Development of superabsorbent soy protein-based bioplastic matrices with incorporated zinc for horticulture

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.9738 Development of superabsorbent soy protein-based bioplastic matrices with incorporated zinc for horticulture M. Jiménez-Rosado1*, V. Perez-Puyana2, F. Cordobés1, A. Romero2, A. Guerrero1 1Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011, Sevilla, Spain 2Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012, Sevilla, Spain. *MERCEDES JIMÉNEZ-ROSADO Departamento de Ingeniería Química Escuela Politécnica Superior, Universidad de Sevilla, 41011, Sevilla (Spain) E-mail: [email protected] Phone: +34 954557179 Fax: +34 954556441 This article is protected by copyright. All rights reserved. ABSTRACT Background The use of superabsorbent materials in horticulture has spread recently. These materials, which can retain water and release it as crops need it, have great advantages such as the efficient use of water in periods of drought. However, these superabsorbent materials are made of synthetic polymers, which present problems of degradability and, sometimes, also toxicity. For this reason, the main objective of this work is the development of biodegradable superabsorbent bioplastic (SAB) matrices using as raw material a soy protein isolate (SPI). In addition, zinc is incorporated into these bioplastic matrices as an essential micronutrient for plants, to increase their added value. Results The incorporation of zinc chelated with 2,2’,2’’,2’’’-(Ethene-1,2- diyldinitrilo)tetraacetic acid (Zn EDTA) (a salt with which the micronutrient is incorporated) into soy protein-based bioplastic matrices improve their superabsorbent capacity and provided a controlled release of water and nutrients to the crops. Conclusions The results show the high potential of these bioplastic matrices for their use in horticulture as superabsorbent materials that can release nutrients in a controlled manner. This article is protected by copyright. All rights reserved. Keywords: bioplastic matrices, superabsorbent, soy, zinc, controlled-released, horticulture This article is protected by copyright. All rights reserved. 1. Introduction In recent years, the effects of global warming have increased considerably due to human activity, mainly due to the increase in carbon dioxide emissions, which have boosted the greenhouse effect1. One of the main impacts of this warming is the change in the periods of rainfall. In this sense, periods of drought become more noticeable and extensive, whereas the periods of rainfall, although scarce, record a large amount of precipitated water, which produces floods and destruction2. These changes are notoriously detrimental to horticulture, since crops need a regular supply of water, increasing the use of irrigation in these crops, which makes them more expensive3. One of the solutions to this problem is the use of superabsorbent polymeric (SAP) matrices, which are buried into farmland4. These materials are capable of absorbing and retaining large amounts of water without disintegrating (from 10 to 1000 times their weight)5, thus being able to retain rainwater and supplying it to plants, directly in the roots, during periods of drought, that is, optimizing the water cycle6. Nowadays, commercial SAP matrices already exist, which are added to the land in order to retain water and release it in a controlled way according to the needs of the crops (Creasorb from Evonik7 or Luquasorb from BASF8). However, these matrices do not have a good biodegradability, so they have to be removed from the field once they have acted in order to avoid toxicity problems. A possible alternative to these SAP matrices is the use of biodegradable superabsorbent This article is protected by copyright. All rights reserved. (bio)polymeric (SAB) matrices, which have a high biodegradability and no toxicity9. Among all the materials that can be used to elaborate SAB matrices, proteins have technological, economic and environmental advantages. On the one hand, there is a considerable potential to modulate their properties due to their great variability in amino acid composition, the wide range of potential formulations and the wide variety of applicable processing techniques10. In addition, they are the most underutilized and undervalued by-product in the agri-food industry, thus their use as SAB materials implies an increase in their added value11. Finally, they can pose an extra contribution of nutrients after their degradation due to their significant nitrogen content. All this makes protein-based SAB matrices have a great potential for their use in horticulture; however, this use is still limited by the lack of knowledge in the formulation and processing thereof. Another advantage that these protein-based SAB matrices could provide for their use in horticulture is the incorporation of micronutrients to crops in a controlled manner. In this sense, the micronutrients would be incorporated into the protein-based SAB matrices thought soluble salts already used in horticulture which could be released from the matrices due to their biodegradation and the release of water, thus being able to be captured by the plant. In this way, there would be an increase in the efficiency of incorporating micronutrients due to they would be incorporated in a controlled manner, avoiding the typical excesses of conventional fertilization, which causes the This article is protected by copyright. All rights reserved. contamination of soil and groundwater by excess nutrients12,13. One of the micronutrients that generate the most pollution problems is zinc. This is due to the great solubility of the salts used for the incorporation of this micronutrient14. In this sense, the incorporation of zinc into the protein-based SAB matrices, and its extrapolation to the other essential micronutrients, would pose an added value, increasing its interest while replacing the commercial SAP matrices. From all the different proteins studied in the literature for the elaboration of protein-based SAB matrices (based on pea15, egg albumen16, crayfish17, rice18, soy19, whey20), soy protein has proven to be the one with the best absorbent capacity, due to its high content of glutamic and aspartic acid, which gives it a good hydrophilic character21. Furthermore, its high lysine content allows the introduction of some carboxylic groups, improving, even more, its hydrophilic character 22. Among the fabrication methods, soy protein-based SAB matrices allow an adequate processing by injection molding, one of the most used techniques for the manufacture of synthetic plastics, which would facilitate its implementation on an industrial scale21,23,24. However, the incorporation of micronutrients generally hinders the processing of these matrices, as it results in a reduction in the strengthening potential of the matrix associated to the corresponding reduction in the protein content25. Therefore, the main objective of the present work is the development of suitable soy protein-based matrices with zinc chelated with 2,2’,2’’,2’’’-(Ethene-1,2- This article is protected by copyright. All rights reserved. diyldinitrilo)tetraacetic acid (Zn EDTA), which is a salt that contains the essential micronutrient for plants. To achieve this objective, the mechanical and morphological properties of matrices with different concentrations of Zn EDTA incorporated throughout the processing stages (blends, bioplastic matrices and final matrices) as well as their water absorption capacity have been evaluated. 2. Materials and methods 2.1 Materials Soy protein isolate (SPI) with min. 910 g·kg-1 protein, 60 g·kg-1 moisture and 30 g·kg-1 ash, which was supplied by Protein Technologies International (SUPRO 500E, Belgium) has been used to obtain protein-based SAB matrices. In addition, glycerol (Gly), provided by Panreac Química Ltd. (Spain) has been used as plasticizer. Finally, zinc chelated with 2,2’,2’’,2’’’-(Ethene-1,2- diyldinitrilo)tetraacetic acid (Zn EDTA) has been used as a salt that provides the selected micronutrient, which was supplied by Trade Corporation International S.A.U. (Spain). 2.2 Preparation of soy protein-based matrices A generic two-stage procedure (Figure 1) was followed to obtain soy proteinbased matrices with Zn EDTA incorporated. The process consisted in an initial homogenization which was carried out with a two-blade counter-rotating mixer Polylab QC (ThermoHaake, Germany). The mixing was performed adiabatically for 10 min, at 50 rpm and at room temperature (25 ± 2 ºC) following the same protocol used in SPI/Gly blends previously studied24,26. A constant ratio (1:1) This article is protected by copyright. All rights reserved. between SPI and Gly and different concentrations of Zn EDTA (50, 100 and 150 g·kg-1) were used. The SPI/Gly blend without Zn EDTA was selected as reference (Ref.). During this mixing, both torque variation (M) and temperature evolution (ΔT = 100·(T-T0)/T0, where T is the temperature at time of measurement and T0 is the initial temperature) were studied. Consecutively, an injection molding of the blends was carried out in order to obtain rectangular bioplastic matrices (60x10x1 cm). Thus, the blends were introduced in a MiniJet Piston Molding System II (ThermoHaake, Germany) where they were injected through a nozzle into a previously heated mold. The parameters in this stage were selected based on the rheological characterization of the blends and previous studies carried out to optimize the water uptake capacity of SPI/Gly systems21: a cylinder and mold temperature of 40 and 70 ºC, respectively, an injection pressure of 500 bar (for 20 s) and a post-injection pressure of 200 bar (for 300 s). Finally, a dehydrothermal treatment (DHT) was carried out before performing the water uptake capacity tests according to the ASTM 180 D570 standard27. In addition, it was found that without this stage the bioplastics’ network is not strong enough and they disintegrated in the water. This treatment consisted of introducing the bioplastic matrices in a conventional oven at 50.0 ± 0.1 ºC for 24 h. 2.3 Characterization of the soy protein-based systems with Zn EDTA This article is protected by copyright. All rights reserved. A complete characterization of the systems was carried out in order to evaluate the suitability of the matrices as superabsorbent materials with incorporated micronutrients. Thus, the blends were evaluated to optimize their processing, as well as the bioplastic matrices before and after a severe treatment with water to study their properties. It is worth mentioning that the matrices after the severe treatment with water were dried by a freeze-drying process to obtain dry matrices that can be characterized. 2.3.1 Thermomechanical tests of the blends. The homogenized blends were mechanically characterized by small amplitude oscillatory compression tests, using a mechanical dynamic analyzer RSA3 (TA Instruments, USA) with a plate-plate geometry of 8 mm in diameter. The purpose of these tests was to define the behavior of the blends with the temperature to select the optimal treatment in the subsequent injection molding. Thus, temperature ramp tests between 25 and 120 ºC were performed, at 1 Hz and a strain within the linear viscoelastic range (0.01%), where the elastic modulus (E’) and the loss tangent (tan δ = E’’/E’) were studied throughout the temperature range selected. 2.3.2 Thermogravimetric analysis (TGA) The thermogravimetric analyses (TGA) were carried out using a thermogravimetric analyzer Q600 (TA Instrument Inc., USA) in order to study the thermomechanical stability of the different raw materials, a homogeneous blend of them and a matrix after water absorption. In these analyses, the This article is protected by copyright. All rights reserved. materials used, only glycerol was totally lost at 600 ºC, leaving 28.7% of SPI and 54.9% of Zn EDTA. Regarding the homogeneous blends of the different components (450, 450 and 100 g·kg-1 of SPI, Gly and Zn EDTA, respectively), different weight losses can be observed, corresponding to each of the raw materials. Thus, the SPI peak is between 200 and 450 ºC and the Gly peak between 200 and 280 ºC. As for the Zn EDTA, a first peak between 50 and 100 ºC is observed, which overlaps with the possible humidity of the sample, and another slightly pronounced peak between 350 and 400 ºC. The first peak observed at 50-100 ºC is the possible cause of the strengthening observed previously in thermomechanical tests. The release of water from the protein linked to the possible breakdown of the Zn EDTA complex would cause the structure to form new bonds (metalloprotein) reinforcing and obtaining an improvement of the elastic modulus (E’). Finally, in the profile of the matrix after the water treatment, the weight loss that corresponds to Gly does not appear, thus it is assumed that all Gly was released to the water due to its great hygroscopic character. Regarding the peaks corresponding to the SPI and Zn EDTA, these still appear, thus these materials are present after water absorption. 3.2.2 Tensile strength measurements The results from tensile strength measurements of the bioplastic matrices with different percentages of incorporated Zn EDTA (50, 100 and 150 g·kg-1), as well as those without Zn EDTA for the reference (Ref.) can be observed in Figure 5. This article is protected by copyright. All rights reserved. Figure 5A shows the relationship between stress (σ) and strain (ε) in the different bioplastic matrices. All the bioplastic matrices had a first elastic zone, where the strain could be recovered over time. In this zone, a lineal relationship was maintained between strain and stress, thus making it possible to obtain the Young’s modulus of the different matrices. Then, a plastic area was recorded, which remained until the breakage of the bioplastic matrices. Once the elastic zone was overcome, the matrices did not recover their shape. The incorporation of 50 g·kg-1 Zn EDTA improved the maximum strain, without modifying the maximum stress reached. However, this improvement was not observed when higher percentages of Zn EDTA were incorporated. As previously mentioned, this fact is possibly due to the action of Zn EDTA as a filler in these bioplastic matrices, which did not cross over with the protein structure. In addition, it can hinder the crosslinking of the protein, which is also in smaller proportion, therefore the bioplastic matrices have worse mechanical properties in these cases. On the other hand, Figure 5B shows the parameters of strain at break (εmax), maximum stress (σmax) and Young’s modulus of the different bioplastic matrices studied without Zn EDTA (Ref.) and with different Zn EDTA concentrations (50, 100 and 150 g·kg-1) to compare their mechanical properties. As can be observed σmax did not show significant differences between the reference bioplastic matrix (Ref.) and bioplastics with 50 g·kg-1Zn EDTA. However, matrices with higher Zn EDTA concentrations (100 and 150 g·kg-1) showed This article is protected by copyright. All rights reserved. lower σmax values, which were more pronounced for the system with the highest Zn EDTA concentration. As for εmax, an increase was observed with the incorporation of 50 g·kg-1 Zn EDTA, which decreased significantly when incorporating larger amounts of Zn EDTA. Finally, the Young’s modulus did not present significant differences in the studied systems except, once again, for the bioplastic matrix that contains 50 g·kg-1 Zn EDTA that improved slightly. This behavior is consistent with the theory that in concentrations above 50 g·kg- 1Zn EDTA, there is a greater amount of filler material incorporated, leading to worse mechanical properties due to this extra material do not allow to form the protein network of the matrices. 3.2.3 Water uptake capacity and soluble matter loss Figure 6 shows both the water uptake capacity and the soluble matter loss. The matrix without salt showed values slightly lower than the limit of the superabsorbent materials (10000 g·kg-1). However, the incorporation of Zn EDTA increased the water absorption capacity of the bioplastic matrices, which was more pronounced at higher percentages, being able to consider these materials as superabsorbents. Although, the bioplastic matrix with 150 g·kg-1 Zn EDTA, which had the best water uptake capacity, was not resistant out of the water, even breaking down, possibly due to its lower amount of protein, which may have caused the matrix to be less structured. Regarding soluble matter loss, there were no significant differences between the different systems, which concludes that Gly is lost in all systems due to its high hydrophilic character, as This article is protected by copyright. All rights reserved. previously confirmed with TGA (3.2.1). In addition, the protein/Zn EDTA loss increased when larger amounts of Zn EDTA were incorporated since these matrices had less Gly, although these systems reached practically the same soluble matter loss. This may be due to the lower strengthening of the matrices, as mentioned above. However, the protein/Zn EDTA ratio that was lost could not be estimated until ICP-AES analyzes were carried out. 3.3 Characterization of soy protein-based matrices with Zn EDTA Once the bioplastic matrices were subjected to a water absorption step, followed by a freeze-drying stage, the morphological and mechanical properties of the final matrices were studied to analyze the possible variation that this stage confers to the matrices. 3.3.1 Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) Table 1 shows both the amount of zinc in the bioplastic matrices before and after the absorption stage, with the latter being measured by ICP-AES. The amount of zinc remaining in the matrices after the absorption stage did not present significant differences between the matrices. Therefore, the zinc released in the water absorption stage was more pronounced as more Zn EDTA was incorporated into the initial bioplastic matrix. This fact may be due to the lower structuring of the matrix, as has already been mentioned, which caused the loss of a greater amount of protein and Zn EDTA, as already observed in the soluble matter loss measurements. This article is protected by copyright. All rights reserved. With respect to these results, it is expected that in all the matrices, most of the zinc would be released in a controlled manner with the water incorporated in the matrix, with this percentage being more pronounced in the matrix with higher Zn EDTA incorporated. Moreover, the small amount of Zn retained in the matrix would be released along with the other nutrients provided by the protein matrix (mainly nitrogen) during its biodegradation. 3.3.2 Scanning electron microscopy (SEM) Figure 7 shows the micrographs taken from the different samples. The images of the bioplastic matrices (Figures 7A, 7C, 7E, 7G) show that the addition of Zn EDTA to the bioplastic matrices made their structure more homogeneous, with less pores in it. This could be due to the greater amount of solid material in the systems, with part of it acting as a filler material. On the other hand, the images captured from the matrices (Figures 7B, 7D, 7G, 7H) show how the passage through the stages of water absorption and freeze-drying made the matrices develop larger and more heterogeneous pores, which is more evident at the higher concentrations of Zn EDTA. This behavior could be due to the greater soluble matter loss there was as more salt (filler material) was incorporated into the matrix, which was anticipated in previous tests. In this sense, the filler material made the structure develop fewer holes in the bioplastic matrices. However, most of this material was lost in the water absorption stage, due to the lower structuring of the protein matrix, making the matrices with greater concentrations of Zn EDTA, present more holes (free This article is protected by copyright. All rights reserved. volume) after absorption. Thus, the matrix obtained after incorporating 50 g·kg-1 of Zn EDTA is the one with the lowest free volume and the most heterogeneous pores. This could explain the improvement of the mechanical properties of the bioplastic matrices when 50 g·kg-1 of Zn EDTA was incorporated, since they have a defined, consolidated and reinforced structure. Furthermore, these matrices possibly have better mechanical properties after absorption. 3.3.3 Mechanical measurements The mechanical properties of the matrices after absorption can be seen in Figure 8. This figure shows the evolution of the elastic (E’) and viscous (E’’) moduli in the frequency range studied for the matrices with different percentages of Zn EDTA (50, 100 and 150 g·kg-1) incorporated, using the matrix without Zn EDTA as reference (Ref.). As can be observed, E’ remained above E’’ for all the frequency range studied. Moreover, E’ had a slight dependence on frequency. Thus, all the matrices showed a basically elastic character. In fact, stronger matrices (higher E’ values) were obtained when Zn EDTA was incorporated. In addition, E’’ presented greater variations, possibly due to the low stability of the matrices after water absorption. Once again, matrices with Zn EDTA incorporated also had higher E’’ values than the reference system. 4. Conclusions This article is protected by copyright. All rights reserved. Superabsorbent soy protein-based matrices have been developed, showing a great potential for their use in horticulture. In addition, essential micronutrients for crops (zinc) can be incorporated in them. The addition of zinc chelated with 2,2’,2’’,2’’’-(Ethene-1,2-diyldinitrilo)tetraacetic acid (Zn EDTA) to the matrices improved their superabsorbent capacity and their mechanical properties up to a concentration of 50 g·kg-1 Zn EDTA. In addition, the microstructure of bioplastic matrices with Zn EDTA incorporated is more homogeneous due to their greater amount of filler material. However, after introducing them into the water absorption stage, these matrices present a greater free volume due to a greater soluble matter loss. Zinc is mostly released with water when the latter leaves the matrix in a controlled manner. However, a small amount is retained in the matrix, to be releasedit during its biodegradation. Thus, there is a first faster release of zinc, when water is incorporated into the crop and a slower second release of zinc along with the rest of the nutrients of the protein matrix conditioned by its biodegradability. This release profile can benefit the crops, since most of the zinc is needed in the growth stage, which occurs in the first days, and a lower quantity is required during maturation. Further studies will be needed required in the future to analyze the release and plan assimilation of water and zinc in normal horticultural conditions. 5. Acknowledgments This article is protected by copyright. All rights reserved. This work is part of a project funded by MINECO/FEDER (Ref. CTQ2015- 71164-P), the authors gratefully acknowledge their financial support. The authors also acknowledge for the pre-doctoral grant from Mercedes Jiménez- Rosado (FPU2017/01718-MEFP) and Víctor Perez-Puyana (VPPI-US). Finally, the authors thanks CITIUS for their access and assistance to the Microanalysis and Microscopy services. 6. References 1 Cox PM, Betts RA, Jones CD, Spall SA, and Totterdell IJ. Acceleration of global warming due to carbon-cycle feedbacks in a coupled climate model. Nature Macmillan Magazines Ltd.; 408:184–187 (2000). 2 Wang X, Auler AS, Edwards RL, Cheng H, Ito E, and Solheid M. Interhemispheric anti-phasing of rainfall during the last glacial period. Quat Sci Rev 25:3391–3403 (2006). 3 Venuprasad R, Lafitte H, and Atlin G. Response to direct selection for grain yield under drought stress in rice. Crop Sci 47:285–293 (2007). 4 Johnson MS and Leah RT. Effects of superabsorbent polyacrylamides on efficiency of water use by crop seedlings. J Sci Food Agric 52:431–434 (1990). 5 Chanda M and Roy K. Plastics Technology Handbook. 4th ed. Boca Raton: CRC Press; 2007. 6 Zohuriaan-Merh MJ and Kabiri K. Superabsorbent polymer materials: a review. Iran Polym J 17:451–477 (2008). This article is protected by copyright. All rights reserved. 7 Evonik. Creasorb: superabsorbents for non-sanitary applications. 2017. Available: https://www.creasorb.com/product/creasorb/en/about/ [16 November 2018]. 8 Chempoint. BASF superabsorbent polymers. 2018. Available: https://www.chempoint.com/products/catalog/basf/basf-superabsorbent- polymers [16 November 2018]. 9 Mortain L, Dez I, and Madec PJ. Development of new composites materials, carriers of active agents, from biodegradable polymers and wood. Comptes Rendus Chim 7:635–640 (2004). 10 Bozell JJ. Feedstocks for the Future: Using Technology Development as a Guide to Product Identification. Feedstocks for the Future p. 1–12 2006. 11 Damodaran S. Food Proteins and Their Applications. New York: Routledge; 1997. 12 Muscanescu A. Organic versus conventional: advantages and disadvantages of organic farming. Econ Eng Agric Rural Dev 13:253–256 (2013). 13 Timilsena YP, Adhikari R, Casey P, Muster T, Gill H, and Adhikari B. Enhanced efficiency fertilisers: A review of formulation and nutrient release patterns. J Sci Food Agric 95:1131–1142 (2015). 14 Alloway BJ. Micronutrients and Crop Production: An Introduction. In: Alloway BJ, ed. Micronutrient Deficiencies in Global Crop Production Dordrecht: Springer Netherlands; p. 1–39 2008. This article is protected by copyright. All rights reserved. 15 Perez-Puyana V, Felix M, Romero A, and Guerrero A. Development of pea protein-based bioplastics with antimicrobial properties. J Sci Food Agric 97:2671–2674 (2017). 16 Jones A, Zeller M, and Sharma S. Thermal, mechanical, and moisture absorption properties of egg white protein bioplastics with natural rubber and glycerol. Prog Biomater 2:12 (2013). 17 Felix M, Romero A, Cordobes F, and Guerrero A. Development of crayfish bio-based plastic materials processed by small-scale injection moulding. J Sci Food Agric 95:679–687 (2014). 18 Félix M, Lucio-Villegas A, Romero A, and Guerrero A. Development of rice protein bio-based plastic materials processed by injection molding. Ind Crop Prod 79:152–159 (2016). 19 Liu W, Misra M, Askeland P, Drzal LT, and Mohanty AK. ‘Green’ composites from soy based plastic and pineapple leaf fiber: fabrication and properties evaluation. Polymer (Guildf) 46:2710–2721 (2005). 20 Montaño-Leyva B, Silva GGD da, Gastaldi E, Torres-Chávez P, Gontard N, and Angellier-Coussy H. Biocomposites from wheat proteins and fibers: Structure/mechanical properties relationships. Ind Crops Prod 43:545–555 (2013). 21 Fernández-Espada L, Bengoechea C, Cordobés F, and Guerrero A. Thermomechanical properties and water uptake capacity of soy proteinbased bioplastics processed by injection molding. J Appl Polym Sci This article is protected by copyright. All rights reserved. Accepted Article Figure 4: Thermalgravimetric analysis tests for raw materials (SPI, Gly and Zn EDTA), a homogeneous blend of them (450, 450 and 100 g·kg-1 of SPI, Gly and Zn EDTA respectively) and a matrix after water absorption test (A) and their corresponding derivative signals (B). 296x209mm (300 x 300 DPI) This article is protected by copyright. All rights reserved. Accepted Article Figure 5: Tensile strength measurements for soy protein isolate/glycerol/Zn EDTA bioplastics at different Zn EDTA contents (Ref., 50, 100 and 150 g·kg-1): (A) Stress-strain curves and (B) Parameters: maximum stress (σmax), strain in the roture (εmax) and Young’s Modulus. Columns with different letters are significantly different (p ≤0.05). 290x203mm (300 x 300 DPI) This article is protected by copyright. All rights reserved. Accepted Article Figure 6: Water uptake capacity and soluble matter loss for soy protein isolate/glycerol/Zn EDTA matrices at different Zn EDTA contents (Ref., 50, 100 and 150 g·kg-1). Columns with different letters are significantly different (p ≤0.05). 290x203mm (300 x 300 DPI) This article is protected by copyright. All rights reserved. Accepted Article Figure 7: Scanning Electron Microscopy (SEM) micrographs of bioplastic matrices and matrices with different concentration of Zn EDTA (50, 100 and 150 g·kg-1). The matrix without salt is used as reference (Ref.). 296x209mm (300 x 300 DPI) This article is protected by copyright. All rights reserved. Accepted Article Figure 8: Dynamic frequency sweep tests for soy protein isolate/glycerol/Zn EDTA matrices with diffetent Zn EDTA percentages (Ref., 50, 100 and 150 g·kg-1). 296x209mm (300 x 300 DPI) This article is protected by copyright. All rights reserved. Accepted Article Tables Table 1: Amount (g·kg-1) of zinc (Zn2+) in the final matrix determined by ICP-AES measurements. Columns with different letters are significantly different (p ≤0.05). g·kg-1 Zn EDTA incorporated in the blends g·kg-1 Zn2+ in bioplastic matrices g·kg-1 Zn2+ in matrices Ref. - - 50 9.14a 0.87 ± 0.30b 100 18.28c 0.88 ± 0.66b 150 27.42d 0.88 ± 0.24b This article is protected by copyright. All rights reserved.