Development of crayfish bio-based plastic materials processed by small-scale injection moulding

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Journal of the Science of Food and Agriculture (2015) vol.95 n.4 DOI: 10.1002/jsfa.6747 Copyright: 1999-2025 John Wiley & Sons El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is the peer reviewed version of the following article: Félix, Manuel; Romero, Alberto; Cordobés, Felipe; Guerrero, Antonio: Development of crayfish bio-based plastic materials processed by small-scale injection moulding, which has been published in final form at https:// doi.org/10.1002/jsfa.6747. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 Development of crayfish bio-based plastic materials processed by small-scale injection moulding Crayfish bio-based plastic materials processed by injection moulding Manuel Felix*, Alberto Romero, Felipe Cordobes, Antonio Guerrero Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Sevilla, Spain Abstract BACKGROUND: Protein has been investigated as a source for biodegradable polymeric materials. This work evaluates the development of plasticized crayfish (CF) bio-based plastic materials by means of injection moulding as a fully biodegradable alternative to conventional polymer-based plastics, using glycerol (GL) as plasticiser. The effect of using different additives, namely sodium sulphite (SS) or bisulphite (BS) as reducing agents, urea (U) as denaturing agent and L-cysteine (LC) as crosslinking agent, is also analysed. RESULTS: The addition of any additive always yields an increase in energy efficiency at the mixing stage, but its effect on the mechanical properties of the bioplastics is not so clear and even dampened. The additive developing a greater effect is LC, showing higher Young’s modulus values and exhibiting a remnant thermosetting potential. Thus, processing at higher temperature yields a remarkable increase in extensibility. CONCLUSION: This work illustrates the feasibility of CF-based green biodegradable plastics, thereby contributing to find potential value-added applications for this byproduct. 2 Keywords: Bioplastic; Crayfish Protein; Mixing blends; Rheology; Tensile strength test. _______________________ *M. FELIX Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, 41012 Sevilla (Spain) E-mail: [email protected] Phone: +34 954557179; fax: +34 954556447. Introduction The freshwater red-swamp crayfish (Procambarus Clarkii) was introduced in Europe in the early 60s. Since then this species has undergone a fast widespread growth due to its resistance to fungal disease as well as to favourable weather conditions, abundant food and a lack of predators1. This fact has driven to the development of a strong local crayfish industry at the marshes of the Guadalquivir River in Spain. This development has also led to the generation of a big amount of crayfish (CF) surpluses, as it is frequently the case in the fish and shellfish industry2. According to FAO's recent estimations the total world output of fish represented 156 million tonnes in 2011. Unfortunately, a high proportion of this amount is not eventually used for human food consumption. Thus, according to the USDA, up to 45% of fish and shellfish that enters the USA retail food market is not eaten and, as a consequence, end as wastes. Therefore, search for any value added application to these surpluses is becoming a real priority. 3 As for CF surpluses concerns, some applications based on the functional properties of CF protein fraction have been previously assessed. Thus, interfacial properties of crayfish protein isolate (CFPI) have been studied3-5 in order to address the good performance of CF protein in emulsion stabilization.6,7 Thermal properties of CF flour8 and CFPI in aqueous solution have been also studied in order to assess thermal-induced enhancement of emulsion stability 9 and the potentials in the manufacture of surimi-like products based on its ability to form gel 10,11. A currently attractive way to valorise these by-products is through their use as renewable resources in the manufacture of “green materials”, replacing hardly degradable plastic materials from oil-based synthetic polymers. Nowadays, some important applications for bioplastics are beginning to emerge in the areas of packaging, food production, pharmaceutics, electronics, automotive industry and biomedicine. Thus, among other applications bioplastics can be used in food packaging, fruit coating, encapsulation, textiles, absorbent materials or tissue engineering12-14. This wide variety of potential applications, for which possessing suitable physicochemical properties is essential, allows us to envisage an increasingly use of biobased-plastic materials. Thus, according to European Bioplastics, the production capacity for bioplastics is predicted to increase from approximately 700,000 tons in 2010, to 1.7 Mtons by 2015 12. However, like other biobased innovations, bioplastics have struggled to achieve market share such that at present bioplastics constitute less than 0.5% of world plastics consumption 15. Therefore, it is still necessary to intensify the efforts in R+D and innovation in this field. Some biopolymers can directly replace synthetically derived materials in traditional applications or simply possess unique properties that could open up a range of new commercial opportunities. Thus, cellulose, starch, polysaccharides and protein has 4 become increasingly competitive in recent years as substitutes for petrochemicals, in view of the increase in oil production costs. Both environmental and economic factors are expected to entail the development of new plastic materials such as those using high protein-containing byproducts16. In this respect, proteins are exceptionally versatile materials, both in the sources from which they can be obtained and in the wide variety of possible modifications, which can be helpful in tailoring their properties to the particular requirements of a specific application. They present significant advantages in that proteins are derived from a sustainable resource and can be processed in much the same way as conventional synthetic polymers13. However, proteins are generally mixed with a plasticizer in order to reduce intermolecular forces among polymer chains, increasing mobility and reducing the glass transition17. Traditionally, protein films18-20 are processed by casting method, however classical polymer processing techniques (compression moulding or extrusion) are being increasingly used in this field21-24. Among them, injection moulding is a fairly attractive operation that has not received due attention yet. Thus, studies on protein-based biodegradable polymeric materials processed by injection moulding are scarce.25,26 Typically in this process, polymeric materials are subjected to suitable thermal conditions, being injected at high pressure into the mould cavity. Optimization of processing conditions is essential to achieve the desire properties of the final product. This is particularly relevant in protein-based materials that require thermoplastic mixing with a proper plasticizer but show a predominant thermoset character upon injection moulding 26. Besides, additives such as reducing agents may be helpful in order to reduce the average molecular weight of protein aggregates, thus facilitating both mixing and moulding processes. Thus, some authors studied the influence of reducing agents on the 5 properties of thermo-moulded wheat gluten bioplastics 27, soy protein isolate 28-30, starch 31 or flax fibers 32. The effect of adding a denaturing agent, such as urea has also been analysed on bioplastics processed by extrusion 33 or compression moulding 34-36. As for the use of L-cysteine as crosslinking agent, Sun et al. 37 have recently evaluated its performance on thermomolded gluten-based plastics. However, no information about crayfish-based bioplastics with or without reducing, denaturing or crosslinker agents has been found. The overall objective is to evaluate the potential development of plasticized crayfish bio-based plastic materials by means of a conventional and highly versatile polymer processing technique such as injection moulding as an alternative to moulded materials based on polymers derived from fossil fuel. A further objective is to analyse the effect of using different additives on the properties of CF-based products, using glycerol (GL) as the plasticiser. The additives assessed in this study are sodium sulphite (SS) or bisulphite (BS) as reducing agents, urea (U) as denaturing agent and L-cysteine (LC) as crosslinking agent. A small-scale plunger-type injection moulding machine is used in this study to obtain CF-based specimens from CF/GL/additive blends, previously mixed by means of a mixing-rheometer that allows recording torque and temperature over mixing. Rheological and Differential Scanning Calorimetry measurements of these blends are also carried out in order to obtain information that may be used in the selection of suitable processing parameters for injection moulding operations (temperature and residence time in the pre-injection cylinder as well as the temperature of the mould). Experimental Materials 6 Crayfish flour (CF) was obtained from ALFOCAN S.A. (Isla Mayor, Seville, Spain). The protein content was determined in quadruplicate as % N x 6.25 using a LECO CHNS-932 nitrogen micro analyser (Leco Corporation, St. Joseph, MI, USA) being 65 wt.%. GL, used as plasticizer, and additives: SS, BS, U and LC were purchased from Panreac Química, S.A. (Spain). Sample preparation Blends with different CF/GL ratios were manufactured by a thermomechanical procedure which includes two stages: Initially, selected blends, containing 700 g kg−1 CF and 300 g kg−1 glycerol (denoted 70/30), were mixed in a two-blade counter-rotating batch mixer Haake Polylab QC (ThermoHaake, Germany) at 25ºC and 50 rpm for 60 or 20 min, monitoring the torque and temperature during mixing. Secondly, the dough-like materials obtained after mixing were subsequently processed by injection moulding using a MiniJet Piston Injection Molding System II (ThermoHaake, Germany) to obtain bioplastic probes. Two types of moulds were used to prepare the probes: a 60×10×1 mm rectangular shaped mould for both DMTA experiments and transparency measurements and a Dumpbell type probe defined by ISO 527-2:1993 for Tensile Properties of Plastics. Free sulfhydryls. Free sulfhydryl groups of protein samples were determined using the method developed by Beveridge et al. 38. Samples were suspended (10 g L-1) in 0.086 mol L-1 Tris-HCl – 0.09 mol L-1 glycine – 4 mmol L-1 EDTA – 8 mol L-1 urea – pH 8 buffer. Dispersions were stirred at 25 ºC during 10 min at 500 rpm in a thermomixer and then centrifuged at 15,000 xg (10 min, 10 ºC). Supernatant was incubated with Ellman’s reagent (4g DTNB L-1 methanol). Absorbance at 412 nm was measured in a Genesis-20 spectrophotometer (Thermo Scientific, USA). The molar 7 extinction coefficient of NTB (13,600 M-1 cm-1) was used. Protein concentration of extracts was determined by the Bradford method. Characterization of blends The most suitable processing variables such as temperatures in the pre-injection cylinder or in the mould were selected after performing temperature ramp and DSC measurements. Rheological measurements. Dough-like materials were characterized by Small Amplitude Oscillatory Shear (SAOS) measurements, using a controlled-strain rheometer (ARES), in order to select the optimum conditions for injection moulding. The geometry used has been a plate and plate geometry (dia: 25 mm) with a rough surface and a gap between plates of 1 mm. Low viscosity Dow Corning 200 fluid has been used as sealant to avoid sample drying. Strain sweep SAOS tests were also performed in order to establish the linear viscoelasticity range. Temperature ramp tests were carried out at 5 ºC min-1 from 20 to 100 ºC and time sweep tests were performed for 1800 s at a selected constant temperature. Linear viscoelastic properties (G’, G’’) were monitored at a constant frequency of 6.28 rad s-1. All the systems studied had the same thermorheological history before performing any rheological test. Characterization of bioplastics Dynamic Mechanical Temperature Analysis (DMTA). DMTA tests were carried out with a RSA3 (TA Instruments, New Castle, DE, USA), on rectangular probes using dual cantilever bending. All the experiments were carried out at constant frequency (6.28 rad s-1) and strain (between 0.01 and 0.3%, within the linear viscoelastic region). The selected heating rate was 3ºC min−1. All the samples were coated with Dow Corning high vacuum grease to avoid water loss. 8 Tensile strength measurements. Tensile tests were performed by using the Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA), according to ISO 527-2:1993 for Tensile Properties of Plastics. Tensile stress and elongation at break were evaluated from at least three duplicates for each product using type IV probes and an extensional rate of 100 mm·min−1 at room temperature. Statistical analysis At least three replicates of each measurement were carried out. Statistical analyses were performed using t-test and one-way analysis of variance (ANOVA, p0.05) by means of the statistical package SPSS 18. Standard deviations from some selected parameters were calculated. Results and Discussion Thermoplastic mixing of blends Figure 1 shows both torque and temperature profiles as a function of mixing time for blends obtained at different CF/GL ratios, as well as their visual appearance after mixing. These results put forward the remarkable dependence of these parameters on the CF/GL ratio. Thus, a rapid increase in torque up to a maximum value takes place for the system having less amount of plasticizer, denoted as 80/20, which represents the CF/GL weight ratio. This evolution is readily followed by an asymptotic decrease towards a plateau value. In contrast, the system 70/30 shows a moderate growth in torque showing no maximum value but a slow tendency to the plateau value. The profile of the 60/40 system shows only a slightly initial increase in torque, being dominated by a constant torque value over mixing time. In general, the temperature evolution is very similar to the torque profile where an increase takes place excepting for the lowest CF/GL ratio. Both increases 15 different additives (BS, SS, U and LC) for specimens moulded at 100ºC for 200s, whereas Figure 5B shows the effect of different moulding conditions for LC containing specimens. As may be observed in Figure 5A, all the specimens show similar profiles for E’ and also for E’’ (data not shown), undergoing a remarkable decrease with increasing temperature that tends to reach a plateau value at high temperature that in some cases evolves to an eventual increase in E’, indicating a certain thermosetting potential. DMA profiles for the additive-free, U and BS specimens do not display any significant difference, in spite of the effect observed on the viscoelastic properties of their corresponding blends (Fig. 4A). SS containing specimens show lower E’ values at low temperature but higher values in the high temperature region as compared to the reference (additive-free) system. This system also evolves in a more gradual way than the other bioplastic specimens, particularly at high temperature showing no thermosetting potential region. This behaviour may be associated to the ability of SS to impair disulphide bridges. Thus, the results obtained after application of the Beveridge et al. 38 procedure indicate that the concentration of free sulfhydryl groups in CF is raised from 19  3 mmol kg-1 to 300  17 mmol kg-1 after addition of 3g SS kg-1 protein. As for LC specimens, they show a rather similar DMA profile at low and medium temperature but also shows an apparent increase in E’ at high temperature, which reveals that this system still exhibits a marked remnant thermosetting potential for further processing. This increase may be a consequence of different crosslinking reactions involving SS bond formation, SH-SS interchange 44 but also non-disulphide bonds. Thus, Rombouts et al. (2011) 45 reported a heat-induced reduction in ε-amino groups, beimg most likely the result of isopeptide bond formation in combination with Maillard and/or other heat- 16 induced reactions. These authors also found that lysineand glutamine-containing peptides from high molecular weight glutenin subunits also induced the formation of isopeptide bonds, thereby demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine residues. It is interesting to note that CF protein is rich in glutamine and lysine aminoacids46. In order to explore this potential LC-containing specimens were processed using different moulding conditions over the packing stage, increasing either the packing time in the mould at 100ºC and 20 MPa (up to 600s) or the moulding temperature at 20 MPa over 200 s (up to 130ºC). DMA results for these new LC-containing specimens are shown in Figure 5B. As may be observed, the increase in moulding time does not lead to any noticeable change in the DMA profile, which suggest that the time selected for the former specimens is long enough to complete the crosslinking stage. On the other hand, an increase in moulding temperature leads to a plateau value for E’ in the high-temperature region. However, this change in moulding conditions does not drive any particular enhancement in the viscoelastic bending properties below 60ºC. This behaviour is somehow unexpected since occurrence of the above-mentioned thermosetting potential typically involves an enhancement of mechanical properties 21,47,48. A possible explanation for this lack of enhancement may be found in the fact that some degradation of specimens has been observed at 130ºC and above. Probably, this degradation is related to other components (e.g. lipids content) rather than protein but it may produce some alterations of mechanical properties. Thus, using a defatted CF protein concentrate instead of CF flour would be a better choice in order to process at higher temperatures. 17 Uniaxial tensile strength measurements Figure 6A displays the results of stress-strain curves obtained from tensile strength measurements for additive-free and additive-containing specimens at an Additive/CF ratio. All the curves exhibit a similar behaviour which consist of an initial linear elastic behaviour of high constant stress-strain slope yielding high values for the Young’s Modulus (E), followed by a deformation stage with a continuous decrease in the stressstrain slope. A second constant slope is reached at the end of the plastic deformation stage. All the curves eventually reach a maximum value for the stress (max) and the strain at break (εmax). Only LC leads to an apparent enhancement of the tensile strength profile, also leading to a slightly shorter strain value. The values of these parameters (E, max and εmax) and their corresponding standard deviations are plotted in Figure 6B for the additivefree and additive-containing specimens at 3 g kg-1 Additive/CF ratio. This figure puts forward once again that additive LC is the only one that improves parameters E and max over the additive-free system, under the same processing conditions. On the other hand LC-added specimens exhibit lower values for εmax. The rest of additives lead to similar or even lower values of the three parameters. Figure 7 also shows the values for LC-added specimens moulded at longer time or higher temperature. As may be observed, an increase in the packing time from 200 s up to 600 s does not yield any noticeable change in tensile parameters. This result indicates that after 200 s packing time the sprue is already closed by the solidified blend. On the other hand, an increase in the mould temperature leads to remarkable changes in tensile parameters. Thus, the maximum stress and above all the strain at break undergo an apparent increase in value (ca. 12 and 70%, respectively), 18 whereas the Young modulus clearly decreases (ca. 30%) by increasing the mould temperature from 100 to 130ºC. Interestingly, it is the maximum elongation the property that undergoes the most remarkable enhancement by favouring heat-induced crosslinking. In this way, the material exhibits higher toughness, in spite of being less strong. In fact, as stated by Lagrain et al. 49, increasing the elongation at break of glassy, amorphous polymers typically goes at the expense of the elastic modulus. This compensation may also affect to the bending elastic properties of the bioplastic, thus explaining the small dependence of DMA profiles on moulding temperature. Moreover, this behaviour is similar to that one found for other elastomeric materials such as rubber-based blends 50 and is consistent with the results from DMA measurements that show an extension of the rubbery plateau obtained at high temperature. Conclusions From the experimental results, it may be concluded that monitoring the torque over mixing of protein-based flour, additives and plasticizer it is useful to select the more suitable conditions (e.g. mixing time and formulation) in terms of energy efficiency. Characterization of the rheological properties of blends (particularly their dependence on temperature) is also important to select suitable operation conditions for injection moulding processing. The addition of reducing agent (BS or SS), denaturing agent (U) or crosslinking promoter (LC), always yields an increase in energy efficiency (i.e. a decrease in REI value) at the mixing stage, leading to a remarkable reduction in the linear viscoelastic properties of blends. In contrast, the effect of the additives on the mechanical properties of the final bioplastic material is not so clear, particularly for the results from DMA measurements. The additive 19 developing a greater effect is LC, which provides specimens showing a higher value for the Young’s modulus, as well as a remnant thermosetting potential for further processing at high temperature. This potential typically involves an enhancement of mechanical properties 21,47,48,51. However, in this study it is the maximum elongation the property that is remarkably enhanced by increasing the thermosetting temperature. This moulding temperature-driven enhancement is clearly at the expense of the Young’s modulus, such that the effect on the bending elastic modulus is dampen. The present work put forward the feasibility of developing CF-based green biodegradable plastics, thereby finding potential value-added applications for this protein concentrate byproduct. Acknowledgements This work is part of a research project sponsored by Andalousian Government, (Spain) (project TEP-6134) and by “Ministerio de Economía y Competitividad” from Spanish Government (Ref. MAT2011-29275-C02-02/01). The authors gratefully acknowledge their financial support. The authors also acknowledge to the Microanalysis Service (CITIUS-Universidad de Sevilla) for providing full access and assistance to the LECOCHNS-932 equipment. References (1) Geiger, W; Alcorlo, P; Baltanas, A; Montes, C. 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