Optimization of Processing Conditions for Rice Bran-based Bioplastics Through Extrusion and Injection Molding

ORIGINAL ARTICLE Journal of Polymers and the Environment (2025) 33:512–527 https://doi.org/10.1007/s10924-024-03377-4 From the contamination of oceans with persistent plastic wastes to the harmful effects of microplastic ingestion by wildlife, the detrimental effects of conventional plastics on biodiversity and ecosystem health are becoming increasingly evident [2]. Bioplastics are a promising and sustainable alternative proposed by the scientific community to deal with this environmental crisis. Derived from renewable resources such as plant starches, agricultural byproducts, or microbial fermentation, bioplastics present a compelling solution to mitigate the ecological footprint associated with conventional plastics [3]. The use of biobased and biodegradable plastics (particularly derived from waste and byproducts of the agro-food industry) emerges as a cutting-edge strategy to address the environmental challenges associated with conventional plastics [4]. In contrast to other types of bioplastics, those based on agricultural raw materials offer significant advantages. Firstly, harnessing waste and byproducts from this source Introduction Conventional plastics have emerged as a significant threat to the delicate balance of our ecosystems in today’s environmental landscape [1]. The ubiquitous use of traditional plastics in various industries has led to widespread pollution, adversely impacting land and aquatic environments alike. María Alonso-González [email protected] 1 Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, Sevilla 41011, Spain 2 Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, Sevilla 41012, Spain 3 Department of Chemical Engineering Materials Environment, University of Rome La Sapienza and UdR INSTM, Via Eudossiana 18, Rome 00184, Italy Abstract Conventional plastics pose environmental threats due to their non-biodegradable nature and their reliability on fossil resources, leading to the exploration of sustainable alternatives. In this sense, biodegradable bioplastics derived from renewable resources offer a promising solution to mitigate ecological impacts. This study focuses on the combination of extrusion and injection molding for the development of rice bran-based bioplastics. Being a by-product from the rice industry rich in starches and proteins, rice bran is an abundant and non-expensive resource that contributes to an enhanced waste management and represents a step forward in integrating the principles of a circular economy. This study delves into the optimization of processing conditions through a Design of Experiment approach. For this purpose, the number of extrusion steps, cylinder and mold temperatures, and injection pressure were investigated. The results showed that two extrusion steps led to a significant increase of approximately 22.8% in Young’s modulus and 37.5% in tensile strength compared to a single extrusion cycle. This enhancement was attributed to the facilitation of starch gelatinization and biopolymer-plasticizer interactions (achieving thermoplastic starch and protein plasticization). Similarly, manipulation of injection temperatures and pressure had notable effects on tensile properties, highlighting the complex interplay between processing parameters. In particular, when using cylinder and mold temperatures of 110 °C and 180 °C, respectively, along with 800 bar, it was possible to achieve a further enhancement in tensile properties, with an increase of 97.1% in Young’s modulus and over 100% in tensile strength. Overall, this research underscores the importance of understanding the relationship between processing conditions and biopolymer interactions for bioplastic production. Keywords Rice bran · Circular Economy · Bioplastics · Extrusion · Injection Molding · Design of Experiment Accepted: 1 August 2024 / Published online: 3 August 2024 © The Author(s) 2024 Optimization of Processing Conditions for Rice Bran-based Bioplastics Through Extrusion and Injection Molding MaríaAlonso-González1,2,3· ManuelFelix1· AlbertoRomero2· ClaudiaSergi3· IreneBavasso3· FabrizioSarasini3 1 3 Journal of Polymers and the Environment (2025) 33:512–527 not only reduces reliance on non-renewable resources but also tackles the issue of agricultural waste management by transforming them into valuable raw materials with specific applications [5]. Furthermore, their effective biodegradation in specific environments, such as composting, provides an additional solution to the long-term plastic waste accumulation problem. In this regard, bioplastics derived from agricultural waste not only represent a more sustainable alternative but also constitute a crucial step towards building a more environmentally respectful future [6]. The selection of rice bran as a primary raw material for bioplastic production is supported by its numerous advantages based on its chemical composition and availability. Rice bran is an abundant byproduct of rice milling processes, more than 60 million metric tons were produced in 2017 [7], providing a sustainable source that simultaneously addresses the issue of agricultural waste disposal [8] and it is aligned with the principles of circular economy, turning an otherwise underutilized byproduct into a valuable resource for sustainable materials [9]. In this sense, rice bran presents a noteworthy repository of proteins and starches, both of which serve as biopolymers with immense potential for bioplastic development [10, 11]. Through appropriate processing techniques, such as thermomechanical treatments, these proteins and starches can be effectively transformed into versatile bioplastic materials [12, 13]. Among the many processing techniques, extrusion and injection molding play key roles. Extrusion is a widely employed method that allows the efficient and continuous production of bioplastics, making it versatile for a range of applications [14, 15]. On the other hand, injection molding is particularly advantageous for producing intricate and customized bioplastic products [16, 17]. Both extrusion and injection molding techniques offer scalability and reproducibility, making them integral to the industrial-scale manufacturing of bioplastics. By harnessing these techniques, the mechanical properties and geometrical precision of bioplastics can be optimized, paving the way for their widespread adoption as eco-friendly alternatives in diverse applications. However, current bioplastics often face significant limitations, including narrow processing windows and poor mechanical properties, such as lower tensile strength and ductility compared to conventional plastics. Additionally, they exhibit lower barrier properties, which can affect their effectiveness in protecting against moisture and gases. Furthermore, although regulations could benefit them, the higher production costs and limited scalability of bioplastics present challenges for widespread adoption and competitiveness in the market [18]. For these reasons, in-depth research is required to overcome potential disadvantages. Previous investigations were focused on the optimization of the rice bran and plasticizers to be employed [19–22] and the influence of some injection molding parameters has been already addressed [23, 24]. This study aims to optimize the processing conditions for the development of rice bran-based bioplastics by combining extrusion and injection molding. Processing parameters such as the number of extrusion cycles, the temperature of the cylinder, the temperature of the mold and the injection pressure were evaluated and investigated through a Design of Experiment (DoE) approach to establish a relationship between the processing conditions and the final properties and to tailor those regarding specific applications. To this end, a comprehensive characterization encompassing mechanical, thermal and chemical and microstructural analysis was conducted on the systems, using mechanical properties as the main parameters for system optimization. The novelty of this study lies in the use of a whole by-product from the agro-food industry, specifically rice bran, instead of isolated proteins or starches. By employing only a defatting treatment, this research addresses the complexity arising from the interactions between the various phases present in rice bran. Therefore, an investigation into optimizing processing conditions via extrusion and injection molding, using a design of experiments approach, is crucial to understand how this complex material behaves. Experimental Materials Rice bran (RB) from indica rice, provided by Herba Ricemills (San José de la Rinconada, Seville, Spain), was used as the raw material for this study. The composition of the rice bran, as determined by A.O.A.C. methods [22], was found to be 16 wt% moisture, 13 wt% ashes, 2 wt% lipids, 17 wt% proteins, 28 wt% fiber (comprising cellulose and hemicellulose), and 24 wt% starch. It is important to note that the rice bran underwent a steaming thermal treatment, a process also carried out by Herba Ricemills. Glycerol (Gly), used as the plasticizer, was sourced from Sigma-Aldrich (USA). Distilled water (W) was used as needed in the experimental procedures. Defatted Rice Bran (DRB) Upon initial reception, the lipid content in the rice bran exceeded 20 wt%. However, to achieve a reduced lipid content of less than 2 wt%, a defatting procedure was implemented using the same methodology detailed in prior studies [22]. Briefly, rice bran (< 500 μm) was mixed with hexane in a proportion of 1:10 w/v. The resulting suspension underwent magnetic stirring at 25 ºC for 24 h, followed 1 3 513 Journal of Polymers and the Environment (2025) 33:512–527 by centrifugation at 5,000 rpm for 10 min. The solid fraction was then separated from the supernatant, containing the hexane phase and the extracted lipids. The separated solid fraction was subsequently dried in a fume hood. This defatting process was repeated twice. After defatting, the rice bran was ground and sieved to a size smaller than 200 μm (70 mesh) for the balanced properties of this fraction [20], resulting in the development of the DRB system. Bioplastics Preparation Dry-blend Preparation The samples were produced through a two-step procedure, beginning with the formulation of a dry-blend by combining DRB, Gly, and W. This preliminary dry-blend approach, achieved through different drying steps along with the addition of the plasticizers, facilitates subsequent extrusion, resulting in a final material with minimal exudation phenomenon, that is, migration of the plasticizer over time. This favourable outcome is primarily attributed to the compatibility of protein and polysaccharide chains and the polyols used as plasticizers (Gly in this case), as reported by Chivrac et al. [25]. To this end, the DRB underwent overnight drying at 70 ºC in an oven AG-System from Fratelli Galli (Italy) to eliminate free water (approximately 9 wt%, depending on ambient conditions). Subsequently, the dried powder was subjected to mechanical stirring at 100 rpm using an Argo Lab AM20-D stirrer (Italy), incorporating Gly gradually until a uniform mixture was achieved. The blend was then dried at 110 ºC in the oven for 1 h, facilitating the volatilization of bound water through diffusion exchange with Gly molecules interacting with protein and starch macromolecules. After cooling, water was added to the dry-blend following the same procedure, slowly introducing it into the mixture under mechanical stirring. This resulted in a formulation comprising 60 wt% DRB, 30 wt% Gly, and 10 wt% W, ensuring an optimal moisture content (dry-blends). Extrusion The dry-blends were introduced into the hopper of a parallel co-rotating twin-screw extruder (Thermo Scientific Process 11, Thermo Fisher Scientific, Waltham, MA, USA), with 11 mm fully segmented screws and a L/D ratio of 40. This extruder was equipped with 8 heating zones and a standard screw configuration with conveying elements (helix pitch of 1 L/D) interspaced among three mixing sections obtaining alternating elements with 0º and 90º (length ¼ L/D). The screw speed (15 rpm) and temperature profile were set based on previously optimized conditions (results not published yet), maintaining a constant feeding rate. The temperature profile was tailored for an extruder with 8 heating zones, with temperatures set at 90–100–110–120–120–110– 105–100 °C, from hopper to die. The extrusion process was conducted once and twice to evaluate the potential benefits of a second run, providing an extended residence time for biopolymer plastification. Injection Molding Following extrusion, the pellets underwent injection molding (Haake MiniJet II Pro, Thermo Fisher Scientific, Waltham, MA, USA) under varying processing conditions, including adjustments to the cylinder temperature (Tc), mold temperature (Tm) and applied pressure (P), where all the specimens remained in the mold for 200 s. By these means, tensile dogbone specimens were obtained. In order to optimize the injection molding phase and to maximize the mechanical properties of the resulting bioplastics, the aforementioned parameters were selected, exploiting the DoE technique and changing them on two levels, low (–) and high (+), to obtain a 23 full factorial design. Figure S1 shows an image of the final bioplastics, while Table 1 details the low and high levels of the three parameters that were used in the full factorial design as well as the designation of the different obtained systems. In this way, 110 ºC and 140 ºC were chosen as low and high levels for the temperature of the cylinder after observing that increasing the temperature of the cylinder from 50 ºC to 110 ºC resulted in improved mechanical properties. This improvement is likely ascribed to the overstepping of the bioplastic glass transition temperature, a critical point at which the material undergoes a transition from a rigid to a more flexible state. The role of this transition in enhancing mechanical properties has been established in previous works, providing supporting evidence for our observed temperature-dependent effects. For the temperature of the mold, 150 ºC and 180 ºC were selected as low and high levels, based on a previous study that demonstrated the beneficial effect of increasing the temperature of the mold on the mechanical properties Table 1 Designation of the different processed biopolymers System Extrusion Injection molding Steps Tc (ºC) Tm (ºC) P (bar) E1 150 150 500 E2 250 150 500 E2I1 2110 (–) 150 (–) 500 (–) E2I2 2110 (–) 150 (–) 800 (+) E2I3 2140 (+)150 (–) 500 (–) E2I4 2140 (+)150 (–) 800 (+) E2I5 2110 (–) 180 (+)500 (–) E2I6 2110 (–) 180 (+) 800 (+) E2I7 2140 (+) 180 (+)500 (–) E2I8 2140 (+) 180 (+) 800 (+) 1 3 514 Journal of Polymers and the Environment (2025) 33:512–527 tests were conducted using a DSC 214 Polyma instrument (Netzsch, Selb, Germany) in a nitrogen atmosphere (40 mL/ min), employing a heating/cooling rate of 10 ºC/min. The calorimetric tests involved heating/cooling ramps ranging from − 40 ºC to 300 ºC. Key temperatures, such as the glass transition temperature (Tg) and melting temperature (T′m) were obtained from the DSC curves. To assess the thermal stability of the samples, thermogravimetric analysis (TGA) was performed. The specimens were placed in alumina crucibles and subjected to heating from 25 ºC to 800 ºC at a heating rate of 10 ºC/min in an inert nitrogen atmosphere (40 mL/min). All experiments were conducted using a TG209 F1 Libra instrument (Netzsch, Selb, Germany). Fourier Transform Infrared Spectroscopy (FTIR) The FTIR analysis was conducted on the raw material, namely, the untreated DRB, and on one of the samples processed at 150 °C and another at 180 °C as Tm (systems E2I1 and E2I5, respectively). The equipment used was an Invenio X spectrometer (Bruker) to obtain absorbance spectra between 4000 and 750 cm−1. Scanning Electron Microscopy (SEM) Micrographs of the bioplastics fracture surface were taken by SEM to assess the microstructure of the different specimens and the failure mechanism. To this end, a field-emis- sion scanning electron microscope (FE-SEM) MIRA 3 by Tescan (Brno, Czech Republic) was used. The samples were coated with a thin layer of gold by sputtering to prevent charging and they were then observed at 10 kV acceleration voltage. Statistical Analyses Statistical analysis was used to disclose the relationship between the injection molding parameters selected and the resulting mechanical properties and it was divided in two different steps. At first, inferential statistic was used to determine whether the injection molding parameters selected as independent variables, i.e., Tc, Tm and P, have an actual effect on the dependent variable, i.e., tensile modulus, tensile strength and elongation at break, by meeting the criteria for statistical significance. The analysis was performed by applying the F-test (ANOVA) and selecting a p-value < 0.05 as a statistically significant limit. This means that a p-value lower than 0.05 confirms that the parameter under consideration had an actual effect on the dependent variable. The analysis was performed with the R-studio platform, of rice bran-based bioplastics [23]. Nevertheless, the temperature was not increased beyond 180 ºC because further increments led to samples that were completely burnt, easily breaking upon demolding, as can be seen in Figure S2. Finally, 500 bar and 800 bar were selected as low and high levels of injection pressure to establish a comparison with previous studies and address the suitability of bioplastics formation driven by high injection pressure [26]. Regarding the extrusion procedure, designation of the systems is based on E1 for 1 step and E2 for 2 steps. Since E2 was selected based on the mechanical properties of the resulting samples (injected at Tc = 50 ºC, Tm = 150 ºC and P = 500 bar), the rest of the systems where the injection molding conditions were changed are designated as E2I1-E2I8. Characterizations Tensile Tests Tensile tests were carried out on the samples using a Zwick/ Roell Z010 universal testing machine. Measurements were performed at room temperature based on the ISO 527-2 standard [27] using a strain rate of 1 mm/min, a load cell of 1 kN, a grip-to-grip separation of 53 mm and an extensometer with a 30 mm gauge length. Stress-strain curves were obtained, which were used to calculate the values of Young’s modulus (E), tensile strength (σmax) and strain at break (εmax) for the different systems developed. At least three specimens were tested for each system. Dynamic Mechanical Thermal Analysis (DMTA) DMTA tests were performed on a DMA850 rheometer (TA instruments, USA) with rectangular probes measuring 10 mm × 8 mm × 1.5 mm using the film clamp in tension mode. Firstly, the linear viscoelastic range was determined by strain sweep tests at a constant frequency of 1 Hz within a deformation range from 0.01–1%. From this test, the critical deformation was determined, and a suitable deformation was selected within the linear viscoelastic range (LVR) to carry out the following tests. Frequency sweep tests between 0.5 Hz and 20 Hz at room temperature and temperature ramps between − 10 ºC and 160 ºC at a constant frequency of 1 Hz were carried out. From these tests, the values of the storage modulus (E′), the loss modulus (E″) and the tan δ = E″/E′ were obtained as a function of frequency and temperature. Thermal Characterization Differential Scanning Calorimetry (DSC) was employed to examine the thermal characteristics of the systems. The 1 3 515 Journal of Polymers and the Environment (2025) 33:512–527 an increase of 22.8% in stiffness and 37.5% in strength was observed. For this reason, two extrusion steps were selected for the rest of the tests used to optimize the injection molding conditions. The longer processing time under temperature, shear forces and the presence of plasticizers could improve starch gelatinization and the creation of more biopolymerplasticizer interactions [28, 29] with both the starch and protein fractions, resulting in a material more suitable to be manufactured by thermo-mechanical methods. On the other hand, the effects of increasing the cylinder’s temperature were tested while keeping the other two parameters, Tm and P, constant. In this way, the Tc was increased from 50 ºC (E2) to 110 ºC (E2I1) and to 140 ºC (E2I3) for Tm = 150 ºC and P = 500 bar. In this case, the increasing Tc from 50 ºC resulted in enhancements in both σmax and εmax, improving from 1.1 to 1.6 MPa and from 4.0 to 6.9%, respectively when using Tc = 110 ºC, the Young’s modulus remaining at 70 MPa. The beneficial effect was not observed when further increasing the Tc to 140 ºC, revealing similar values for σmax (1.8 MPa) and εmax (7.0%). As E decreased to 42 MPa, no further increases for the Tc were proposed. Similar tendencies were observed when increasing the Tc for both Tm = 150 and 180 ºC and P = 800 bar (comparing E2I2 to E2I4 and E2I6 to E2I8), with higher εmax but lower E and σmax. At higher temperatures there is more mobility of the biopolymer chains because the glass transition has fully developed so it is easier for them to flow and completely fill the mold cavity, leading to more compact bioplastics with enhanced mechanical properties [30]. Indeed, the temperature increase is beneficial only to some extent because too high temperatures will accelerate crosslinking before filling the mold cavity and even plasticizer evaporation decreases the processability of the material. For these reasons, increasing the temperature of the cylinder from 110 ºC to 140 ºC does not lead to clear improvements when balancing the 3 parameters under study. The only improvement with Tc = 140 ºC is observed when using Tm = 180 ºC and P = 500 bar (E2I5 to E2I7) where the high temperature combined with the lower P led to plasticizer evaporation, resulting in stiffer bioplastics with higher load capacity but reduced flexibility. When studying the effect of P, it is possible to observe that when the cylinder is set at 140 ºC, crosslinking begins to take place within the material before entering the mold and the processability decreases, making it more difficult to uniformly fill the mold cavity regardless of the applied P. Therefore, no significant advantages are observed between the systems processed at 500 bar (E2I3 and E2I7) and those processed at 800 bar (E2I4 and E2I8). On the other hand, when Tc = 110 ºC, a beneficial effect of P can be observed but only for high mold temperatures, i.e., 180 ºC. With good processability, the raw material will flow easily from the cylinder to the mold, where P will be a determining factor when applying a linear model (lm), which also accounted for the interactions of injection molding parameters. The second step of the analysis entailed the implementation of the response surface technique through the software Matlab® version 2020a to identify the manufacturing parameters that maximize the mechanical properties of the bioplastic. Considering that the full factorial design was set only on two levels, i.e., an upper and a lower one, a linear approximation function was used to describe the response surface. Based upon the results obtained from inferential statistics also, some of the interactions of the injection molding parameters were considered in the approximation functions leading to the Eq. (1): Y(T c ,T m ,P)=a+b×T c +c×T m +d×P+e×T c ×P+f×Tm×P+g×Tc×Tm×P (1) where Y is the mechanical property under consideration, Tc the cylinder temperature, Tm the molding temperature and P the injection pressure. Results and Discussion Tensile Tests and Statistical Analysis Tensile properties of the different studied systems are gathered in Table 2. At first, the effect of extrusion step number was investigated (E1 and E2 systems for one pass or two passes through the extruder, respectively) and it was found that the longer residence time given by the second extrusion resulted in improved Young’s modulus and tensile strength without any detrimental effect upon elongation at break. In this way, under the same injection molding conditions, the samples submitted to two extrusion steps revealed average values of E and σmax of 70 MPa and 1.1 MPa compared to the 57 MPa and 0.8 MPa exhibited by the system submitted to only a single extrusion step, with an almost constant elongation at break (4.0% for E2 and 3.9% for E1). Therefore, Table 2 Mechanical properties (Young’s modulus (E), tensile strength (σmax) and strain at break (εmax)) of the different biopolymers System E (MPa) σmax (MPa) εmax (%) E1 57 ± 4 0.8 ± 0.1 3.9 ± 0.6 E2 70 ± 4 1.1 ± 0.1 4.0 ± 0.2 E2I1 70 ± 4 1.6 ± 0.1 6.9 ± 0.6 E2I2 65 ± 2 1.5 ± 0.1 6.0 ± 0.3 E2I3 42 ± 6 1.8 ± 0.3 7.0 ± 2.0 E2I4 30 ± 7 1.6 ± 0.1 7.2 ± 0.9 E2I5 56 ± 5 1.2 ± 0.1 6.8 ± 0.8 E2I6 138 ± 18 2.5 ± 0.2 5.7 ± 0.6 E2I7 95 ± 8 1.9 ± 0.1 6.1 ± 0.3 E2I8 67 ± 1 1.9 ± 0.1 6.6 ± 0.5 1 3 516 Journal of Polymers and the Environment (2025) 33:512–527 optimization of the injection molding process depends on a complex balancing of phenomena and mechanisms. For example, bioplastic flow is helped by temperatures higher than glass transition, but it is also hindered by excessively high temperatures, promoting crosslinking and fostering plasticizer removal before injection. Otherwise, void content must be minimized and reduced by better mold filling but hindered by plasticizer evaporation, i.e., phenomena that must be balanced as a function of the applied temperatures. Interestingly, neither the processing parameters selected nor their interactions have any effect on the elongation at break. This can be likely ascribed to the ashes inside the feedstock whose chemical composition and morphology are not affected by any of the injection molding parameters and acting as stress intensifiers always determine a brittle response of the bioplastic. Based on the results obtained, the surface response analysis was used to optimize the manufacturing parameters to maximize both tensile modulus and strength by applying a linear function that accounted for the ternary interaction of the three parameters and the binary interaction of P with the mold is set at 180 ºC (E2I5 and E2I6). At these temperatures, plasticizers evaporate rapidly, but aided by high P, the material continues to flow and distribute throughout the cavity, eliminating possible defects in the final piece and leading to higher E and σmax. Although this phenomenon can occur at 150 ºC (E2I1 and E2I2), this lower temperature leads to a lower number of biopolymeric interactions and less plasticizer evaporation. Thus, regardless of the P, there will be a lower degree of compaction during injection. Additionally, in both cases, the εmax decreases as a consequence of the plasticizer losses. The effect of Tm, as happened with Tc and P, will be explained in terms of the other two parameters that will have a direct influence. Thus, increasing the mold temperature from 150 °C to 180 °C at a lower P (500 bar) does not show a clear benefit in the mechanical properties of bioplastics due to the higher plasticizer losses. For Tc = 110 °C, there are no improvements in E or εmax, while σmax decreases (E2I1 and E2I5). For Tc = 140 °C, although there is a certain improvement in E, σmax remains unchanged, and εmax decreases (E2I3 and E2I7). On the other hand, the use of higher P (800 bar) provides a much clearer trend. An improvement in the stiffness and load-bearing capacity of the specimens (E and σmax) is observed, accompanied by a loss of ductility (εmax). This represents a better compromise of properties, especially well-balanced when using Tc = 110 °C (E2I2 and E2I6), resulting in a system with the best values of E and σmax, with a slightly lower εmax still close to those of the other systems. This is achieved through a combination of an appropriate temperature in the cylinder, sufficiently high for proper flow but not too high to initiate crosslinking, as well as a high Tm and P to allow proper mold filling with good compaction and temperature-induced thermoforming [30]. All these conclusions were further corroborated by inferential statistics, whose results are summarized in Tables 3, 4 and 5. Both Tm and P display strong statistical significance on both tensile stiffness and strength, as well as their interaction. This is coherent with the different degrees of heatinduced protein and starch crosslinking achieved by the bioplastics depending on the mold temperature and on its ability to fill the mold as a function of the applied P. On the contrary, Tc proved to be determinant only for tensile modulus, while its interaction with P is fundamental for both stiffness and strength. Indeed, the capability of the bioplastic to flow into the mold does not depend only on the applied P, but also on its viscosity, which is strictly correlated to the cylinder temperature, thus explaining the strong importance of the interaction of these two parameters. Finally, the ternary interaction between the three processing parameters displays a high statistical significance for both tensile stiffness and strength, thus proving that the Table 3 Statistical significance of the effects of the injection molding parameters and their interactions on bioplastic Young’s modulus Parameter F value Pr(> F) Tc12.12 0.003 Tm65.36 4.83·10−07 P 5.51 0.032 Tc × Tm1.31 0.268 Tc × P 39.71 1.05·10−05 Tm× P 16.58 8.87·10−04 Tc × Tm× P 15.24 0.001 Table 4 Statistical significance of the effects of the injection molding parameters and their interactions on bioplastic tensile strength Parameter F value Pr(> F) Tc1.90 0.186 Tm13.10 0.002 P 14.86 0.001 Tc × Tm0.36 0.557 Tc × P 28.78 6.32·10−05 Tm× P 38.41 1.28·10−05 Tc × Tm× P 17.79 6.52·10−04 Table 5 Statistical significance of the effects of the injection molding parameters and their interactions on bioplastic elongation at break Parameter F value Pr(> F) Tc2.33 0.15 Tm2.79 0.11 P 1.69 0.21 Tc × Tm1.42 0.25 Tc × P 1.46 0.24 Tm× P 0.33 0.57 Tc × Tm× P 0.45 0.50 1 3 517 Journal of Polymers and the Environment (2025) 33:512–527 improving material flowability; at the same time, the higher the cylinder temperature, the higher the progressive crosslinking of the bioplastics, which acts in the opposite direction in terms of viscosity. In light of this, a higher injection P ensures a better flowability of the material already at lower Tc, thus allowing the prevention of bioplastic crosslinking in the cylinder, while at lower P, the higher viscosity deriving from the higher proximity to the glass transition temperature makes the higher Tc a preferred choice. In this way, the highest P and the lowest cylinder temperature are the best choices among the two aforementioned configurations. Dynamic Mechanical Thermal Analysis (DMA) Figure 3 depicts the frequency sweep tests conducted on the E1 and E2 systems (resulting from changing the number of passes through the extruder). It is worth mentioning that the rest of the systems displayed similar values for the both Tm and Tc. The results obtained are shown in Figs. 1 and 2 for tensile modulus and strength, respectively. The main outcome is that a P of 800 bar, a cylinder temperature of 110 °C and a mold temperature of 180 °C allow for the maximum tensile stiffness and strength, but some further conclusions can be drawn. To begin with, the best mechanical performance is achieved nearly always for the highest value of mold temperature, i.e., 180 °C, regardless of P and Tc and this can be likely ascribed to the higher crosslinking degree induced in the bioplastic [31]. Another important outcome is related to the interaction of two parameters, namely, the injection P and the cylinder temperature. When working with higher P, the best results are achieved with the lowest cylinder temperature, while a higher Tc is necessary when working with lower P. This can likely be ascribed to two competing phenomena. The higher the cylinder temperature, the farther the bioplastic from its glass transition temperature, thus Fig. 1 Surfaces response for tensile modulus 1 3 518 Journal of Polymers and the Environment (2025) 33:512–527 The viscoelastic moduli as a function of temperature for the systems obtained for different injection molding conditions are presented in Fig. 4A. In this figure, only the systems where the injection conditions (Tc, Tm and P) were modified are represented, as the viscoelastic moduli of the E1 and E2 systems followed the same trend as the rest of the systems. Thus, their behavior with temperature is represented by these systems, while the change induced by using two extrusion steps instead of one is that discussed in Fig. 3, with the E2 system exhibiting slightly higher E′ and E″ than the E1 system. Once again, it is possible to observe that E′ is above E″ for all the studied samples, regardless of the temperature, implying that the bioplastics have a predominantly elastic character (Fig. 4A). In this study, all systems exhibit similar behavior, with their moduli remaining closely aligned and even overlaid throughout the entire temperature range, frequency range studied, indicating no discernible differences in viscoelastic moduli (E′ and E″) for the different injection conditions evaluated. Therefore, E1 and E2 systems are chosen as representatives to elucidate the effect of a second extrusion step on the bioplastics developed. Firstly, all systems exhibit a predominant elastic character, with higher storage (E′) than loss modulus (E″), a typical behavior for polymeric materials [32]. Furthermore, both viscoelastic moduli increase with increasing frequency, showing a certain dependency on this parameter. Although the tensile properties of the bioplastics were notably altered by the second extrusion step, there is only a small enhancement of the rheological properties. This is evident from the slightly higher viscoelastic moduli of the E2 system compared to the E1 one. Such enhancement could be attributed to the longer residence time in the extruder during the second step, facilitating starch gelatinization and promoting increased protein chain interactions [28, 29]. Fig. 2 Surfaces response for tensile strength 1 3 519 Journal of Polymers and the Environment (2025) 33:512–527 thermosetting potential (i.e., there is no recovery of the viscoelastic values at elevated temperatures), indicating, on the one hand, that it is not possible to achieve further enhancement in rheological properties with higher injection molding temperatures and, on the other hand, that the bioplastics developed behave as thermosets [34]. The ability of these materials to maintain their viscoelastic moduli at elevated temperatures, coupled with their high E′ and E″ values, suggests potential suitability for substituting conventional polymers in specific applications [35]. supporting the statement that significant differences among samples cannot be identified. In terms of the evolution of the viscoelastic moduli with temperature, it is noticeable that the viscoelastic moduli decrease in all cases as the temperature rises from − 10 ºC to approximately 100–120 ºC due to increased polymer chain mobility promoted by certain structural relaxation [33]. Subsequently, for further temperature increase, the viscoelastic moduli remain stable up to 160 ºC, reaching a plateau. 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