Reinforcement of soy protein-based bioplastics as potential sustainable packaging solutions

Reinforcement of soy protein-based bioplastics as potential sustainable packaging solutions Carmen María Granados-Carrera a,* , Daniel Castro-Criado a , Mercedes Jim´ enez-Rosado b , Alberto Romero a,* , Víctor Manuel Perez-Puyana c a Department of Chemical Engineering, Faculty of Chemistry, University of Seville 41012, Seville, Spain b Department of Applied Chemistry and Physics, Faculty of Biological and Ambiental Sciences, University of Le´ on, 24071, Le´ on, Spain c Department of Engineering and Materials Science and Transportation, University of Seville 41092, Seville, , Spain ARTICLE INFO Keywords: Bioplastics Agri-food waste Soy protein Crosslinking Injection moulding ABSTRACT Due to the substantial amount of plastic waste in the environment, scientists are seeking new alternatives to traditional plastics. Bioplastics are considered to be important in addressing this issue despite their significant drawbacks, such as poor mechanical properties and higher costs. In order to reduce their price, agri-food waste and by-products can be used as raw materials (e.g., soy protein), promoting a circular economy; and by incorporating different reinforcement methods, it is possible to develop materials with improved mechanical and barrier properties. The aim of this work is to improve the properties of soy protein/glycerol injected bioplastics by incorporating different biopolymers (gelatin and saccharose) or applying different crosslinking methods (physical, chemical or enzymatic crosslinking through thermal treatment, glyoxal or transglutaminase, respectively). These materials were evaluated by physicochemical, mechanical, and functional tests. The results confirmed an improvement in the mechanical properties of the reinforced protein-based bioplastics, showing an increase in their stiffness and a decrease in their deformability, reducing their capacity to absorb water. In any case, these results support the modification of the properties compared to the reference systems. 1. Introduction Agricultural and food waste, commonly known as agri-food waste, has a negative impact on our daily lives as well as on the environment, society and economy. Globally, around 1.3 billion tonnes of food are lost before human consumption, which represents one-third of the total production, leading to social impacts such as nutrient loss and world hunger (Capanoglu et al., 2022; Matei et al., 2021). However, the valorisation of agricultural and food wastes and by-products can be a potential solution to achieve a green circular economy (J˜ ogi and Bhat, 2020; Paini et al., 2022; Valencia et al., 2021). Hence, these wastes can be transformed into value-added products, such as bioplastics, to address the large number of plastics accumulated in ecosystems (Ben-Othman et al., 2020; Tsang et al., 2019; Valencia et al., 2021). Plastics are used in a wide range of daily applications. The packaging industry is particularly noteworthy, accounting for 39.7 % of total demand, making this sector one of the largest consumers due to the increase in purchases of manufactured and packaged products (Fogt Jacobsen et al., 2022; Foschi and Bonoli, 2019). Specifically, in the case of food packaging, which is exposed to external deterioration methods such as mechanical forces or water vapour, among others, one of the main considerations is the lower permeability to avoid contact with oxygen and work as a barrier (Harnkarnsujarit et al., 2021; Merino et al., 2022; Santana et al., 2021). Nowadays, in particular, approximately 95 % of the plastics used are derived from non-renewable resources (Shaikh et al., 2021), are single-used and encourage the accumulation of waste in landfills, contributing to almost 50 % of the total global plastic waste (Rahardiyan et al., 2023; Shang et al., 2023). The main drawback of these materials is that they accumulate in the ecosystems and persist for centuries without degradation due to their high resistance to microbial degradation (Lavagnolo et al., 2024; Lim et al., 2023; Yin and Woo, 2024). Therefore, scientists are focusing on the development of new biodegradable and eco-friendly plastics that can replace conventional plastics (Kumari et al., 2023; Pascoe Ortiz, 2023), focusing on the application of these biodegradable plastics in food packaging and agricultural sectors (Shaikh et al., 2021). Bioplastics have been developed * Corresponding authors. E-mail addresses: [email protected] (C.M. Granados-Carrera), [email protected] (D. Castro-Criado), [email protected] (M. Jim´ enez-Rosado), alromero@ us.es (A. Romero), [email protected] (V.M. Perez-Puyana). Contents lists available at ScienceDirect Future Foods journal homepage: www.elsevier.com/locate/fufo https://doi.org/10.1016/j.fufo.2024.100524 Received 13 October 2024; Received in revised form 13 November 2024; Accepted 11 December 2024 Future Foods 11 (2025) 100524 Available online 14 December 2024 2666-8335/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). using different raw materials such as starch, sugar cane or corn (Ali et al., 2023; Jayakumar et al., 2023; Kong et al., 2023), and methods such as casting, thermomoulding, 3D-printing, among others (Adorna et al., 2022; Carvajal-Pi˜ nero et al., 2019; Dey et al., 2023). Currently, they are considered as an important way to achieve sustainable development goals, such as reducing reliance on fossil fuels, minimising the use of toxic chemicals, and moving towards recycling (Ahsan et al., 2023). However, one major drawback of bioplastics is their cost, which ranges from 1.14 to 21.50 € per kilogram, compared to 0.57 to 1.59 € per kilogram for conventional plastics. This cost difference makes bioplastics less competitive and limits their applicability (Gong et al., 2024; Jim´ enez-Rosado et al., 2020; Karan et al., 2019; Patria et al., 2024). In order to reduce their cost, agri-food waste can be used as raw material without compromising food safety (Gong et al., 2024; Morone et al., 2019). Agri-food waste and by-products are rich in protein (Bagnani et al., 2024; J˜ ogi and Bhat, 2020), which promotes its valorisation to develop bioplastics as a high-added value product (Castro-Criado et al., 2024). In this context, soy protein isolate (SPI) is an encouraging raw material due to its favourable properties such as biodegradability or good absorption capacity (Cuadri et al., 2017; Rani et al., 2021; Song et al., 2011; Yan et al., 2021), as well as its hydrophilic character (as a consequence of the high presence of acidic amino acids of aspartic acid and glutamic acid in its composition) (Jim´ enez-Rosado et al., 2019; Lamaming et al., 2021; Yamada et al., 2020). On the other hand, another drawback associated with bioplastics is their poor mechanical properties and poor water resistance (Lusiana et al., 2019; Othman et al., 2021; Zhang et al., 2022), being necessary for its application in food packaging an improvement of the properties since the moment the food within it till the end of the consumption (Merino et al., 2022). However, these drawbacks can be overcome by using different reinforcement strategies (Awadhiya et al., 2016; Ishak et al., 2020). Chemical crosslinking can be used to impart mechanical stiffness to protein structures, and this can be developed by numerous agents such as aldehydes (e.g. glyoxal, glutaraldehyde or formaldehyde) or acids (e.g. citric acid) due to the existence of multiple multifunctional groups (´ Alvarez-Castillo et al., 2021; Marqui´ e, 2001), highlighting glyoxal due to its ability to produce bioplastics with excellent mechanical and thermal properties (Ishak et al., 2020; Z´ arate-Ramírez et al., 2014). On the other hand, physical crosslinking, such as heat treatment, can be used to promote the formation of covalent bonds and increase tensile modulus and strength by reducing elongation (Du et al., 2016; Jim´ enez-Rosado et al., 2020; Perez-Puyana et al., 2022; Zhang et al., 2023); moreover, this method highlights for its facility and the modification that promotes in water absorption capability and the viscoelastic properties of the matrix (´ Alvarez-Castillo et al., 2018; Xie et al., 2022). Another alternative can be the incorporation of additives, which can also improve certain functional properties (Kong et al., 2023). For example, gelatin is a protein that can increase mechanical resistance, tensile strength, hydrophilicity, swelling capacity and transparency, as shown in previous studies (Galus, 2017). Similarly, other studies have focused on the incorporation of other biodegradable polymers, such as propylene glycol alginate (PGA), which promotes the optimisation of water resistance through the formation of covalent complexes (Zhang and Serventi, 2020). Polysaccharides such as chitosan (CH) can also be added due to their hydrophobicity, high interfacial tension and their improvement of the water barrier behaviour (Haghighi et al., 2019). Moreover, disaccharides, such as saccharose, can promote an increase in intermolecular interaction and tensile strength, as seen in previous studies (Wang et al., 2022). Finally, the incorporation of enzymatic crosslinkers, such as transglutaminase, promotes a higher tensile strength and surface hydrophobicity (Mohammad Zadeh et al., 2018; Wang, 2022). Therefore, this study focused on the evaluation of different methods of reinforcing soy protein-based bioplastics by incorporating different biopolymers (gelatin and saccharose) or applying different crosslinking methods (physical, chemical or enzymatic crosslinking through thermal treatment, glyoxal or transglutaminase, respectively). Later, the physicochemical, thermomechanical and functional properties were studied. Finally, the main novelty of this study is the comparison between the results obtained by different crosslinking methods on soy-based bioplastics. 2. Materials and methods 2.1. Materials Soy protein isolate (SPI, 91 wt% protein and 6 wt% moisture) used as raw material was supplied by Protein Technologies International (SUPRO 500E, Belgium). Food gelatin (G, 90 wt% protein) and saccharose (S, purity ≥95 %), used as reinforcing biopolymers, were supplied by Manuel Riesgo Ltd. (Spain) and Sigma Aldrich S.A. (Germany), respectively. Glycerol (Gly), which was provided by Panreac Química Ltd. (Spain), was used as plasticiser. Finally, glyoxal (Glyx) and transglutaminase (Tgase, enzymatic activity of 100 units/g) were provided by Panreac Química Ltd. (Spain) and BDF Ingredients (ProbindTX-Tgasa, Spain), respectively. 2.2. Preparation of bioplastics The bioplastics were processed by injection moulding, which consists of two stages: a mixing stage and an injection moulding stage. First, a two-blade counter-rotating rheometer mixer Polylab QC (ThermoHaake, Germany) was used to homogenise the different raw materials present in the bioplastics. In this case, the conditions imposed were an angular speed of 50 rpm for 10 min under adiabatic conditions starting from room temperature (25 ±2 ◦C). During mixing, the torque and temperature generated were followed and different protein/plasticiser/additives ratios were analysed to select a suitable proportion, as shown in Table 1. Subsequently, an injection moulding stage was carried out using a MiniJet Piston Injection Moulding System (ThermoHaake, Germany), in which the previously obtained blends were placed in a cylindrical prechamber at 40 ◦C and then forced through a nozzle using a plunger (600 bar for 20 s) to flow into the cavities of a mould (rectangular, 60 × 10 ×1 mm 3 ) at 90 ◦C, where the bioplastics were subjected to a densification stage at 200 bar for 300 s (Jim´ enez Rosado, 2022). It is worth noting that the systems containing saccharose were processed at 70 ◦C with injection and post-injection pressures of 500 and 300 bar, respectively, to avoid saccharose degradation and enhance water absorption of the systems (Abd-Elrahman and Ahmed, 2009; Jim´ enez-Rosado et al., 2021). Furthermore, the bioplastics subjected to physical crosslinking underwent an additional step in a conventional oven (Memmert, Germany) Table 1 Composition of the different bioplastic matrices. System Percentage (wt%) Reinforcement SPI Gly Tgase Glyx S G Ref 50 50 – – – – – G-5 45 50 – – – 5 Aditive G-10 40 50 – – – 10 Aditive G-20 30 50 – – – 20 Aditive S-5 47.5 47.5 – – 5–Aditive S-10 45 45 – – 10 –Aditive S-20 40 40 – – 20 –Aditive HT-50 50 50 – – – – Heat treatment HT-120 50 50 – – – – Heat treatment Glyx-1 49.5 49.5 –1– – Chemical Glyx-3 48.5 48.5 –3– – Chemical Tgase-0.1 49.95 49.95 0.1 – – – Enzimatic Tgase-0.2 49.90 49.90 0.2 – – – Enzimatic C.M. Granados-Carrera et al. Future Foods 11 (2025) 100524 2 for 24 h at different temperatures (50 and 120 ◦C) (´ Alvarez-Castillo et al., 2018; Fern´ andez-Espada et al., 2016). 2.3. Characterization of samples 2.3.1. Physicochemical properties 2.3.1.1. Fourier transform infrared spectroscopy (FTIR). A Fourier Transform Infrared Spectroscopy (FTIR) analysis was conducted using a Hyperion 100 spectrophotometer by Bruker, USA. This device was utilized to identify the various chemical bonds present in the bioplastic matrices. The spectra were depicted in a wavenumber spectrum ranging from 4000 to 400 cm −1 and then processed using Jasco Spectra ManagerTM software, version 2. 2.3.1.2. Water contact angle (WCA). The water contact angle was measured using an optical tensiometer (Attention TL 10, KSV, Finland) and the sessile drop method. A 2 µL drop of milli-Q grade water was placed on the bioplastic samples’ surface, and the droplet’s shape was analysed for 10 s. The water contact angle was measured on both sides of the droplet, and the average value was calculated. 2.3.2. Mechanical properties 2.3.2.1. Dynamic flexure test. Dynamic-mechanical analyses were carried out to evaluate the linear viscoelastic response of the different bioplastics. Two tests were performed in flexural mode using a mechanical-dynamic analyser RSA3 (TA Instruments, USA) with a dual cantilever geometry at room temperature (25 ±2 ◦C): •Strain sweep tests were performed between 0.002 and 1 % of strain at a constant frequency of 1 Hz to determine the linear viscoelastic range (LVR), where the elastic and viscous moduli remain independent of the applied deformation. From these tests, the critical deformation (%) was determined, which marks the limit of the LVR. •Frequency sweep tests were developed between 0.02 and 20 Hz, applying a constant strain within the viscoelastic range. From this test, the elastic (E’) and viscous (E’’) moduli and the loss tangent (tan (δ)=E’’/E’) were obtained in the established frequency range. From these tests, E’ 1 and tan (δ) 1 were used as the corresponding values of E’ and tan (δ) at 1 Hz to facilitate comparison between different systems. 2.3.2.2. Tensile tests. Tensile tests until breakage were carried out in an Insight 10 kN Universal Testing Machine (MTS, USA) to study the strength of the bioplastics. The working conditions of the tensile test were set according to the ISO 527 2.2 (2012) standard. In this test, the bioplastics were subjected to an increasing axial force at a rate of 5 mm/ min. The applied stress is measured by the strain of the bioplastic until it breaks. This test provides data on Young’s modulus, maximum stress and strain at break. 2.3.3. Functional properties 2.3.3.1. Water uptake capacity and soluble matter loss. Water uptake capacity (WUC) and soluble matter loss (SML) were carried out according to the procedure previously used by Jim´ enez-Rosado et al. (Jim´ enez-Rosado et al., 2019). Thus, the different bioplastics were immersed in a closed vessel with 30 mL of distilled water for 24 h Then, WUC was calculated using Eq. (1), where w 2 refers to the weight of the bioplastic after water absorption and w 3 refers to the weight of the dry bioplastic after the absorption (dried in an oven at 120 ◦C for 2 h). WUC (%) = w2−w3 w3 ⋅100 (1) Finally, SML can be calculated using Eq. (2), in which w 1 refers to the weight of the dry bioplastic before the test. SML (%) = w1−w3 w1 ⋅100 (2) 2.3.3.2. Biodegradability. The biodegradability of the systems which incorporate gelatin as a blending component, was measured by burying the bioplastics at room temperature in a composting medium (2:1 cropland: compost, the same ratio of inert/organic materials as specified by ISO 20,200 (Paulson et al., 2001)). Specifically, these systems were chosen due to the heterogeneities present in their bioplastic samples to evaluate if there was any relation with biodegradation. At least three rectangular bioplastics from each system were evaluated by digging them up for visual evaluation on different days, and photographs were taken of the systems for this purpose. The test was considered ended when no portion larger than 1 mm of the bioplastic could be unearthed (establishing the total biodegradation time). 2.4. Statistical analysis The different measurements were performed at least in triplicate. In order to obtain representativeness in the systems, a statistical analysis was carried out using a comparison of means test (Student’s t-test) and an analysis of variance (ANOVA) with a 95 % confidence level (p < 0.05). This analysis was carried out using the SPSS18 Excel statistical package (Microsoft, USA). 3. Results and discussion 3.1. Preparation of bioplastics The first step of the thermochemical procedure for the preparation of bioplastics matrices is mixing, in which the different raw materials were introduced into a mixer with different reinforcement methods. Fig. 1 shows the evolution of torque (M) and the profiles of the percentage ratio between the increase in the initial temperature (100⋅(T-T 0 )/T 0 ). In this case, the profiles are similar without showing significant differences in the torque associated with the different blends. Thereby, in all cases, the profile shows a fast increase at the beginning in torque, followed by a decrease till a constant value, which is maintained during the rest of the process. Thus, the reinforced bioplastics with gelatin and a post-heat treatment show similar behaviour, decreasing torque while the concentration of these compounds or temperatures increases in the matrix. However, in the rest of the systems, there is not a bright change, highlighting the systems Glyx-1, which possesses an apparent variation, maybe due to the enhancement in the properties with limited concentrations of glyoxal. The system with a 20 wt% of gelatin (G-20) shows a minimum torque, followed by a maximum torque in the system with a 1 wt% of glyoxal (Glyx-1). On the other hand, all the thermal profiles display a similar evolution, consisting of a moderate increase in temperature while the torque values increase. Thus, generally, thermal profiles shown an exponential shape whose ratio is higher while most of the concentration increases. 3.2. Characterization of samples 3.2.1. Physicochemical properties 3.2.1.1. Fourier transform infrared spectroscopy (FTIR). Fig. 2 plots the results obtained for the FTIR profile of the different bioplastic samples developed. Analysing the differences between the systems is possible by determining the bonds comprising each sample, as each bond absorbs at different wavenumbers. Thus, all the systems presented a profile similar to the reference system. The main peaks and their wavenumber are displayed in Table 2. Firstly, a band between 3500 and 3000 cm-1 (with C.M. Granados-Carrera et al. Future Foods 11 (2025) 100524 3 a maximum peak at approximately 3266 cm-1) showed the stretching of NH bonds present in amides A and B and the stretching of OH bonds (Türker-Kaya and Huck, 2017). The 2928 and 2876 cm −1 bands corresponded to CH 2 stretching (asymmetric and symmetric) and the 1620 cm −1 peak corresponded to C =O strain, all of them presented in the protein chains (Türker-Kaya and Huck, 2017). Nonetheless, the most remarkable bands of the proteins are those represented in 1628, 1550 and 1228 cm-1, which represented amide I, II and III, respectively, highlighting these amide bands which showed a maximum value when a heat treatment of 120 ◦C for 24 h was applied as a consequence of the chemical modification that suffered the protein during the manufacturing process (Jim´ enez-Rosado et al., 2022). Finally, other bands shown at 1393 and 1100–974 cm −1 are related to CH 2 and C – O-C, respectively (Baker et al., 2014; Perez-Puyana, 2023). 3.2.2. Mechanical properties 3.2.2.1. Dynamic flexure tests. The flexural properties of the different bioplastics obtained from the strain sweep tests are represented by the critical strain values (Table 3). The strain profiles are also shown in Figure S1. In all cases, the critical strain values are significantly lower than those obtained in the reference systems. The addition of gelatin worsens the critical strain (lower values) when its proportion increases, possibly due to the large difference in particle size of both proteins (in the case of SPI and gelatin, their particle size was 10 – 100 and 250 – 600 µm, respectively). The addition of saccharose and transglutaminase (enzymatic crosslinking) has a similar effect but without significant differences in the concentration used. Finally, the heat treatment and the addition of glyoxal (physical and chemical crosslinking, respectively) Fig. 1. Evolution of mixing torque and percentage ratio between the increase in temperature and the initial temperature (100⋅(T-T 0 )/T 0 ) of SPI blends with different reinforcement methods: (A) Gelatin (5, 10 and 20 wt% shown as G-5, G10 and G-20, respectively), (B) Saccharose (5, 10 and 20 wt%, shown as S-5, S-10 and S-20, respectively), (C) Thermal treatment (50, 120 ◦C shown HT-50 and HT-120, respectively), glyoxal (1 and 3 wt%, shown as Glyx-1 and Glyx-3, respectively) and transglutaminase (0.1 and 0.2 wt% shown as Tgase-0.1 and Tgase-0.2, respectively). Ref. refers to a reference system without any treatment or additive included. Fig. 2. FTIR profiles of SPI blends with different reinforcement methods: (A) Gelatin (5, 10 and 20 wt% shown as G-5, G10 and G-20, respectively), (B) Saccharose (5, 10 and 20 wt%, shown as S-5, S-10 and S-20, respectively), (C) Thermal treatment (50, 120 ◦C shown HT-50 and HT-120, respectively), glyoxal (1 and 3 wt%, shown as Glyx-1 and Glyx-3, respectively) and transglutaminase (0.1 and 0.2 wt% shown as Tgase-0.1 and Tgase-0.2, respectively). Ref. refers to a reference system without any treatment or additive included. Table 2 FTIR measurements for the different system developed. Name in the spectrum Correspondent peak (cm −1 ) Assignation Fig. 2.AFig. 2.BFig. 2.C A Amide A 3289 3266 3267 N-H strain B Amide B 2928 2934 2945 Asymmetric strain CH 2 C CH 2 2852 2876 2876 Symmetric stretching CH 2 D Amide I 1620 1628 1628 C =O strain E Amide II 1529 1550 1520 N – H bending F CH 2 1417 1393 1384 CH 2 folding G Amide III 1215 1228 1237 N-H bending H C – O – C 1106–964 1101–974 1111–974 C-O-C stretching Table 3 Parameters obtained from flexure tests: Critical strain, E ′ and tan δ at 1 Hz (indicated as E ′ 1 and tan (δ) 1 ). System Critical strain (%) E’ 1 (MPa) tan (δ) 1 (-) Ref 1.000 ±0.05 820 ±140 0.27 ±0.02 G-5 0.700 ±0.90 120 ±3 0.29 ±0.01 G-10 0.600 ±0.30 231 ±19 0.27 ±0.01 G-20 0.200 ±0.05 216 ±88 0.39 ±0.01 S-5 0.315 ±0.01 1500 ±46 0.27 ±0.01 S-10 0.315 ±0.01 1370 ±134 0.28 ±0.01 S-20 0.315 ±0.01 1334 ±57 0.29 ±0.01 HT-50 0.780 ±0.01 251 ±88 0.28 ±0.02 HT-120 0.150 ±0.05 321 ±439 0.13 ±0.01 Glyx-1 0.320 ±0.01 2340 ±469 0.23 ±0.01 Glyx-3 0.170 ±0.20 2030 ±577 0.22 ±0.03 Tgase-0.1 0.310 ±0.01 2900 ±126 0.24 ±0.01 Tgase-0.2 0.310 ±0.01 2740 ±21 0.25 ±0.01 C.M. Granados-Carrera et al. Future Foods 11 (2025) 100524 4 were the ones that affected the critical strain the most, with the lowest values being achieved. This behaviour may be due to a greater crosslinking between the chains that stiffens the bioplastics, lowering their elastic condition (D¨ orrstein et al., 2018; Jim´ enez-Rosado et al., 2022). Frequency sweep tests are plotted in Fig. 3 in order to assess the stability of the bioplastics. All the systems have a predominant solid character (E’ >E’’), showing certain instability in their mechanical resistance at high frequencies (short recovery times). It should be noted that this slope is lower in the case of chemical and physical crosslinking, showing greater resistance and being consistent with the values obtained for critical strain. Table 3 also shows the values of E’ and tan (δ) at 1 Hz (E’ 1 and tan (δ) 1 ) to improve the comparison between the systems. As can be seen, the incorporation of gelatin in the bioplastics promotes a decrease in E’ 1 and a greater dependence of viscoelastic moduli on frequency. The results show that there is a slight variation in the viscoelastic properties as a function of the gelatin concentration due to the particle size, as previously mentioned. This reduction in E’ 1 was also observed in the bioplastic subjected to a heat treatment. On the other hand, the addition of saccharose, glyoxal or transglutaminase to the samples causes an increase in E’ 1 ; however, as the amount of saccharose additive in the bioplastics increases, E’ 1 decreases, suggesting that these components improve the mechanical properties by strengthening the structure, but only up to a certain point. As for the loss tangent, no significant differences are observed, except for two specific cases: the G20 system, which has the highest value, showing the loss of part of its solid character; and the HT-120 system, which has the lowest value, being the most rigid system. These results are consistent with the ones obtained in previous studies where the incorporation of a post-thermal treatment promotes a significant increase in E’ values as well as a decrease in loss tangent, originating system more stable (Perez-Puyana, 2023). The incorporation of glyoxal can be translated as a drastic increase in E’ values and the addition of transglutaminase promotes a reduction in the elongation of the systems (Cui et al., 2017; Perez-Puyana et al., 2022). 3.2.2.2. Tensile tests. The tensile properties of the different bioplastics are shown in Fig. 4. In all cases, the stress-strain curves show an initial linear elastic region where there is a constant slope between stress and strain. Later, this linear region is followed by a plastic deformation phase, which shows a decrease in the stress-strain slope when these bioplastics exceed the elastic limit, promoting the fracture of the sample. In particular, this behaviour is similar to the one demonstrated in previous studies of soy protein-based bioplastics by other authors (Fern´ andez-Espada et al., 2016; Gamero et al., 2019), existing a minimization of the strain at break and an enhancement of stiffness with most of the reinforcement methods as shown in other studies (Boey et al., 2022). Table 4 shows the values of Young’s modulus, maximum stress and strain at break of the different systems. It is evident that the addition of reinforcements reduces the deformability of the systems. This reduction is more pronounced when a biopolymer such as gelatin or saccharose is added, possibly due to the lack of homogenisation between the biopolymers as shown in other studies where the incorporation of keratin into a SPI-bioplastic, enhances the resistance of the material but, as a result of the heterogeneity, there is a reduction in elongation (Wang, 2022). Furthermore, in the study developed by Omrani-Fard et al., the incorporation of gelatin into a whey protein bioplastic modified the properties of the resulting bioplastics (Omrani-Fard et al., 2020). However, the system with the lowest strain at break is HT-120, likely because the heat causes a denaturation of the soy protein, weakening the bioplastic structure. This behaviour has been already described in previous studies in which there is a deterioration in the properties as a result of high temperatures (Baimark et al., 2021; Jerez et al., 2007). This reduction is linked to an increase in Young’s modulus for each system, indicating that the reinforcement leads to higher stiffness and lower deformability. Notably, the HT-50 system is an exception, as it shows no significant differences compared to the reference system and results in a lower Young’s modulus due to the lower rigidity observed in dynamic tests. 3.2.3. Functional properties 3.2.3.1. Water uptake capacity and soluble matter loss. Firstly, the water contact angle was measured to study the hydrophilicity or hydrophobicity of the different samples created. In this case, the measurements were really difficult to do as a consequence of the existence of constant variation in the value due to the existence of high absorption. In all cases, the water contact angle was <57◦, which was the value associated with the reference systems and without presenting significant differences between systems. These results indicate that the bioplastics produced have a moderate hydrophilic property. This hydrophilic behaviour may be advantageous for applications where biodegradability and interaction with the aqueous environment are more relevant such as fresh food packaging (Audebrand et al., 2013), packaging of products in low humidity conditions (Jin and Zhang, 2008) and coating applications for food or agricultural products (Krochta and Mulder-Johnston, 1997). Moreover, the measurements of water uptake capacity and soluble matter loss are represented in Fig. 5. Reinforcements generally reduce the water absorption capacity of soy bioplastics, which is less pronounced when enzymatic (transglutaminase) or physical (heat treatment) crosslinking is incorporated. These results are consistent with what has been observed for the mechanical properties since a lower stiffness allows a greater degree of swelling, which allows more water to be absorbed (´ Alvarez-Castillo et al., 2018; Cuadri et al., 2016; Kuraishi et al., 2001). Thus, in this case, the decrease in the values of water uptake capacity is related to the increase in the stiffness of the systems, avoiding the incorporation of more water, and not due to the water contact angle previously mentioned because systems were more Fig. 3. Frequency sweep test of SPI blends with different reinforcement methods: (A) Gelatin (5, 10 and 20 wt% shown as G-5, G10 and G-20, respectively), (B) Saccharose (5, 10 and 20 wt%, shown as S-5, S-10 and S-20, respectively), (C) Thermal treatment (50, 120 ◦C shown HT-50 and HT-120, respectively), glyoxal (1 and 3 wt%, shown as Glyx-1 and Glyx-3, respectively) and transglutaminase (0.1 and 0.2 wt% shown as Tgase-0.1 and Tgase-0.2, respectively). Ref. refers to a reference system without any treatment or additive included. C.M. Granados-Carrera et al. Future Foods 11 (2025) 100524 5 hydrophilic with the different reinforcement methods, being able to absorb large amounts of water. Thus, the incorporation of gelatin into a bioplastic matrix promotes a decrease in water uptake capacity, creating more hydrophobic systems or with a water-repellent capacity, as shown in previous studies (Chen et al., 2023). In the same way, in the case of the addition of sugar, an increase in sugar content can lead to a decrease in water uptake capacity due to a reduction in the hydrophilicity as a result of the restriction in the movement of the polysaccharide chains (Halimatul et al., 2019). As for SML, all systems have a similar behaviour, losing all the plasticizer and part of the protein that is solubilized. The HT-120 system has the lowest loss of material, which is due to the loss of water not considered during its thermal treatment. 3.2.4. Biodegradability Fig. 6 depicts the biodegradation of samples containing gelatin, which is an essential factor for evaluating the effect on the pollution of the environment and can be affected by multiple abiotic and biotic factors (Abe et al., 2024). Specifically, the inclusion of these biopolymers in the matrix leads to a significant increase in degradation speed as the gelatin concentration rises. This may be due to the system’s heterogeneity, making it more susceptible to microbial attack. Thus, as described in previous studies, the presence of heterogeneous surfaces enhances degradability as a result of a weak network formed (Liang et al., 2022). The results show that after 20 days in burial conditions, these bioplastic materials undergo severe degradation, resulting in reduced thickness and weight. Furthermore, the bioplastic samples have become significantly more fragile, making them susceptible to manual breakage. 4. Conclusion Soy-protein-based bioplastics were successfully developed, combining different reinforcement methods and demonstrating a generally enhanced modification in the mechanical, physicochemical and functional properties in comparison to the reference systems. Particularly, the addition of gelatin and saccharose improves the mechanical properties while decreasing the critical strain and WUC. Therefore, in the case of heat treatment, bioplastics show a higher stiffness and lower deformability, as well as a decrease in WUC. Finally, chemical and enzymatic crosslinking (the addition of glyoxal and transglutaminase, respectively) promotes a similar reinforcement, causing the highest Young’s modulus, specifically appreciable when the contest in the additive increases. In addition, the incorporation of the different reinforcement methods promotes the formation of bioplastics with an increased stiffness and a lower deformability, obtaining reinforced materials with better mechanical properties. However, the reinforced bioplastics show a lowering in the water uptake capacity and the soluble matter loss as a result of the minimisation in stiffness, as well as the incorporation of hydrophobic components in the networks. Thus, the systems with the best properties were G-5, S-10 and HT-120. For this purpose, future studies should focus on the evaluation of the thermal properties of bioplastic materials and an analysis of their microstructure by microscope techniques, as well as proving their suitability as packaging material by, for instance, evaluating the migration of the materials into food simulants. Moreover, in the case of the addition of protein as an additive, there is a potential for optimisation in the size of particles to further enhance the reliability of these bioplastics. Ethical statement Authors declare that the present research does not involve any human or animal study. Author’s name Affiliation Carmen María Granados-Carrera University of Seville Daniel Castro-Criado University of Seville Mercedes Jim´ enez-Rosado University of Le´ on Alberto Romero University of Seville Víctor Manuel Perez-Puyana University of Seville CRediT authorship contribution statement Carmen María Granados-Carrera: Writing – original draft, Fig. 4. Stress-strain curves of SPI blends with different reinforcement methods: (A) Gelatin (5, 10 and 20 wt% shown as G-5, G10 and G-20, respectively), (B) Saccharose (5, 10 and 20 wt%, shown as S-5, S-10 and S-20, respectively), (C) Thermal treatment (50, 120 ◦C shown HT-50 and HT-120, respectively), glyoxal (1 and 3 wt%, shown as Glyx-1 and Glyx-3, respectively) and transglutaminase (0.1 and 0.2 wt% shown as Tgase-0.1 and Tgase-0.2, respectively). Ref. refers to a reference system without any treatment or additive included. Table 4 Maximum tension, strain at break and Young’s modulus of SPI blends with different reinforcement methods. System Maximum tension (MPa) Strain at break (mm/ mm) Young’s Modulus (MPa) Ref 1.10 ±0.20 1.09 ±0.12 27 ±2 G-5 1.43 ±0.24 0.13 ±0.08 38 ±7 G-10 1.88 ±0.28 0.20 ±0.06 53 ±11 G-20 1.70 ±0.18 0.23 ±0.05 35 ±7 S-5 1.20 ±0.20 0.16 ±0.03 31 ±7 S-10 1.08 ±0.05 0.16 ±0.03 33 ±2 S-20 0.45 ±0.06 0.07 ±0.01 24 ±6 HT-50 2.50 ±0.10 1.16 ±0.17 18 ±1 HT-120 6.00 ±1.40 0.02 ±0.01 343 ±53 Glyx-1 2.60 ±0.20 0.58 ±0.03 56 ±8 Glyx-3 3.50 ±0.30 0.54 ±0.04 45 ±6 Tgase0.1 1.80 ±0.10 0.34 ±0.03 53 ±2 Tgase0.2 1.80 ±0.30 0.24 ±0.03 54 ±9 C.M. Granados-Carrera et al. Future Foods 11 (2025) 100524 6 Visualization, Software, Methodology, Investigation, Data curation. Daniel Castro-Criado: Writing – original draft, Software, Investigation, Data curation, Conceptualization. Mercedes Jim´ enez-Rosado: Writing – review & editing, Visualization, Methodology, Formal analysis, Conceptualization. Alberto Romero: Writing – review & editing, Supervision, Resources, Funding acquisition, Formal analysis. Víctor Manuel Perez-Puyana: Writing – review & editing, Validation, Resources, Project administration. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fig. 5. Water uptake capacity (WUC) and soluble matter loss (SML) of SPI blends with different reinforcement methods: (A) Gelatin (5, 10 and 20 wt% shown as G-5, G10 and G-20, respectively), (B) Saccharose (5, 10 and 20 wt%, shown as S-5, S-10 and S-20, respectively), (C) Thermal treatment (50, 120 ◦C shown HT-50 and HT120, respectively), glyoxal (1 and 3 wt%, shown as Glyx-1 and Glyx-3, respectively) and transglutaminase (0.1 and 0.2 wt% shown as Tgase-0.1 and Tgase-0.2, respectively). Ref. refers to a reference system without any treatment or additive included. Fig. 6. Biodegradability of SPI/Gly bioplastics combined with gelatin. C.M. Granados-Carrera et al. 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