Biodegradability

The added value of these bioplastics is their degradation after their use without releasing toxic substances for crops, which eliminates the need for their removal. This quality was evaluated by burying the bioplastics in farmland and irrigating them daily with 20 mL of water (intensive irrigation simulation of 20 L water/m2). The samples were unearthed at different times, evaluating their disintegration using direct observation.

The used farmland was a special commercial substrate for orchards and fruit trees (Compo, Barcelona, Spain) which contains the ideal ratio of nutrient to the correct crop growth and does not contain microorganisms or pathogens that can alter the tests.

### *2.5. Statistical Analysis*

All specimens were visually analysed prior to testing. In this way, those that presented defects were discarded from the study. The discarded specimens represented less than 10% of the specimens made.

Each analysis was carried out at least three times for each system and all the data were reported with their standard deviation statistically assessed using analysis of variance and Tukey's post hoc test with 95% confidence level (*p* < 0.05) using the statistical package SPSS 18 (Excel, Microsoft, Redmond, WA, USA). The significant differences have been reported with different letters in the corresponding figures.

### **3. Results and Discussion**

*3.1. Mechanical Properties*

### 3.1.1. Flexural Properties

Figure 1 shows the flexural properties of different bioplastics. All the systems have similar profiles in elastic modulus (E') and loss tangent (tan δ), regardless of the micronutrient loading and mould temperature used. In this way, E' increases with frequency at a rate that tends to become constant at high frequency, giving rise to a slope lower than 0.19. This behaviour may be due to an extension of the links that recover instantaneously, not leaving the linear range of deformations in the interval studied. This behaviour is similar to those obtained in other works with similar protein-based bioplastics, looking like a typical response from these materials [35,36].

**Figure 1.** Flexural properties of bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at different mould temperatures: 70 ◦C (**A**), 90 ◦C (**B**) and 110 ◦C (**C**). Elastic modulus (E') and loss tangent (tan δ) profile in frequency interval.

The effect of the nanoparticle content on the elastic modulus depends on the mould temperature. Among the different mould temperatures used, 70 ◦C (Figure 1A) showed no significant differences between the different ZnO percentages used. However, this difference was more remarkable when the mould temperature used was 90 ◦C and 110 ◦C (Figures 1B and 1C, respectively). In this sense, a low micronutrient load (1.0 wt% and 2.0 wt%) increased the E' values, while higher loads (4.5 wt%) reduced it. In this case, at these temperatures, the incorporation of ZnO nanoparticles always induces an increase in the frequency dependence which is more apparent at 90 ◦C. The enhancement with micronutrient load, which is modulated by the mould temperature, could be attributed to the interaction between the nanoparticles and the bioplastic, as reported by other au-

thors [37,38]. At low temperatures (70 ◦C), the nanoparticles, being in small concentrations, did not affect the bioplastics. However, the nanoparticles interacted with each other and with the biopolymeric chains when the temperature increased, improving the mechanical properties of the bioplastics [37]. Nevertheless, this improvement reached a limit, showing no increase in E' values with 4.5 wt% nanoparticles. It can be kept in mind that a higher content of nanoparticles also involves a reduction in the protein content available that may impair the development of the protein network. Therefore, when the amount of filler material increased, it worsened the crosslinking between the biopolymer chains, thus limiting their mechanical properties. Similar behaviours have already been reported in previous studies, where filler materials improved the mechanical properties up to a certain concentration, reducing them at higher concentrations [39,40]. On the other hand, the bioplastics with 0 wt% and 1.0 wt% nanoparticles presented a slight increase in E' values when higher mould temperatures are used, which is the common behaviour in these materials [41]. However, this increase is not observed at higher nanoparticle concentrations (2.0 wt% and 4.5 wt% ZnO).

Regarding tan δ, all systems presented similar values, between 0.2 and 0.35. This indicates that all bioplastics had a strong solid character that was enhanced either with the incorporation of nanoparticles or with the increase of temperature. This behaviour is characteristic in protein-based bioplastic, being found in other works. Thus, Yue et al. (2012) also found this behaviour in cottonseed protein [42], and the pea protein-based bioplastics processed by Perez et al. (2016) show this solid character [43]; Gómez-Heincke et al. (2017) obtained similar results with rice, potato and wheat gluten proteins [44].

### 3.1.2. Tensile Properties

The tensile properties of bioplastics are shown in Figure 2. Firstly, the maximum stress (Figure 2A) increased when higher temperatures were applied, being more notable when the maximum stress started from lower values (0 wt% and 4.5 wt% nanoparticles). Furthermore, 1.0 wt% and 2.0 wt% nanoparticles increased the maximum stress at the same temperature, while 4.5 wt% nanoparticles decreased it. This behaviour is similar to those obtained with flexural properties, although for this parameter it is only significant at the lowest temperature. This evolution reaffirms the hypothesis of some detrimental effect on the development of the protein network caused by an excess of nanoparticles. The strain at break (Figure 2B) shows a similar behaviour as in maximum stress, although in this case, the effect is significant for all temperatures, and 1.0 wt% nanoparticles had significantly the highest values in this case.

On the other hand, Young's modulus presented a different behaviour, which is opposite for the lowest mould temperature. Thus, an increase of mould temperature or content of nanoparticles caused a decrease in Young's modulus, showing no significant differences once the minimum value was reached. This suggests that there is a minimum value of Young's modulus that is not lost regardless of how the bioplastic is processed.

Finally, it is worth mentioning that all bioplastics show strong enough mechanical properties for the suitable transport, storage and distribution of the product, not altering its final functionality and facilitating its production. It is also interesting to point out that these ZnO-containing bioplastics show better mechanical properties than those formulated with zinc sulphate, which is an advantage in this sector [28].

**Figure 2.** Tensile parameters of bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at different mould temperatures (70 ◦C, 90 ◦C and 110 ◦C). (**A**): maximum stress. (**B**): strain at break. (**C**). Young's modulus. Different letters in the bars mean that the values are significantly different (*p* < 0.05).

### *3.2. Morphological Properties*

The morphological properties of the bioplastics with 1.0% ZnO nanoparticles are shown in Figure 3 as the representative behaviour of all systems. Nevertheless, Figure S3 shows the morphology of the rest of the systems. Firstly, the macrographic images of the systems (Figure 3A–C) show that all the bioplastics are homogeneous, presenting a colour change with the increase of temperature. This colour change could be attributed to a higher degree of crosslinking generated by Maillard reactions, which colours the systems towards a tanner brown as the temperature increases. This change has already been observed in previous works [45,46]. However, a temperature change not only changes the macrographic appearance of the bioplastic, since differences in the microstructure are also noticed. In this way, structural differences can be seen in micrographic images obtained using a secondary electron detector (Figure 3A'–C'). The bioplastics processed at 70 ◦C were the only ones that presented porosity in their macrostructure, with cracks appearing in those processed

at 90 ◦C and, above all, at 110 ◦C. This closure of the microstructure caused by temperature is due to the higher degree of crosslinking generated in these temperatures and has already been reported in previous works [35,41].

**Figure 3.** Macrographs (**A**–**C**) and micrographs of bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at different mould temperatures (70 ◦C, 90 ◦C and 110 ◦C), using a secondary electron detector and a scattered electron detector before ((**A'**–**C'**) and (**A"**–**C"**), respectively) and after water uptake capacity (WUpC) tests (**A"'**–**C"'**).

> Furthermore, the distribution of ZnO nanoparticles in the bioplastics can be seen using a scattered electron detector (Figure 3A"–C"). All the systems present a homogeneous distribution of nanoparticles in the bioplastic, which appears as lighter areas within the dark matrix that makes up the bioplastic. These areas with different tonality were corroborated using EDXA as nanoparticles (white) and protein matrix (black) (Figure S4). As can be observed, the increase of mould temperature generates an effect of nanoparticles sintering, which join together, increasing their size [47], and even forming rings in the direction of injection. This behaviour of the ZnO nanoparticles with temperature could explain the structure observed by the secondary electron detector, since an increase in nanoparticle size makes it difficult to join the biopolymeric chains, causing the observed cracks to appear.

### *3.3. Functional Properties*

### 3.3.1. Water Uptake Capacity

Figure 4 shows the water uptake capacity (Figure 4A) and soluble matter loss (Figure 4B) of the different bioplastics. As can be seen, the increase in both temperature and ZnO nanoparticles percentage reduced the water uptake capacity of the bioplastic matrices, causing them to lose their superabsorbent quality. This behaviour could be due to the lower free volume and the greater crosslinking of the systems when mould temperature or nanoparticle percentage is increased, reducing the bioplastics' space to interact with water, forming hydrogen bonds that retain it. This is corroborated using the SEM images

after the water uptake capacity tests (Figure 3A"'–C"'), which show a decrease in pore size and depth. In addition, other research works have already reported this isolated behaviour when increasing the mould temperature [41] or the amount of additive incorporated [37,48] in similar bioplastics. Moreover, it is also worth mentioning that these bioplastics improve the water uptake capacity generated by systems studied for the same purpose, where microstructure salts were incorporated instead of nanoparticles, improving their functionality [27,28].

**Figure 4.** Water uptake capacity (**A**) and soluble matter loss (**B**) of the bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at different mould temperatures (70 ◦C, 90 ◦C and 110 ◦C). Different letters (a,b, . . . , g) in the bars mean that the values are significantly different (*p* < 0.05).

Regarding the soluble matter loss, there were no significant differences between the systems. This indicates that, even if the structure changes, the bioplastics always maintain their integrity by only releasing the incorporated plasticizer (glycerol) and part of the soluble protein.

### 3.3.2. Nanoparticle Release

The profile of water release of nanoparticles is shown in Figure 5. However, only bioplastics processed with a mould temperature of 90 ◦C were shown as representative. As can be seen, all the systems present a quick release at short test times, probably due to the greater difference in concentrations between the system and the medium. This release stabilizes over time until reaching the maximum release time. Furthermore, all profiles adapt to the Korsmeyer-Peppas model with an *n* between 0.1 and 0.3, which indicates that several processes, such as diffusion, disintegration, etc., simultaneously occur during the release, none of them being predominant [49].

Regarding the maximum release time (Table 1), the higher the concentration of nanoparticles, the more prolonged the release over time. This indicates that all the incorporated nanoparticles are released in a controlled way. In addition, this maximum release time is higher than that found when microstructured salts are used instead of nanoparticles [27,50], which indicates that the release is better controlled on this occasion.

**Figure 5.** Accumulation of conductivity in the water release tests of bioplastics with different ZnO nanoparticle concentrations (1.0 wt%, 2.0 wt% and 4.5 wt%) processed at a mould temperature of 90 ◦C.

**Table 1.** Maximum release time of bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at a mould temperature of 90 ◦C and degradation time of bioplastics with different ZnO nanoparticle concentrations (0 wt%, 1.0 wt%, 2.0 wt% and 4.5 wt%) processed at different mould temperatures (70 ◦C, 90 ◦C and 110 ◦C).


### 3.3.3. Biodegradability

Finally, the degradation time of each bioplastic matrix is indicated in Table 1. As can be seen, higher mould temperatures lead to more durable bioplastics, probably due to the better mechanical properties observed with increasing temperature, as is reported in previous works [51]. However, the incorporation of nanoparticles in the bioplastics caused this degradation to be faster, except for the systems with 1.0 wt% nanoparticles. This behaviour could be due to the fact that, when the nanoparticles are released, there are more free holes where the bioplastic is more susceptible to degradation, thus accelerating this process. This behaviour has already been reported by Abdullah et al. (2020) [52]. Figure S5 shows the physical appearance of a bioplastic with 1.0 wt% nanoparticles processed at 110 ◦C. The appearance of bioplastics is similar in all cases, although the lightness of this decomposition changes. It should be noted that, in all cases, the bioplastics decompose into their primary elements (mainly nitrogen), serving as a supplementary fertilizer for the crop and not requiring its removal after use. Furthermore, the degradation time of bioplastics could be modified through the incorporation of nanoparticles and a change in mould temperature, making them very versatile, thus they could be used in all types of horticultural crops.

### **4. Conclusions**

To sum up, soy protein-based bioplastics have shown their grea<sup>t</sup> capacity to hold ZnO nanoparticles and release them in a controlled way. In this sense, two novel lines of grea<sup>t</sup> interest in horticulture (bioplastics and nanobiofertilization) have been brought together, generating interesting synergies between them, and improving the devices investigated so far. Thus, a controlled release biodegradable device is achieved that presents functionality both to release water and fertilizers, as well as to be used as a long-lasting pesticide, having an enhanced efficiency in plants. In addition, these bioplastics have grea<sup>t</sup> versatility to change their characteristics by modifying their composition and processing parameters. In this way, they can be used in different crops, not being necessary to remove them after use. Nevertheless, this is only an initial study, requiring further research to evaluate the functionality of these bioplastics in real and large-scale crops.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2073-436 0/13/4/486/s1, Figure S1: Yield (Ki, A) and crystal size (B) of nanoparticles processed at different ZnCl2 concentrations (0.2, 0.5 and 1.0 M) and ZnCl2:NaOH ratios (0.5, 1.0 and 2.0); Figure S2: Temperature increment (A) and torque variation (B) of raw materials mixed at different nanoparticle concentrations (0, 1.0, 2.0 and 4.5 wt%); Figure S3: Macro and micrographs of bioplastics processed with 2.0 and 4.5 wt% ZnO nanoparticles at different mould temperatures (70, 90 and 110 ◦C); Figure S4: EDXA analyses of the different coloured zones in a bioplastic matrix with nanoparticles incorporated; Figure S5: Biodegradability photographs of bioplastics with 1.0 wt% ZnO nanoparticles processed at 110 ◦C.

**Author Contributions:** Conceptualization, M.J.-R., V.P.-P. and A.R.; methodology, M.J.-R.; validation, V.P.-P., P.S.-C. and A.R.; formal analysis, M.J.-R.; investigation, M.J.-R.; resources, A.G.; data curation, M.J.-R. and V.P.-P.; writing—original draft preparation, M.J.-R., P.S.-C. and V.P.-P.; writing— review and editing, A.R.; visualization, P.S.-C.; supervision, A.R. and A.G.; project administration, A.R.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the "Ministerio de Ciencia e Innovación" of the Spanish Government and FEDER (UE), gran<sup>t</sup> number RTI2018-097100-B-C21.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors acknowledge the "Ministerio de Educación y Formación Profesional" for the PhD gran<sup>t</sup> of M. Jiménez-Rosado (FPU2017/01718). The authors also thank CITIUS for granting access to and their assistance with the microscopy service.

**Conflicts of Interest:** The authors declare no conflict of interest.
