiffness or intermolecular forces will decrease the molecular mobility, therefore increasing the Tg (Figure 1). Most synthetic plastics have a Tg below 100 °C, whereas proteins with less than 5% water show glass transition temperatures close to or above their decomposition temperatures (Table 2).44 Above the Tg, further heating to above the softening point (or flow temperature, Tf) results in a low viscosity material, which can easily be processed. It previously been shown that the difference between Tg – Tf for casein, soy and gluten-based biopolymers are in the order of 40 °C.9
Table 2. Glass transition temperatures of dry proteins.
| ||°C|| |
|Wheat gluten||162||5, 9, 45, 46|
|Corn gluten meal||178||17|
Consequently, the use of plasticizers or other additives to increase chain mobility has become essential to prevent protein degradation and increasing processability.47 Figure 2 illustrates how plasticization and heating above a material's glass transition and softening point will allow it to become shaped into a marketable product; plasticization reduces both Tg and softening point and when the softening point is below the decomposition temperature, the material should be easily processable.
Plasticizers improve processability by interposing themselves between the polymer chains and alter the forces holding the chains together.17 This occurs through two mechanisms, lubrication and increasing free volume. Small molecules are easily incorporated into the protein matrix, shown by the high plasticizing effect of water and glycerol.17, 43 Water is considered a natural plasticizer of proteins and is used extensively in protein extrusion. Its small size allows it to easily maneuver through small openings between chains. When plasticizers are compared based on the mass fraction in a bioplastic, low molecular mass compounds such as water, will be present at larger numbers compared to high molecular mass compounds. Every plasticizer molecule can interact with a protein chain, which implies that at equal mass fractions, water is normally more efficient than other plasticizers.
Hydrophilic compounds such as polyols, carbohydrates and amines also interact with polar amino acids. Some examples are glycerol, sorbitol, saccharose, urea, TEG and polyethylene glycol. Other substances such as, amphiphilic plasticizers will interact with the hydrophobic amino acids within the protein, examples include fatty acids and phthalates and some surfactants.48
Plasticizers are added to proteins to reduce their processing temperature, by increasing molecular mobility and decreasing viscosity. Plasticizers act by reducing hydrogen bonding, van der Waals, or ionic interactions that hold polymer chains together, through forming plasticizer-polymer interactions. Sufficient plasticization will reduce the SME input, resulting in less disrupted and better quality products.5
Polar Compounds as Plasticizers
The effect of moisture content on the glass transition temperature has been extensively studied.9, 12, 17, 25, 46, 49–55 Water reduces the glass transition temperature and reduces the temperature at which secondary interactions between protein chains form. During extrusion, water acts as a dispersion medium, plasticizer and solvent, influencing melting and viscosity of the extrudate and the deformation of dispersed particles.44 Water has a low molecular mass and very low Tg (−135 °C), making it a very efficient plasticizer for proteins. In Table 3 the effect of moisture content on the Tg of various protein bioplastics are shown. It can be seen that only a small amount is required to bring about a large reduction in Tg. Water enters the protein network and interacts with protein chains by hydrogen bonding with easily accessible polar amino acid side chains, preventing protein-protein interactions and thereby leading to plasticization.
Table 3. Water effect on glass transition temperature of various protein sources.
|Protein source||Tg at percentage Water||Analysis technique||Ref.|
|Zein||139||70||40||10||<0|| || ||DSC||49|
|Casein||210||140||90||70||50||40||25||DMTA, PTAc), DSC||9|
Another very common plasticizer for proteins is glycerol. Small and water soluble, it shows a similar plasticizing effect as water, easily penetrating the folded protein's surface to interact with polar amino acids.17 The most important difference between glycerol and water is glycerol's higher viscosity, which has been shown to induce viscous heat dissipation during mixing of wheat gluten.19 Hernandez-Izquierdo et al.47 also found that during extrusion of whey protein, water, and glycerol mixtures, higher glycerol contents induced viscous heat dissipation and higher SME requirements.
Glycerol and water are effective plasticizers, due to their low molecular mass and ability to interact with polar residues. However, studies have observed plasticizer migration during storage of protein-based plastics.56 In dry conditions, unbound water evaporates readily over time, reducing its plasticizing effect.30, 57–59 Low permanence of a plasticizer in the product is very important ensuring consistent properties as to when it was produced.
General consensus is that hydrophobic interactions govern the associations of protein chains during extrusion. Polar plasticizers, like glycerol and water, are unable to interact with the hydrophobic areas in the polypeptide chain. Hydrophobic areas will only be able to interact with each other, and not with the polar plasticizer, forming densely packed structural domains.44 Amphiphilic plasticizers have a similar chemical makeup to proteins, containing both polar and non-polar groups.
Proteins are stabilized mainly by hydrogen bonds, but hydrophobic interactions also play an important role. Di Gioia and Guilbert17 studied the efficiencies of polar and amphiphilic plasticizers in reducing the Tg of corn-gluten-meal (CGM). The Tg for dry CGM was found to be above 150 °C. The plasticizing efficiency, based on decreasing the Tg, was compared on the basis of volume and molar fraction of plasticizer, theoretical hydrogen bonds supplied by the plasticizer (TH) and percentage hydrophilic groups of the plasticizer (%HG) (Table 4). CGM has approximately 37.9%HG, distributed in an amphiphilic pattern. It was found that on a molar or TH basis, amphiphilic plasticizers were more efficient than polar plasticizers. It was also shown that Tg scaled linearly with the molecular mass and %HG of the plasticizers tested. With increasing the molecular mass, plasticizer efficiency increased, whereas with increasing %HG, plasticizer efficiency decreased, except for dibutyl tartrate which has a %HG very close to that of CGM (37.4 compared to 37.9), making it more compatible than the other plasticizers.
Table 4. Physiochemical characteristics of plasticizers.17, 19
|Plasticizer||Molecular mass||Hydrogen bonds||Hydrophilic groups|
|g · mol−1||%|
Pommet et al.19 tested various plasticizers for the production of thermoplastic wheat gluten materials. Preliminary screening of 23 plasticizers at 30 wt.-% was conducted.19 Plasticizers with few hydrophilic groups were found to be incompatible with wheat gluten, not forming a consolidated material.19 The influence of water, glycerol, 1,4-butanediol, lactic and octanoic acid on Tg, was measured using DMTA. It was found that on a molar basis, lactic acid had the highest plasticizing efficiency, whereas octanoic acid was the least efficient, mainly because of its hydrophobicity.19 Lactic acid contains a hydroxyl and carboxyl group, favoring more interactions with different amino acid side chains, compared to the other plasticizers. An additional benefit of the acidic conditions was the prevention of excessive aggregation because disulfide bond formation is favored in alkaline conditions.19 Water, glycerol, and 1,4-butanediol had the same plasticizing effect.
Orliac et al.60 investigated a variety of plasticizers for the production of sunflower-based thermo-molded films. Poly(ethylene glycol), poly(propylene glycol), and tetraethylene glycol did not form homogeneous melts with the sunflower protein (molecular mass above 190). The smaller molecular mass plasticizers, glycerol, propylene glycol, ethylene glycol, diethylene glycol, and TEG, produced good homogeneous melts with the sunflower protein.60 Glycerol and TEG were the only plasticizers that did not migrate out of the sunflower plastics over three-month aging.60 In contrast with studies on other proteins, glycerol must have formed sufficient interactions with the sunflower protein, reducing the tendency to migrate out of the material.60
In Table 1, the amino acid content of CGM and wheat gluten is shown. CGM has ≈10% more hydrophobic groups and ≈10% less hydrogen bonding capacity than wheat gluten. This affects the types of plasticizers that will be efficient for either protein source. Water and glycerol will always be most efficient when compared to higher molecular mass plasticizers based on weight fraction. However, a plasticizer that has similar TH and %HG compared to the chosen protein, will have better plasticization efficiency. In light of this, on a molar basis, water may not always be the most efficient plasticizer.
An efficient plasticizer will have a low melting point, low volatility, and good compatibility with the protein. The plasticizing efficiency has been reported to be generally proportional to the molecular mass and inversely proportional to the percentage hydrophilic groups of the plasticizer.11 A plasticizer that works for one protein may not necessarily be successful in another protein system because of the wide range of amino acid sequences possible (Table 1). For that reason, it is important to carry out preliminary research into compatible plasticizers, and their rheological effect on the protein network.