Development of soy protein-based matrices containing zinc as micronutrient for horticulture

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Industrial Crops and Products on Jiménez‐Rosado, M., Perez‐Puyana, V., Cordobés, F., Romero, A., & Guerrero, A. (2018). Development of soy protein-based matrices containing zinc as micronutrient for horticulture. Industrial Crops and Products, 121, 345-351. Available at: https://doi.org/10.1016/j.indcrop.2018.05.039 ” Development of soy protein-based matrices containing zinc as micronutrient for horticulture M. Jiménez-Rosadoa,*, V. Perez-Puyanaa, F. Cordobésb, A. Romeroa, A. Guerrerob a Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012, Sevilla, Spain b Departamento de Ingeniería Química, Universidad de Sevilla, Escuela Politécnica Superior, 41011, Sevilla, Spain *CORRESPONDING AUTHOR Mercedes Jiménez-Rosado [email protected] Facultad de Química, Universidad de Sevilla 41012 Sevilla Spain ABSTRACT Protein-based bioplastics may be regarded as novel biopolymer matrices based on renewable natural components. Due to technological, economic and environmental benefits, these biopolymer matrices are highly attractive for the incorporation and subsequent release of micronutrients that are essential for the development and health of plants, avoiding the typical excesses of conventional fertilizers. In addition, soy protein isolate (SPI) seems to be an adequate resource for the manufacture of natural-based superabsorbent materials due to its hydrophilic character and excellent processability when combined with a plasticizer. The objective of this work is to develop soy proteinbased bioplastic matrices loaded zinc sulphate monohydrate with potential applications in horticulture. With this aim, the micronutrient loading level and water absorption are the most important properties to assess. The effect of the presence of a selected micronutrient (zinc) at different concentrations in the soy protein-based matrix was assessed, evaluating the mechanical properties, water uptake capacity, microstructure and loading level of zinc. The results confirm that important amounts of an essential micronutrient for a plant (Zn) can be incorporated into bioplastic matrices, modifying water absorption, mechanical and microstructural properties. In any case, the results obtained in this work open up a great potential for the use of this matrices as a supplying source of micronutrients for horticultural crop applications. Keywords: Bioplastics; Soy protein; Zinc; Micronutrients; Horticulture 1. INTRODUCTION Plastics are one of the most important and most used consumer products in human life. Almost everything used in society is made of plastic or contains plastic, so plastics are used in a great variety of applications, such as, packaging, construction, electronic devices, houseware, etc. Due to this great use, the worlds production of plastics reached 322 million tons in 2015, when China led the market (27.8% of production), followed by Europe (18.5%) (Plastics Europe, 2016). However, this high production of plastic products has significant disadvantages derived from their low biodegradability, which leads to high contamination, and their extremely low renewability, since most of them come from fossil sources (Bledzki and Jaszkiewicz, 2010). In 2014, recycling and recovery of plastic reached 69.2%, while the rest of this percentage is still discarded in landfills, causing garbage accumulation (Plastics Europe, 2016). For these reasons, plastics are being gradually replaced by bioplastics. The term bioplastics refers to either those polymeric materials which are obtained from renewable sources, or those which exhibit a natural degradability that helps eliminate garbage accumulations. A superabsorbent matrices due to its hydrophilic character derived from its high content of glutamic and aspartic acid, which make it more hydrophilic than other proteins, whereas Cuadri et al. (2018, 2017, 2016) benefit from its high content in lysine to introduce some functional carboxylic groups, thereby enhancing its hydrophilic character. There are several soy products with different protein content. Among them, the soy protein isolate (SPI) contains at least 90 wt.% protein; although more expensive, it gives the best results due to the greater amount of protein and less moisture (Mo et al., 1999). Plasticizers are low molecular weight agents that have the ability to increase protein chain mobility, reducing the number of inter and intramolecular interactions and decreasing the glass transition temperature. Glycerol (Gly) is the most widely used from this group (Bourny et al., 2017; Guerrero et al., 2010; Matveev et al., 2000). A wide variety of additives are often used to improve the processability and/or the final properties of bioplastics (Bourny et al., 2017). The properties of these soy-based matrices (bioplastic) can be controlled by the SPI/Gly ratio, the processing conditions and technologies used. Protein-based bioplastics can be processed by using existing processing technologies, from the physicochemical to thermomechanical methods. Among these thermomechanical techniques, injection moulding is one of the most important and suitable process for systems that may exhibit a mixed (thermoplastic and thermoset) character such as proteins. However, this technique needs a previous mixing process in order to obtain a readily injectable proteinplasticizer blend (Reddy, 2015). In the agricultural sector, the use of these materials would bring great advantages due to their null toxicity and high biodegradability (Guo et al., 2015), as well as the extra contribution of nutrients after their degradation (Saenghirunwattana et al., 2014). Besides, these materials are able to absorb and retain water from the environment, maintaining their integrity without dissolving, even though they can suffer a marked increase in volume (Fernández-Espada et al., 2016a). All of these advantages make these materials an attractive potential candidate for the incorporation of essential micronutrients for the development and health of plants in horticulture. Micronutrients are elements needed by plants in small quantities. There are seven essential micronutrients in horticulture: Boron, Copper, Iron, Zinc, Manganese, Molybdenum and Chlorine. Among them, Zinc has an important role in the growth and development of plants (Uchida, 2000). The incorporation and subsequent controlled release of micronutrients would contribute to avoid the typical excesses of conventional fertilization, while increasing the efficiency of assimilation by the plant. In addition, being able to store water and dispose it during crop growth would increase water-use efficiency of plants (Mortain et al., 2004). The present work is focused on the development of soy protein-based matrices loaded with a soluble micronutrient for their potential use in horticulture. For this purpose, the most important property to evaluate is the micronutrient loading level followed by the water uptake capacity. Zinc cation was selected as the micronutrient, and it was introduced in salt form in the mixing stage, along with SPI and glycerol. Then, bioplastic systems were obtained by injection moulding, at selected conditions, evaluating the water uptake capacity, the mechanical properties and particularly the zinc loading level. It is worth pointing out that mechanical properties of zinc-loaded bioplastics only have a relative importance for their horticulture applications. However, a minimal level of mechanical properties is necessary to ensure physical stability of these bioplastics. In addition, the microstructure of the freeze-dried matrix and their loading level of zinc have also been studied. 2. EXPERIMENTAL 2.1 Materials Soy protein isolate (SPI, with min. 91 wt.% protein and 6 wt.% moisture) was supplied by Protein Technologies International (SUPRO 500E, Belgium). Glycerol (Gly) was used as a plasticizer and zinc micronutrient was incorporated into the matrix as zinc sulphate monohydrate (ZnSO4·H2O, MN), which were both purchased from Panreac Química Ltd. (Spain). 2.2. Preparation of bioplastic matrices The procedure for the preparation of matrices consisted of several stages, as can be seen in Fig. 1. Fig. 1. Processing stages for the preparation of soy/glycerol and soy/glycerol/micronutrient matrices. F: soy, glycerol (and micronutrient) flow. B: moulding blends. BP: bioplastics after injection moulding. DBP: bioplastics after dehydrothermal treatment. W: water. WGM: water with glycerol and micronutrient which leave the matrix. WPM: matrix with water. DPM: matrix without water. First of all, SPI and Gly (ratio 1:1) with different percentages of ZnSO4·H2O (0.0, 2.5, 5.0, 10 and 15 wt.%), maintaining this SPI/Gly (1:1) ratio constant (Table 1), were mixed in a two-blade counter-rotating mixer Polylab QC (ThermoHaake, Germany) at room temperature and 50 rpm for 10 min, under adiabatic conditions, following the same protocol as in previous studies (Felix et al., 2016; Fernández-Espada et al., 2016a). A detailed description of this equipment and its operational conditions may be found in the literature (Dealy, 1983; Van Wazer et al., 1963). At this stage, torque (M) and temperature (T) measurements were taken during mixing time. Table 1. Percentages and nomenclature of the different soy/glycerol/micronutrient (SPI/Gly/MN) blends in the mixing stage. In the second stage, SPI/Gly or SPI/Gly/MN blends (B) obtained after mixing were subsequently processed by injection moulding using a MiniJet Piston Injection Moulding System II (ThermoHaake, Germany) to obtain soy protein-based bioplastic matrices (BP). In this stage, the processing parameters were selected on the basis of a previous study (Fernández-Espada et al., 2016a), taking into account the specific requirements of this current study. As a result, the selected values were: cylinder temperature (40 °C), mould temperature (90 °C), injection pressure (600 bar, 20 s) and post-injection (holding) pressure (200 bar, 300 s). These moulding conditions will be referred to as 40/90. Subsequently, the bioplastic matrices were subjected to a dehydrothermal treatment that consisted in a heating stage in a conventional oven at 50 °C for 24 h to obtain dried bioplastic matrices (DBP). This stage involves some network strengthening and, therefore, is necessary to maintain the physical integrity of the matrices in the subsequent stage of absorption with water. The next step was an absorption process performed in a closed vessel with 300 mL of distilled water (W) for 24 h. In this stage, the glycerol, with some micronutrient, and even protein, was released into the water (WGM). Finally, the wet protein matrices after absorption (WPM) were introduced into a freeze-drying equipment (LyoQuest, Tesltar, Spain) in order to remove the water and obtain the dry protein matrix (DPM). 2.3. Characterization of bioplastic matrices 2.3.1. Tensile strength measurements Tensile tests were carried out in a dynamic mechanical analyser RSA3 (TA Instruments, USA), with a modified protocol of the norm ISO 527-2:1993 (“ISO 570-2:1993, Plastics. Determination of tensile properties. Part 2: Test conditions for moulding and extrusion plastics,” 1993). In this respect, bioplastic matrices (BP) were subjected to increasing axial stress until breakage. Thus, a crosshead speed of 1 mm/min was selected, and at least three replicates were tested for each sample. The stress produced (σ) against the applied strain (ε) was recorded. Besides, Young’s modulus, maximum stress (σmax) and maximum strain (εmax) were obtained. 2.3.2. Dynamic mechanical analysis Dynamic Mechanical Analyses were performed in bending mode and were carried out in a dynamic mechanical analyser RSA3 (TA Instruments, USA). In the bending tests, BPs were subjected to a small amplitude oscillatory flexural strain. Firstly, a strain sweep test between 0.002% and 2%, at a constant frequency of 1 Hz, was performed to determine the linear viscoelastic region in which elastic moduli and viscous moduli remained independent of the imposed strain. Subsequently, frequency sweep tests (0.02–20 Hz and room temperature) were carried out within the linear viscoelastic region. These tests are frequently performed to associate the viscoelastic properties with the unchanged matrix microstructure (Fernández-Espada et al., 2016a; Perez-Puyana et al., 2016). The elastic (E’) and the viscous (E”) moduli as well as the loss tangent (tan (δ)) were measured across the frequency range. In addition, E’ and tan (δ) were obtained at a constant frequency of 1 Hz (E’1 and tan (δ1)) in order to perform a suitable comparison of the systems. 2.3.3. Water uptake capacity and soluble matter loss Water absorption tests were carried out according to a modification of the ASTM D570 norm (2005) (ASTM D570-98: Standard Test Method for Water Absorption Of Plastics, 2005), using rectangular DBP specimens (55 × 10 × 1 mm) after the dehydrothermal stage. Then, DBP samples were immersed into 300 mL of distilled water for 24 h. The water uptake capacity was calculated as follows: Water uptake capacity = (wWPM-wDPM)/wDPM Where wDPM is the weight of the matrix after the freeze-drying stage (DPM) and wWPM is the weight of the matrix after the absorption stage (WPM). Finally, soluble matter loss was estimated as: Soluble matter loss= (wDBP-wDPM)/wDBP Where wDBP is the weight of the matrix after the dehydrothermal stage (DBP). 2.3.4. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) ICP-AES measurements were performed using an ICP SpectroBlue TI (Spectro, Germany). The bioplastic matrices before (DBP) and after absorption and drying stages (DPM) were subjected to a previous treatment of microwave digestion. To do this, a known sample weight was taken and digested with the help of acids (HNO3 and H2O2 in a ratio 7:1), finally bringing the solution to a specific volume. In addition, water after absorption (WGM) was subjected to a previous gravity filtering treatment. After previous treatments, all the samples were aspirated through a nebulizer passing through a nebulization chamber where they became an aerosol. An argon stream transported the aerosol to the plasma torch, where it was subjected to a temperature around 6000 K, dissociating it into free atoms and ions with characteristic wavelengths, which were measured and recorded. 2.3.5. Scanning electron microscopy (SEM) The microstructure of the selected matrices (DPM) was determined as described by Orawan et al. (2006). The samples sputter-coated with palladium-gold were mounted on a bronze stub. The specimens were observed using a Philips XL-30 Scanning electron microscope (the Netherlands) at an acceleration voltage of 8 kV and a resolution of ×50 and ×200 magnification. Then, the pore size was measured using ImageJ software, which is a public domain Java image processing program. 2.4. Statistical analysis Al least three replicates of each measurement were carried out. The statistical analysis was performed using t-test and one-way analysis of variance (ANOVA, p < 0.05) by means of the statistical package SPSS 18. Standard deviations from some selected parameters were calculated. 3. RESULTS AND DISCUSSION 3.1. Preparation of bioplastic matrices As can be seen in Fig. 1, mixing was the first stage in the thermochemical processing of the protein-based bioplastic matrices studied. SPI, Gly and ZnSO4·H2O (MN) were introduced into the mixer in different percentages. Fig. 2 shows torque profiles (M) and the evolution of the percentage ratio between the increase in temperature and the initial temperature (100·(T-T0)/T0) as a function of mixing time for different blends. The profiles did not show significant differences in the torque of the different blends (Fig. 2A). All the profiles displayed a fast increase in torque at the beginning, decreasing towards a constant value that was maintained during the rest of the mixing. There was a minimum torque in the system without micronutrients (0% MN), followed by a maximum of 2.5% MN. However, for higher percentages of MN the torque decreased again. On the other hand, all thermal profiles (Fig. 2B) show similar evolution with a moderate increase in temperature, ranging from 10%, for the blend without zinc, to 15% for the blends containing ZnSO4·H2O, regardless of the amount incorporated into the matrix. These results suggest that the zinc salt act as a filler, contributing a moderate increase in the mechanical energy dissipation. The increase in the filler content seems to be compensated by the reduction in protein among the different Zn-containing blends. of an essential micronutrient for a plant (Zn) has been incorporated to the matrix (up to 10 wt.% ZnSO4·H2O). However, the incorporation of the micronutrient Zn, in the form of ZnSO4·H2O, to bioplastic matrices during mixing leads to more rigid and less deformable bioplastic matrices, although these specimens maintain their dimensional stability. In addition, these matrices with Zn involve a significant reduction in water uptake capacity, due to some superimposed effects, including the progressive reduction of protein and glycerol, the negative effect of ionic strength on water absorption and the development of a more integrated enhanced elastic network. In fact, the pore size of these matrices is highly influenced by the presence of the micronutrient since a remarkable decrease in pore size is found when the micronutrient is present. A percentage of ZnSO4·H2O higher than 2.5% does not lead to neither better absorption results (higher water uptake capacity, lower soluble matter loss or higher amount of Zn in the matrix after 24 h absorption,), nor better mechanical properties. Further research is being currently carried out, including the use of different zinc carriers such as carbonate or EDTA quelling agents, as well as new strategies to incorporate the micronutrient into the protein-based matrices, in order to achieve a balance between the zinc loading level and the water uptake capacity. In any case, although a more comprehensive study will be required to optimize the incorporation and release of zinc cations from protein-based matrices, the results obtained in this work open up a great potential for the use of these matrices as a source of supplying micronutrients for horticulture applications. ACKNOWLEDGEMENTS This research is part of a project supported by MINECO, from the Spanish Government (Ref. CTQ2015-71164-P, MINECO/FEDER, UE). The authors gratefully acknowledge their financial support. The authors would also like to thank CITIUS (Universidad de Sevilla, Spain) for providing all access and assistance to Microanalysis and Microscopy Service. REFERENCES References ASTM D570-98 Standard Test Method for Water Absorption Of Plastics (2005) C.S. Araújo, A.M.C. Rodrigues, M.R.S. Peixoto Joele, E.A.F. Araújo, L.F.H. Lourenço Optmizing process parameters to obtain a bioplastic using proteins from fish byproducts through the response surface methodology. Food Packag. Shelf Life, 16 (2018), pp. 23- 30, 10.1016/j.fpsl.2018.01.009 A.K. Bledzki, A. Jaszkiewicz Mechanical performance of biocomposites based on PLA and PHBV reinforced with natural fibres – a comparative study to PP. Compos. Sci. Technol., 70 (2010), pp. 1687-1696, 10.1016/j.compscitech.2010.06.005 V. Bourny, V. Perez-Puyana, M. Felix, A. Romero, A. Guerrero Evaluation of the injection moulding conditions in soy/nanoclay based composites. Eur. Polym. J., 95 (2017), pp. 539-546, 10.1016/j.eurpolymj.2017.08.036 A.A. Cuadri, C. Bengoechea, A. Romero, A. Guerrero A natural-based polymeric hydrogel based on functionalized soy protein. Eur. Polym. J., 85 (2016), pp. 164- 174, 10.1016/j.eurpolymj.2016.10.026 A.A. Cuadri, A. Romero, C. Bengoechea, A. Guerrero Natural superabsorbent plastic materials based on a functionalized soy protein. Polym. Test., 58 (2017), pp. 126-134, 10.1016/j.polymertesting.2016.12.024 A.A. Cuadri, A. Romero, C. Bengoechea, A. Guerrero The effect of carboxyl group content on water uptake capacity and tensile properties of functionalized soy protein- Based superabsorbent plastics. J. Polym. Environ. (2018), 10.1007/s10924-018- 1183-x J.M. Dealy A Practical Guide to Testing and Property Measurement. Vn Nostrand Reinhold Co., New York (1983) European Bioplastics, 2015. Global bioplastics capacities continue to grow despite low oil price [WWW Document]. URL https://www.europeanbioplastics.org/pr_151104/. (Accessed 17 April 2018). M. Félix, J.E. Martín-Alfonso, A. Romero, A. Guerrero Development of albumen/soy biobased plastic materials processed by injection molding. J. Food Eng., 125 (2014), pp. 7-16, 10.1016/j.jfoodeng.2013.10.018 M. Félix, A. Lucio-Villegas, A. Romero, A. Guerrero Development of rice protein bio-based plastic materials processed by injection molding. Ind. Crop. Prod., 79 (2016), pp. 152-159, 10.1016/j.indcrop.2015.11.028 M. Felix, V. Carpintero, A. Romero, A. Guerrero Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding. Polímeros, 26 (2016), pp. 277-281, 10.1590/0104-1428.0044 L. Fernández-Espada, C. Bengoechea, F. Cordobés, A. Guerrero Thermomechanical properties and water uptake capacity of soy protein-based bioplastics processed by injection molding. J. Appl. Polym. Sci., 133 (2016), p. 43524, 10.1002/app.43524 L. Fernández-Espada, C. Bengoechea, F. Cordobés, A. Guerrero Protein/glycerol blends and injection-molded bioplastic matrices: soybean versus egg albumen. J. Appl. Polym. Sci., 133 (2016), p. 42980, 10.1002/app.42980 M. Flieger, M. Kantorová, A. Prell, T. vRezanka, J. Votruba Biodegradable plastics from renewable sources. Folia Microbiol. (Praha), 48 (2003), p. 27, 10.1007/BF02931273 D. Gómez-Martínez, P. Partal, I. Martínez, C. Gallegos Rheological behaviour and physical properties of controlled-release gluten-based bioplastics. Bioresour. Technol., 100 (2009), pp. 1828-1832, 10.1016/j.biortech.2008.10.016 D. Gómez-Martínez, P. Partal, I. Martínez, C. Gallegos Gluten-based bioplastics with modified controlled-release and hydrophilic properties. Ind. Crop. Prod., 43 (2013), pp. 704-710, 10.1016/j.indcrop.2012.08.007 P. Guerrero, A. Retegi, N. Gabilondo, K. De La Caba Mechanical and thermal properties of soy protein films processed by casting and compression. J. Food Eng., 100 (2010), pp. 145-151, 10.1016/j.jfoodeng.2010.03.039 G. Guo, C. Zhang, Z. Du, W. Zou, H. Tian, A. Xiang, H. Li Structure and property of biodegradable soy protein isolate/PBAT blends. Ind. Crop. Prod., 74 (2015), pp. 731- 736, 10.1016/j.indcrop.2015.06.009 ISO 570-2:1993, Plastics. Determination of tensile properties. Part 2: test conditions for moulding and extrusion plastics, 1993. H. Judawisastra, R.D.R. Sitohang, L. Marta, Mardiyati Water absorption and its effect on the tensile properties of tapioca starch/polyvinyl alcohol bioplastics. IOP Conf. Ser. Mater. Sci. Eng. (2017), p. 223, 10.1088/1757-899X/223/1/012066 Y.I. Matveev, V.Y. Grinberg, V.B. Tolstoguzov The plasticizing effect of water on proteins, polysaccharides and their mixtures. Glassy state of biopolymers, food and seeds. Food Hydrocoll., 14 (2000), pp. 425-437, 10.1016/S0268-005X(00)00020-5 X. Mo, X.S. Sun, Y. Wang Effects of molding temperature and pressure on properties of soy protein polymers. J. Appl. Polym. Sci., 73 (1999), pp. 2595- 2602, 10.1002/(SICI)1097-4628(19990923)73:13<2595::AID-APP6>3.0.CO;2-I L. Mortain, I. Dez, P.J. Madec Development of new composites materials, carriers of active agents, from biodegradable polymers and wood. Comptes Rendus Chim., 7 (2004), pp. 635-640, 10.1016/j.crci.2004.03.006 J. Orawan, B. Scoottawat, V. Wonnup Effect to phosphate compounds on gel-forming ability of surimi from bigeye snapper (Priacanthus tayanus). Food Hydrocoll., 20 (2006), pp. 1153-1163 V. Perez-Puyana, M. Felix, A. Romero, A. Guerrero Effect of the injection moulding processing conditions on the development of pea protein-based bioplastics. J. Appl. Polym. Sci., 133 (2016), p. 43306, 10.1002/app.43306 Plastics Europe, 2016. An analysis of European plastics production, demand and waste data [WWW Document]. URL http://www.plasticseurope.es/Document/plastics-the-facts-2016- 15787.aspx?FollD=2. (Accessed 16 January 2018). N. Ramakrishnan, S. Sharma, A. Gupta, B.Y. Alashwal Keratin based bioplastic film from chicken feathers and its characterization. Int. J. Biol. Macromol., 111 (2018), pp. 352-358, 10.1016/j.ijbiomac.2018.01.037 N. Reddy A review on completely biodegradable composites developed using soybased matrices. J. Reinf. Plast. Compos., 34 (2015), pp. 1457- 1475, 10.1177/0731684415573815 P. Saenghirunwattana, A. Noomhorm, V. Rungsardthong Mechanical properties of soy protein based green composites reinforced with surface modified cornhusk fiber. Ind. Crops Prod., 60 (2014), pp. 144-150, 10.1016/j.indcrop.2014.06.010 S. Sharma, I. Luzinov Whey based binary bioplastics. J. Food Eng., 119 (2013), pp. 404-410, 10.1016/j.jfoodeng.2013.06.007 Uchida, R., 2000. Essential Nutrients for Plant Growth: Nutrient Functions and Deficiency Symptoms, in Plant Nutrient Management in Hawaii’s Soils, approaches for Tropical and Subtropical Agriculture. Manoa. J.R. Van Wazer, J.W. Lyons, K.Y. Kim, R.E. Colwell A Laboratory Handbook of Rheology. Interscience Publishers, New York (1963) L.S. Zárate-Ramírez, A. Romero, C. Bengoechea, P. Partal, A. Guerrero Thermo-mechanical and hydrophilic properties of polysaccharide/gluten-based bioplastics. Carbohydr. Polym., 112 (2014), pp. 24- 31, 10.1016/j.carbpol.2014.05.055 J.F. Zayas Water holding capacity of proteins. Springer (Ed.), Functionality of Proteins in Food, Springer, Berlin (1997)