Use of Zinc Oxide Nanoparticles Incorporated in Polybutylene Adipate Terephthalate for Food Packaging. A Focus on the Impact in Functional and Physic-Mechanical Properties and on Migration Thereof

Polybutylene adipate terephthalate (PBAT) and zinc oxide nanoparticles (ZnO NP) are among the most studied when it comes to address biodegradability and antimicrobial properties of materials for food packaging. This work presents a critical review of recent scientific literature with a focus on the impact in functional, physic-mechanical properties,


| Introduction
Bio-based materials, and biodegradables in particular, are generally perceived by consumers as a solution for more sustainable packaging [1].The main applications of biodegradable plastics are currently in (food) packaging, food service ware and shopping bags, that is, low demanding applications, with short contact time and often no need for integrity (sealing).Polybutylene adipate terephthalate (PBAT), a synthetic biodegradable polyester, is the main biodegradable material commercially used today for flexible packaging.PBAT and its blends have raised much attention of researchers and industry.They are targeted in many studies highlighting its biodegradability and dedicating efforts to improving its functionality for food preservation, including antimicrobial active packaging.
Zinc oxide (ZnO) is a metal oxide currently found in daily life applications, including cosmetics (for UV light scattering), medical facilities (e.g., antibacterial paints), dentistry (for blocking microbial leakage) and orthopaedics (as a reinforcing material) [2].ZnO is listed as a Generally Recognised as Safe (GRAS) material by the Food and Drug Administration (FDA) [3].ZnO nanoparticles (ZnO NP) have received a positive safety evaluation from the European Food Safety Authority (EFSA) for packaging applications as transparent ultraviolet light (UV) absorbers based on the absence of a significant migration in particulate forms [4,5].The potential for diverse applications of ZnO NP is due to the variety of shapes, sizes, crystal forms and properties that can be obtained by different synthesis methods [2,6].
ZnO NP have demonstrated antimicrobial properties with potential applications in food preservation after combination in different materials.The incorporation of antimicrobial agents into the food packaging matrix eliminates the direct addition of these to the food in high concentrations.There is a high number of published studies dealing with the synthesis and the antibacterial potential of ZnO nanostructures, indicating the relevance of this topic [7].It is known that the antibacterial activity of ZnO is inversely proportional to the particle size (larger surface area) and directly proportional to the concentration [8].ZnO NP of smaller sizes can easily penetrate bacterial membranes due to their large interfacial area, thus enhancing their antibacterial efficacy.Therefore, controlling the size and concentration of ZnO NP is crucial to achieving the best bactericidal response [9].ZnO morphology is determined by the synthesis conditions and it is essential to control parameters such as solvents, precursor types and physicochemical settings such as temperature and pH [9].
With the increase in nanoparticles (NP) uses and applications, the concern with safety issues also increases.When incorporated in some food packaging matrixes, NP or their constituents can migrate.The impact this will have on the safety and quality of the product is of most importance.The nanomaterials' potential hazard associated with food (oral route) is addressed in the EFSA Guidance [10].However, there is a lack of data to estimate exposure needed for risk assessment [11].NP have a high surface area to volume ratio, and the active surface chemistry of some nanomaterials could give rise to unwanted chemical reactions compared with conventionally sized particles [12].Nanomaterials should be evaluated case-by-case basis and the properties of the NP, such as particle size, physical form and morphology, chemical and photocatalytic activity, should be known and also characterised after being incorporated in the matrix [13].
Polybutylene adipate terephthalate and ZnO are among the most studied when it comes to addressing biodegradability and antimicrobial properties for food packaging.This work presents a critical review of recent developments of PBAT based materials with ZnO NP for food packaging with a focus on the impact on functional, physical and mechanical properties and on migration thereof.The range of concentrations, methods of incorporation in the matrix and the effect of particles size and shape, are addressed, as well as a brief consideration of the impact on the compostability of the materials at the end-of-life.
Most of the works used scanning electron (SEM) and transmission electron microscopy (TEM) to study the morphology and size of the NP. Figure 1   the different synthesis techniques.These can be based on chemical, biological or physical methods.
Table 1 presents examples of ZnO NP shapes obtained by different synthesis methods, according to the literature.Chemical synthesis can occur in liquid phase (e.g., precipitation, sol-gel, hydrothermal and solvothermal, sono-chemical) or in vapour phase (e.g., pyrolysis and inert gas condensation).Physical synthesis includes high energy ball milling and laser ablation.The synthesis of ZnO NP mediated by biological resources and wastes is much less explored [37].Green approaches with plants extracts have received an exponentially growing interest for the ZnO NP synthesis [38].The use of natural components as stabilising agents can be interesting alternative synthesis methods.
A good dispersion of NP in the polymer matrix is critical for the performance of the nanocomposite.Dispersion can be evaluated by analysing the cross-section of the nanocomposite films by TEM and SEM, as well as the compatibility between the polymer and the nanofiller by observing the adhesion at the particle/polymer interface.Excessively dense distribution (too high concentration) would interfere with the continuity of the matrix and negatively affect the properties of the film [39].The concentration of NP influences the dispersibility.Ferreira [40] showed that films with 1 wt% of ZnO NP have better NP dispersion compared with those with 3 and 5 wt% of ZnO NP, which show some aggregations.However, in other studies concentrations up to 10% were reported to be well distributed in PBAT matrix [41].
The neat PBAT film usually exhibits a smooth surface, homogeneous and flexible.When NP were incorporated, the film exhibited a rougher surface and agglomerates on the PBAT matrix [42,43].Increasing the concentration of ZnO increased the size of the dispersed agglomerates of NP.Nanocomposite film surface roughness increased from 98.3 to 135.5 nm by increasing the ZnO NP content from 2 to 5 wt% [43].
ZnO NP can form a crystal in three different forms: hexagonal wurtzite, cubic zinc blende and rocksalt (Figure 2).ZnO hexagonal wurtzite, in which every zinc atom is tetrahedrally coordinated with four oxygen, is thermodynamically the most stable at ambient conditions [44].Cubic zinc blende can be stabilised by growing ZnO on cubic substrates.ZnO will only exist in the rocksalt structure at relatively high pressures [45].ZnO NP usually presents a hexagonal wurtzite structure with very sharp Xray diffraction (XRD) peaks [42,43,46].However, the ZnO can also present broadband without any typical diffraction peak due to water within the nanoparticle structure, which hinders the crystalline structure.Calcination is usually applied to remove residual water and enhance the NP crystallinity [47].
The crystallinity of polymers influences their performance, namely the mechanical and barrier properties, as well as the biodegradation rate.Therefore, it is of interest to understand the effect of ZnO NP incorporation in the PBAT morphology.PBAT films present a semicrystalline structure with a typical XRD broadband pattern.After incorporation of NP, the diffraction pattern showed no significant changes.The crystallinity index (CI) of PBAT increased from 18% to 18.8% and 22.9%, Flower -Talebian et al. [36] Spherical -Rod -respectively, when incorporating 0.5% and 1% of ZnO NP.The ZnO NP presented a CI of 27.5% [47].Therefore, incorporating of ZnO NP seems to increase the crystallinity of the film.It would be interesting to verify this effect on industrially obtained materials as extrusion conditions are known to impact the film crystallinity, due to the inherent chain orientation caused by the die after the extrusion step.
Most of the works reported in the literature have been limited to material development at the laboratory scale only and the solvent-casting method was used to incorporate the ZnO in the film matrix [41-43, 47, 53-57].The average thickness of these films varies between 80 and 140 μm, except for one study that produced thinner films (between 50 and 60 μm) [53].Production methods typically used at industrial level have also been reported, although to a much less extend, namely blown extrusion to obtain films of PBAT with 1 to 5% of ZnO NP [30,50,59] and injection moulding to obtain articles with up to 1.5% of ZnO combined with lignin [46].These industrial moulding techniques follow a step of co-rotating twin-screw extrusion to incorporate the particles in the continuous phase, which is then diluted and mixed during blown extrusion.Melt-blending followed by hot-pressing was also used to produce films and testing strips [58] with a film thickness between 100 and 200 μm.
Although solvent casting is a very practical method for preliminary tests, it would be interesting to explore with more detail the industrially made films and its properties upon incorporation of nanoparticles.
PBAT with ZnO incorporated as a coating over a paper substrate was also studied.The coating solution was applied to kraft paper using a wire bar coater.The thickness of the coating ranged from 52 to 110 μm [52].

| Antimicrobial Activity
ZnO NP have been reported to show antimicrobial activity, reducing the growth of some strains of pathogenic bacteria and spoilage microorganisms, with potential applications, in food preservation.The effects mainly depend on the particle size, shape, concentration and exposure time of the microbial cells [60,61].Three processes have been reported for the action of ZnO NP:release of Zn ions [62], direct contact of ZnO NP with the cells resulting in damaging the microbial cells integrity [63], and the formation of reactive oxygen species (ROS) assisted by the effect of light radiation [64].Some publications indicate that the inhibitory effect of ZnO on Gram-positive bacteria tend to be lower than for Gram-negative.Gram-positive bacteria have a thick cell layer of peptidoglycan in the structure, while Gramnegative bacteria have a thinner layer.Even with an extra layer of lipopolysaccharides in addition to the thin peptidoglycan in Gram-negative bacteria, the cell wall provides less resistance making these bacteria more susceptible to the ZnO effect [65,66].However, it must be recognised that the net effect depends on the species/strain and on several factors that are not always controlled or reported in the literature, making it difficult to generalise.
Escherichia coli is the most common microorganism studied in the PBAT nanocomposites incorporated with ZnO NP alone or in combination with other components such as silver nanoparticles (Ag NP) and lignin [46,47,51,55,58].Generally, the higher the concentration of ZnO incorporated, the greater the antimicrobial effect and the combination of ZnO with other antimicrobial agents was found to be synergic towards inhibiting of the growth of E. coli.The antimicrobial activity against Staphylococcus aureus was also studied and contradictory results were reported.These bacteria were found to be less sensitive than E. Coli to ZnO particles with lignin [56], but other studies reported a higher effect of particles for S. aureus [39,41,42].Another study showed the same inhibitory effect between E. coli and S. aureus when assessed by the zone of inhibition method, with the halos presenting 13 mm of diameter [54].
ZnO in PBAT matrixes showed less antimicrobial activity against the Listeria monocytogenes (Gram-positive) than against the Gram-negative bacteria E. coli [52,53].Seray [43] demonstrated that the inhibitory effect was higher for  Bacillus subtilis compared with Pseudomonas aeruginosa and S. aureus.

PBAT/TPS films with an intermediate concentration of 4%
ZnO NP showed better antimicrobial effects against total viable count, lactic acid bacteria and yeast and mould, compared with the films containing lower (1-3%) or higher (5%) ZnO NP [50].
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by microdilution method.ZnO NP with reduced graphene oxide (rGO) showed the lowest MIC values against E. coli and S. aureus (3.75 μg mL −1 ) when incorporating the maximum content of rGO (5%).The MBC value was consistent with results decreasing from 25 to 6.25 μg mL −1 for both pathogens with the increase of rGO concentration.Xiao [56] showed that the MIC of lignin-ZnO (LZn) hybrid particles, for the same pathogens, is lower than lignin nanoparticles (LNP) due to the presence of ZnO.It also demonstrated that the antibacterial activity of the hybrid particles gets higher, with the increasing content of ZnO NP.
In practically none of these studies, the different morphologies and sizes of ZnO NP were compared.Usually, only one type of ZnO NP were incorporated into PBAT (sometimes commercially available particles) and the particles were not fully characterised.
It is important to note that many of these studies applied different methods to assess antimicrobial activity, such as the disc diffusion assay and the viable colony count method, among others, making the comparison between them difficult.Soares Silva et al. [65,66] provided a more comprehensive study using different species and strains, and different methods to assess antimicrobial activity, making comparison possible.
Most of the studies have determined the effect of ZnO NP and not of the effect when the NP is incorporated in the film and/ or in films produced by industrial processes, where the actual performance may be different.Additionally, the behaviour of the films in situ (i.e., when actually in contact with the food) may differ from the behaviour in vitro.Only a few works have studied the impact of these materials in contact with real foods [48,49,51,67].A recent study combined ultraviolet-C lightemitting diodes (UV-C LED) irradiation and antimicrobial PLA/ PBAT packaging film with ZnO NP and tested its performance as packaging for fresh-cut vegetables.The films showed higher in vitro antibacterial activity against E. coli than S. aureus, although the activity was considered to be relatively low due to the slow release of ZnO entrapped by the polymer.Nevertheless, the combined system showed a significant synergistic effect in reducing the bacteria during storage, with a reduction of about 2 log CFU g −1 of E. coli in onion, cabbage and carrot after 7 days of storage at 10°C [48,49].PBAT/PBS films incorporating ZnO at levels higher than 2.7% were shown to delay discoloration of red pigments in packaged minced pork due to efficient inhibition of microbial growth and UV blocking [51].The incorporation of ZnO NP (together with titanium dioxide) into low-density polyethylene (LDPE) films has also been reported.The bactericidal efficiency tests resulted in the prevention of E. coli growth in fresh calf minced meat [67].

| Other Functionalities
The antioxidant activity of packaging films is important for extending the shelf-life of many packaged foods.Films of PBAT/PLA with 3% ZnO NP exhibited limited antioxidant activity, with results of 4.4% for the 2,2′-azino-bis(3-ethylbe nzothiazoline-6-sulfonic acid) (ABTS) test and 6.9% for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity [48,49].Films of PBAT with LZn hybrid particles incorporated at levels of 1%, 2% and 3% showed a radical scavenging activity value (RSA) via DPPH assay, respectively, of 10.6%, 17.6% and 32.5%, relative to the PBAT [56].However, it is not possible to discriminate the effect of the ZnO itself from the lignin, which is known to be antioxidant.PBAT/TPS films with incorporation of ZnO NP showed a reduction of approximate 50% of the lipid oxidation value determined by the TBARS test (thiobarbituric acid reactive substances) in relation to the control [50].No significant differences were verified for the films with different concentrations of ZnO NP incorporated (1-5%).
However, the study does not include information regarding the conditions of exposure to light of the experiment.

| Effect of Size and Shape on Functional Properties of Films
The functional properties of ZnO NP depend on their physical properties, namely on morphology, particle size and specific surface area.These are determined by the synthesis method and conditions used to obtain the particles.Several authors reviewed the physical characteristics of ZnO NP of different morphologies and sizes obtained by different methods [48,49,68,69].
However, only a few studies have assessed the impact of the morphology and size of ZnO NP on their antimicrobial activities.These two parameters are often not independent.Usually, the spherical particles are reported to be more active.The spherical particles are almost always the smallest particles in the studies and consequently have the highest surface area-to-volume ratio.
When studies compared NP with different morphologies, generally those that were more toxic to microorganisms were also smaller.Although morphology is a very important parameter, the antimicrobial mechanisms of ZnO NP are size-dependent and in general, when the particle size decreases, its antibacterial activity increases [48,49,53,69].
Stanković [70] studied the influence of ZnO NP shape and size against E. coli and S. aureus, concluding that spherical particles (with around 30 nm) demonstrated the highest microbial cell reduction rate when compared with hexagonal prism particles (1 nm in length and 100 nm in diameter) and ellipses particles (length of 500-600 nm and diameter of 100 nm).Particles with cuboidal/rod shape of ZnO NP (size of 40-45 nm) were found to have higher antibacterial activity as compared with spherical (size of 60-180 nm) and hexagonal NP (size of 63 nm) [38].However, in this specific study [38], the spherical shape presents a wide range of sizes (60-180 nm), and the cuboidal/rod shape is the smallest (40-45 nm).
The effect on the antimicrobial activity was the most studied, but the photocatalytic activity was also addressed and largely influenced by the shape [38].The photocatalytic activity (PCA) was studied through the degradation of methyl orange dye at 465 nm over time (Figure 3).Spheric-shaped particles showed the lowest PCA for all doses.The spherical-and hexagonalshaped particles showed higher PCA at intermediate doses, a pattern different than the one presented by the cuboidal NP that increased with the increase of the mass of NP in the range tested.The different morphologies and active surface area may cause different degradation patterns due to interstitial space that mediate the degradation rate.
Most of these studies focused on the activity of the particles per se.However, the incorporation of the particles in the packaging matrix (e.g., a film) may also play a role because the physical properties may interfere with the mechanism of action of the ZnO.Tamimi [71] studied the effect of particle morphology (spherical and rod shape) on some properties, including the antibacterial properties of the particles incorporated in starch-based film.Spherical particles not only revealed the best antibacterial effect but also had the best behaviour on mechanical, physicochemical and barrier properties.Rod shape particles showed the highest impact in UV transmission.
Not addressing the impact of different morphologies and sizes in the final properties of the film is a major limitation regarding the studies of functional properties of ZnO/PBAT packaging matrix.

| Thermostability
Most of the biopolymers exhibit low thermal stability because of their weak chemical bonding.The incorporation of fillers, namely metal and metal oxides, is a common approach to improving the thermal properties of nanocomposite films [72].
Most of the studies reported that thermal stability is enhanced by the incorporation of the ZnO NP, compared with that of pure PBAT, resulting in an increase of the temperature of the thermal degradation, although no significant differences are observed when comparing different percentages of ZnO NP incorporation [59].The endothermic peaks of the differential scanning calorimetry (DSC) thermograms (melting) were recorded at higher temperatures for PBAT/starch incorporated with ZnO NP [39] and PBAT film with ZnO/rGO [42].
However, opposite results were also reported indicating that the addition of ZnO NP reduces the thermal stability of PBAT and that the effect increased with the percentage of ZnO [47,55].The effect was attributed to the increase of the oxidative decomposition of the polymer at the ZnO NP interface at high temperatures, accelerating the thermal degradation of the composites.

| Mechanical
The mechanical behaviour of the nanocomposites is one of the most important properties for packaging applications.The incorporation of nanoparticles in the matrix affects mechanical properties of the film because of interface interactions between the NP and the polymer molecules [39,72].
In general, the incorporation of NP improves the mechanical properties [41,51,53,54].The tensile modulus and the yield strength of the film were improved with the incorporation of lignin-ZnO hybrid particles [56] and with the incorporation of ZnO with rGO [42].Possible synergistic effects of two nanoparticles incorporation, adding Ag NP, were also reported [39,47].
The effect of NP concentration on the mechanical properties is not linear, and some studies show an optimal intermediate concentration for which the mechanical properties are the best [57,58].The tensile strength and elongation at break values decreased when ZnO was added up to 3%, showing a minimum strength, and increased with the addition of ZnO in concentration from 3% to 5% [50].These results suggested that adding NP into the polymer matrices at a certain concentration decreased the adhesion between the polymer network, causing reduced mechanical strength, while the agglomeration of NP at higher concentrations of NP reduced the dispersion of finer nanoparticles [55].The elongation at break of the PBAT film decreased with the increase in the ZnO content (1, 3 and 5 wt%) [55].The tensile strength before the break point increased with the addition of ZnO NP but the film showed a breakpoint at lower elongations when ZnO is incorporated.The yield strength seems to decrease with the incorporation of 1 wt% but then tends to increase for higher loads.Nevertheless, these conclusions are tentative because the results dispersion was not provided so the statistical significance cannot be evaluated.

| Optical
Many packaged foods are significantly affected by light, which can cause organoleptic changes, browning and oxidative degradation.Quality changes in foods result from light exposure, particularly UV light, which has high energy.UV irradiation, especially in UV-A (320-400 nm) and UV-B (290-320 nm) regions, is one of the key factors affecting food spoilage and nutrient loss, which greatly accelerated oxidation [50,73].
ZnO NP is a white powder that can reduce the transmission and transmittance and, therefore, enhance the optical properties of the biodegradable films.The UV-light shielding ability and transparency of the composite films can be determined by measuring the transmittance at 280 nm and 660 nm [53].
Studies on the light transmission of PBAT/TPS films indicated that the addition of ZnO NP reduced the light transmission, mainly in the UV region (below 380 nm): films containing 4 and 5% of ZnO present less light transmission than films with 1% ZnO NP incorporation, and when compared with the neat PBAT/TPS film [50,59].Similar results were obtained with films of PBAT/ PBS in UV region below 280 nm [51].So, the incorporation of ZnO NP clearly provided UV barrier properties as the NP dispersion prevented light transmission through films.The increase in the ZnO NP incorporation improved the haze and decreased the transmittance of the PBAT film: the haze and transmittance of neat PBAT film is 17.3 and 86.4,respectively, and after incorporation of 5% ZnO NP, the haze value increased to 81.5% and the transmittance decreased to 74.6% [55].However, other studies reported that the incorporation of ZnO NP did not affect the transmission in the UV light region, meaning that the high UV barrier properties of PLA/PBAT film were derived from PBAT polymer [53].
The UV-VIS spectra of neat PBAT/PLA blend film demonstrated high absorption of light at wavelengths below 300 nm, decreasing with the increase of the wavelength, confirming the transparency to visible light [53].PBAT and PLA/PBAT films showed absorption at wavelength lower than 300 nm and upon ZnO NP was incorporated, the composite films exhibited strong absorption peaks in the region of 360-376 nm due to the absorption of light by ZnO NP [39,43,53].The light absorption of both neat and PLA/PBAT-ZnO NP films gradually decreased above 400 nm, but the light absorption intensity of PLA/PBAT-ZnO NP composite film was higher than that of neat film.This can be due to the crystalline properties of ZnO NP that cause light scattering, which may result in lower light absorption in the visible light range.On the other hand, some reflection of the light can occur at the interface between ZnO NP and the matrix due to the large specific surface area of ZnO NP [53].
In the visible light wavelength, the incorporation of ZnO NP decreased the transmission values in line with the reduction of transparency in this region of the spectra [53].Furthermore, the transparency to visible light of the nanocomposite films is highly affected by the distribution of the fillers while aggregations and molecular arrangements of the NP structures modify the amorphous-crystalline fraction of the polymers [74].
The colour of the PBAT films is also affected by the incorporation of NP like TiO 2 , SiO 2 and ZnO.The Hunter colour values L, a, b (according to ASTM D2244) showed an increment in (a*) redness and (b*) yellowness [53,54].

| Permeability and Wettability
Permeability to water vapour (WVP) and oxygen (OP) are barrier properties that greatly affect the quality and shelf life of the packaged products.One of the main goals when adding nanomaterials to biopolymers used for food packaging is to improve their barrier to moisture and oxygen.There are many factors that affect permeability: shape, size and the compatibility between the polymer matrix and the nanoparticle, as well as microstructures and morphology of the matrices [39,41].
PBAT itself has low barrier properties with reported values for oxygen permeability of 84.0 cm 3 mm m −2 day −1 bar −1 determined at 23°C and for water vapour permeability of 3.1 g mm day −1 m −2 determined at 23°C and 50% of relative humidity [75].These values correspond to 9.7 × 10 −12 cm 3 m m −2 s −1 Pa −1 and 25 g m m −2 s −1 Pa −1 , respectively, when normalised.At the barrier measuring temperature, the material is well above its glass transition temperature (ca.−34°C) where high polymer chain mobility and free volume allow for high rate of gas transfer.
The barrier properties of PBAT/ZnO NP nanocomposites have been addressed in a few studies, with matrices containing PBAT with other components, such as PLA [53,54] or TPS [50].Results from different studies were compiled in Table 2 after uniformisation of the units.The films were obtained by different processes (blown extrusion or solvent casting) and the conditions for the permeability measurements (temperature and relative humidity) were also different.For these reasons, the comparison between the results is difficult.
Publications show that the WVP and OP of the nanocomposite films produced in the laboratory by solvent casting are slightly reduced with the incorporation of ZnO NP as compared with neat PBAT film [41,42,53,54].However, in other works a slight increase in WVP was recorded when adding ZnO NP [48,49].The incorporation of 1% ZnO NP in the PLA/PBAT matrix decreased the WVP value by ca 20%, from 192 to 152 g m −2 day −1 [54].This has been attributed to the increase in tortuosity of the water molecules path increasing the crossing time through the composite film layer [50,58].However, measurements in blown extruded films showed that the addition of ZnO NP increases the value of WVP from 1.4 × 10 −12 g m m −2 s −1 Pa −1 to between values ranging from 1.7 and 2.4 × 10 −12 g m m −2 s −1 Pa −1 when ZnO NP is incorporated at 1-5% [50].
Some studies show that the effect of concentration is not linear and that there is an optimal concentration of incorporated ZnO NP for which barrier properties present the best performance.WVP results increased from 45 to 60 × 10 −12 g m m −2 s −1 Pa −1 when incorporating ZnO NP between 0-1%, although lower values are obtained in the intermediate concentrations [58].
The results for the impact on OP of adding ZnO NP to the PBAT film also depend on the film production technique.For films produced by extrusion, an increase in OP was observed of ca 15% in values measured at 23°C and 50% RH [50] and an increase of 50% in values measured at 38°C and 90% RH [39], upon incorporation of 1% of NP. Results from other authors show a positive effect of adding ZnO NP in films produced by solvent casting with a decrease in OP of 5-10% [41,48,49,54] when incorporating ZnO NP 1 wt %.
It is recognised the difficulty to compare results from these studies because of the different experimental conditions (film production, temperature, relative humidity, type of matrix and components incorporated).Additionally, the precision in the different concentrations of incorporation (e.g., differences of Contact angle (CA) is a measure of the surface wettability and hydrophobicity.Films with CA at 90° and above are considered hydrophobic surfaces [76].Usually, PBAT alone is nearly hydrophobic.Adding ZnO NP decreases the value of CA and gives a heterogeneous surface that increases wettability explaining what was mentioned before regarding the WVP and OP results [50,53], but adding a low concentration of nano-ZnO has little effect on the hydrophobicity of the material [57].

| End of Life
PBAT-based films are among the most used in the increasing market for biodegradable plastics.Biodegradable films can be gradually decomposed in soil by microbial enzymatic actions and bioassimilation [77].Biodegradable materials are associated with poor physicochemical and mechanical properties such as low barrier properties, low stability during processing and storage and high-water sensitivity.These properties may not suffice the required protection and shelf-life specifications as under today's standards of distribution and supply chains, resulting in shorter shelf-life and increased food losses.Therefore, the benefits of using biodegradable materials as food packaging need to be critically evaluated using recognised tools, such as life cycle assessment (LCA).Furthermore, it is known that the incorporation of active compounds such as nanoparticles, can improve these properties [78] despite there are limited studies focused on interactions between the polymers/nanocomposites and biodegradability.
The biodegradability of PBAT with ZnO NP incorporated at concentrations varying from 0.2-1% was evaluated following the weight loss in soil burial tests for 120 days.At the lowest level of incorporation, a slight reduction in the degradation rate was observed compared with the neat PBAT.However, the statistical significance of this decrease is not discussed.The increase in the degradation rate with the incorporation of NP is more noticed only at 0.6% and higher levels [58].It is possible that low levels of incorporation yield better dispersibility, which can result in more difficulty to penetrate the matrix by microorganisms.However, it should be considered that studies with sound experimental design are required to obtain results statistically significant.

| Effect on Migration
Consumer tends to consider bio-based materials safer than conventional plastics and these natural-based alternatives appear to be a sustainable alternative to conventional plastics.However, their toxicity was compared, presenting similar results [79], which may be a surprise and a disappointment for many.Most often the bio-based materials are evaluated regarding the environmental aspects (such as carbon footprint, end of life) and the impact of the components on safety issues upon human exposure is often neglected.Migration of nanomaterials from packaging into food is a mass transfer phenomenon and can occur by diffusion, dissolution and abrasion of the packaging surface, causing chemical contamination, affecting the organoleptic properties, and may have negative effects on human health [80].Uncertainties on the possible health effects and long-term safety of NP when used in food packaging is an important concern, as there are still few studies regarding their toxicity associated with the ingestion, absorption, distribution, metabolism and excretion after oral exposure, and potential interactions of nano-based material with food components [81].
NP have a large surface area-to-volume ratio and may exhibit different physicochemical properties than the bulk-sized material.Consequently, the mechanisms of mass transfer, interaction with the supporting materials and with the food, as well as the potential toxicity may be different [1].The migration depends not only on the NP type, concentration, size, shape and polymer viscosity, molecular weight and solubility but also on the migration conditions (contact time, temperature) and food composition [82].
According to Regulation (EU) No. 10/2011 [83] and its amendments safety of NP should be assessed on a case-by-case basis considering the physicochemical characteristics and the toxicological profile of the migrate [1].For metallic NP it is important to determine if the migration occurs in nano or ionic form to understand the toxicological risk.The number of reports on the toxicity of ZnO NP has been increasing in literature with studies addressing cytotoxicity, genotoxicity, hepatotoxicity, pulmonary toxicity, neurotoxicity and immunotoxicity [84].ZnO NP are studied for their antimicrobial effect, and therefore, it can be expected to also have an effect on microbiome human cells.Creating a framework to predict the toxicity of ZnO NP is challenging due to the diversity and complex nature of the available literature and more studies regarding the effects of NP ingestion on the gastrointestinal tract and other organs of human body are needed.
Migration of ZnO NP can occur through the ionic form (Zn 2+ ) or the whole particle (ZnO NP) as schematically represented in Figure 4. Considering the diffusional properties of NP in polymers and the solubility characteristics of the ZnO NP, ESFA concluded that the substance does not migrate in nanoform.Therefore, the safety evaluation should focus on the migration of soluble ionic zinc (Zn 2+ ) from packaging materials.
Even if migration would occur in the particulate form, this would be dissolved immediately into ionic zinc upon contact with acidic foods or stomach acid [4].The specific migration limit (SML) for soluble ionic zinc is 5 mg kg −1 food) set out by the European Regulation.Regarding safety assessment, EFSA recommends that the Commission impose an exposure upper limit of zinc of 25 mg per person per day [4].
Migration experiments indicate that when NP are completely embedded in the host polymer and the contact surface is not altered by mechanical stress, NP do not have potential to migrate into food as they may be considered immobilised [85].In cases where NP are not fully embedded but are protruding from and sticking out of the polymer surface, there is a probability of release into food that needs to be considered for risk assessment.Such cases may occur when NP are incorporated at very high use levels in the food contact layer or when the nanocomposite was manufactured under poor technical production conditions.Therefore, it is important to verify whether the NP is fully embedded or freely present at the food contact surface of the nanocomposite [86].It was concluded that spherical shapes of carbon NP would be the worst-case geometry for diffusion in polymers (quicker diffusion than nonspherical objects due to the geometry) and that only sizes up to 2-4 nm in diameter could migrate [87].
Experimental migration results reported until now are limited and contradictory, reflecting experimental challenges [81].
There are already well-established protocols, especially for the case of metals, to quantify concentration of the elements, such as the wet digestion followed by ICP-MS.However, to distinguish between migration of the dissolved form of the particle and migration of the actual particle elemental techniques only are not enough.Microscopic techniques SEM and TEM, light scattering based such as multiangle light scattering detection (MALLS) are used to characterise the particles after separation by techniques such as Asymmetric-Flow Field Flow Fractionation (AF4) and Centrifugal Field Flow Fractionation (CF3).These altogether are needed to detect and characterise NP and give information on the aggregation state of the particle [81,88].The migration of carbon black NP out of plastic materials was investigated and AF4 and MALLS were used to separate, characterise and quantify the potential release of NP.It was concluded that the carbon black does not migrate into food once it is fully embedded in the plastic matrix [89].Concerning the specific migration (SM) of the Zn 2+ , the contact conditions and food simulants for the migration tests have been established by Regulation (EU) No. 10/2011 [83] and its amendments.It is known that migration of Zn into food simulants may overestimate the migration into real food.
Migration of ZnO NP has been studied using a variety of matrices, including polypropylene (PP) [90], LDPE [91][92][93][94] and polylactic acid (PLA) [95], among others.Migration of Zn 2+ from PBAT matrix has been much less studied.Studies related to the migration of the whole particle of ZnO NP have not yet been reported.Therefore, research applying adequate detection and quantification techniques is needed.Table 3 summarises the characteristics of the studies analysed, regarding PBAT/ZnO nanocomposites.
Migration of Zn 2+ increases with the increase in concentration of the particles in PBAT [43,56].Increasing the % the rGO, which acts as a shield, decreased the Zn migration in all simulants [42].The migration is, as expected, the highest for the acetic acid simulant representing acidic food due to the solubility characteristics and induction of changes in the polymer microstructure [59].
The migration profile of Zn 2+ release from PBAT/ZnO films into food simulants was performed and different mathematical models were fitted to the data to describe the release kinetics [43].The release of Zn 2+ ions occurred progressively without reaching equilibrium (Figure 5).An initial linear migration pattern was observed up to around day 5, with a rate that showed not highly affected by the concentration of ZnO incorporated.However, the concentration did affect particularly the level of migration at the longer contact times.At the end of the experiment, more than 70% of the entire load of Zn 2+ was released from the PBAT matrix due to erosion of the surface.
The Higuchi and Korsmeyer-Peppas models were used to describe the kinetics of the Zn 2+ release from PBAT/ZnO nanocomposite films [43].These models have been used to describe drug release from pharmaceutical dosage forms, particularly for controlled-release or sustained-release formulations.Both models described well the release of Zn ions for all concentrations of incorporation presenting high correlation coefficients (R 2 ).
Most migration studies have been performed with the simulants and research on the migration of NP into actual model foods is limited.The migration of Zn 2+ into a food model (chicken skin) was determined but the matrix was bacterial nanocellulose nanocomposite [65,66].It would be interesting to verify the migration into a real food in contact with PBAT/ZnO packaging films.
Overall migration (OM) is another parameter required for materials food contact compliance, with a European Union's plastic regulation limit of 10 mg dm −2 (corresponding to 60 mg kg −1 food).OM from PBAT/TPS films was the highest into 3% acetic acid and increased linearly with the increase of ZnO NP concentration (0-5%) with values between 0 and 13 mg dm −2 , respectively [59].The migration into ethanol 10% did not increase with the ZnO NP content and the migration into water was not significantly different than that from the film without incorporation of ZnO.The OM values of films containing 5% ZnO exceeded the limit when in contact with 3% acetic acid simulant and therefore, these films have limitations in the application for packaging acid foods [59].Similar results were obtained in  PBAT/starch-based films with ZnO NP: the OM limit was exceeded due to the release of starch, although the migration limit of zinc was also exceeded [96].

| Conclusions
The purpose of this review was to assess the potential of ZnO NP incorporation in PBAT films for food packaging industry, considering the several properties required to protect and preserve the food.A great amount of research was reported regarding the impact on functional, physic-mechanical properties and migration characteristics.In this review, the range of concentrations, incorporation methods and the effect of particles size and shape are addressed as well as a brief consideration of the impact of ZnO NP on the PBAT-based films biodegradability.
All properties of the materials addressed in the studies covered in this review are affected by the incorporation of ZnO NP.In general, the properties of the PBAT/ZnO nanocomposites are improved, but in some cases, it occurs the opposite, or there is an optimum intermediate concentration.Unfortunately, many studies did not provide information on variability between replicates, which highly limits the interpretation of results due to lack of statistical significance.
In most of the studies, the NP are not fully characterised particularly regarding the morphology and size.The polymeric matrix is also not always fully described or consists of blends of different plastics.Therefore, contradiction between results is often found which cannot be completely justified.Very relevant is that most of the studies are based in PBAT materials prepared at laboratory scale by solvent-casting which is highly convenient but not representative of the industrially made films which suffer the impact of the extrusion process and impact in chain orientation.This is known to have an important effect on properties.
On the other hand, other relevant properties such as thermosealing of the films was not addressed in the studies.Therefore, proper validation of the developments is needed.
The active properties (antimicrobial) have been investigated in the studies but applying in vitro strategies, and only a few works have addressed the impact of these materials in contact with real foods.These studies are needed to clarify the efficiency and efficacy of the functional activity of the active materials.
The migration studies of the whole particle or ionic form of ZnO NP incorporate in PBAT are scarce.Further investigations are needed to achieve at an equilibrium of ZnO NP incorporation and its properties be effective but taking in note the concerns regarding the toxicity and health issues when applied in food packaging.

FIGURE 4 |
FIGURE 4 | ZnO NP migration: In the ionic form (Zn 2+ ) or as the whole particle (ZnO NP); corresponding techniques for detection and quantification.

TABLE 1 |
ZnO methods and resultant size and shape.
10991522, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pts.2816by Cochrane Portugal, Wiley Online Library on [15/05/2024].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10991522, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/pts.2816by Cochrane Portugal, Wiley Online Library on [15/05/2024].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 0.2 wt% in series of increasing concentration) is also arguable if one takes into consideration the variability of the industrial processes and the fact that many studies do not provide standard deviations between replicates.

TABLE 2 |
WVP and OP values reported in the literature for PBAT-based films with ZnO NP incorporation.

TABLE 3 |
Studies of migration of NP from PBAT food packaging.