Antioxidant and cell-friendly Fe2TiO5 nanoparticles for food packaging application

This is the peer reviewed version of the paper: Rizzotto, Francesco, Vasiljević, Zorka Ž., Stanojević, Gordana, Dojčinović, Milena P., Janković-Častvan, Ivona, Vujančević, Jelena, Tadić, Nenad B., Branković, Goran, Magniez, Aurélie, Vidić, Jasmina, Nikolić, Maria Vesna, "Antioxidant and cell-friendly Fe2TiO5 nanoparticles for food packaging application" in Food Chemistry, 390 (2022):133198, https://doi.org/10.1016/j.foodchem.2022.133198. This work is licensed under a Creative Commons Attribution Non Commercial No Derivatives 4.0 license . 2 Antioxidant and Cell-friendly Fe2TiO5 Nanoparticles for Food Packaging Application Francesco Rizzotto1, Zorka Z. Vasiljevic2, Gordana Stanojevic2, Milena P. Dojcinovic2, Ivona Jankovic-Castvan3, Jelena D. Vujancevic4, Nenad B. Tadic5, Goran O. Brankovic2, Aurélie Magniez1, Jasmina Vidic1,*, Maria Vesna Nikolic2,* 1 Université Paris-Saclay, Micalis Institute, INRAE, AgroParisTech, 78350 Jouy-en-Josas, France 2 University of Belgrade - Institute for Multidisciplinary Research, 11030 Belgrade, Serbia 3University of Belgrade, Faculty of Technology and Metallurgy, 11000 Belgrade, Serbia 4Institute of Technical Sciences of SASA, 11000 Belgrade, Serbia 5University of Belgrade, Faculty of Physics, 11000 Belgrade, Serbia Correspondence: J.V. [email protected] and M.V.N. [email protected] Alginate solution Fe2TiO5 nanoparticles Alginate/ Fe2TiO5 composite film Biocompatibility tests Strong antioxidant activity Migration test No cytotoxicity 3 Abstract An emerging technology of active packaging enables prolongation of food shelf life by limiting the oxygen transfer and the reactivity of free radicals, which both destruct food freshness. In this work, Fe2TiO5 nanoparticles were synthesized using a modified sol-gel method and evaluated as an enforcement of alginate food packaging film. Pure phase Fe2TiO5 nanoparticles had an average particle size of 44 nm and rhombohedral morphology. Fe2TiO5 nanoparticles induce no cell damage of human Caco-2 epithelial cells and show no inhibitory effect towards growth of a panel of bacterial strains, suggesting good biocompatibility. Films obtained by incorporation of Fe2TiO5 nanoparticles into alginate using the solvent casting method show no migration of iron or titanium ions from films to food simulants again suggesting their safety as a packaging material. Fe2TiO5 nanoparticles also showed strong antioxidant efficiency as determined using the DPPH˙ assay, and confirmed further in a preservation test on fresh fruit. Keywords: Fe2TiO5 nanoparticles; alginate film; composite film; antioxidant active; biocompatibility. 1. Introduction Metal oxide nanoparticles (NPs), with a diameter ranging from 1-100 nm, have gained a lot of attention in the last decade in various fields ranging from catalysis, photocatalysis, sensors, biomedical applications and agri-food industry (Kannan et al. 2020; Nikolic et al. 2021). NPs used as food additives and food-contact materials have potential to improve quality and extend food shelf-live but need to be safe, cost-effective and eco-friendly. Potential risks of nanoparticles have aroused serious concerns of the public and in academia and have emphasized the need for engineering biocompatible inorganic nanomaterials. Plastics are still widely used in food packaging due to their cheapness and longer durability. However, plastics cannot be recycled and cause an environmental crisis in the whole world (Ncube et al. 2020). Active packaging based on biocompatible polymers and metal oxide 4 NPs is under development in order to replace petroleum-based plastics and reduce the usage of preservatives (Peighambardoust et al. 2019; Priyadarshi and Rhim 2020). Alginate, as an alternative to plastics for food packaging, is a biocompatible, non-toxic, and an inexpensive biodegradable water-soluble polysaccharide, composed of mannuronic and glucuronic acid molecules bound by 1–4 glycosidic bonds. It can react with diand trivalent cations forming crosslinks and becoming water-resistant which is of great importance for food packaging (Külcü 2020; Omerović et al. 2021). In addition, alginate exhibits low permeability to oxygen and vapours, flexibility, and good tensile strength (Jost et al. 2014). Metal oxide nanoparticles incorporated into alginate reinforced alginate packaging film through providing a strong antimicrobial activity, UV barrier, ethylene scavenging and smart sensing capacity (Nikolic et al. 2021). For instance, hydrothermally prepared Au-TiO2 nanocomposites were dispersed into alginate solution and showed improved water resistance and antibacterial activity (Tang et al. 2018). Halloysite functionalized with ZnO nanoparticles have been incorporated into alginate films and showed a significant increase in the mechanical, water vapor barrier and UV light barrier properties (Shankar et al. 2018). Recent investigations have also included application of a NiMn2O4/alginate nano-biocomposite in temperature sensing (Dojcinovic et al. 2021), or ZnMgO NPs/alginate in extending shelf life of smoked salmon (Vizzini et al. 2020). Even though the properties of metal oxide NPs are useful for food packaging, there is still a lack of using these nanomaterials regarding their changed physical and chemical properties compared with bulk materials, giving them the potential for causing a health risk to humans (Heo et al. 2020). Since May 2021, one of the most used metal oxide NPs in the food industry, TiO2, has been banned from all food products in the Europe under the recommendation of the European Food Safety Authority (EFSA). Since June 2021, TiO2 is no more considered as safe for use in animal 5 feed. On September 28, 2021 the Section: Novel Food and Toxicological Safety of the Food Chain of the Standing Committee on Plants, Animals, Food and Feed of the European Commission conducted an exchange of views and possible opinions on the timeline of the planned ban of TiO2 food additive (E171). Pure TiO2 NPs were shown to accumulate in the body after ingestion and to be potentially genotoxic (Luo et al. 2020). There is a need to replace TiO2 by safer material for food industry applications. Mixed metal oxide NPs, providing synergic physicochemical properties of their components and decreased toxicity are under intensive investigation for biological applications (Nikolic et al. 2021; Stankic et al. 2016). In the present work, we for the first time evaluate possible applications of Fe2TiO5 as a biocompatible food packaging material for alginate reinforcement. Fe2TiO5 (known as iron titanate or pseudobrookite) is a mixed metal oxide and chemically stable crystal (Vasiljevic et al. 2020a). It naturally occurs in titanium-rich volcanic rocks, and could be a good replacement for TiO2. Fe2TiO5 has been identified as an intermediate between Fe2O3 and TiO2 as it inherits a similar electronic and atomic structure to TiO2 whilst its energy band gap is similar to Fe2O3 (2.2 eV). Recent research has focused on applying Fe2TiO5 as a promising photocatalyst material in natural sunlight for removal of pollutants, such as organic dyes (Vasiljevic et al. 2020a), photoanode for photoelectrochemical water splitting (Lee et al. 2020) or in gas sensing (Nikolic et al. 2018) but has not been evaluated in biological and agri-food applications. In view of promising photocatalytic properties of Fe2TiO5 and proven radical scavenging activity of TiO2 (Ajmal et al. 2019) and Fe2O3 (Dowlath et al. 2021) we anticipate strong radical scavenging activity of Fe2TiO5. Through the combination of one highly reactive metal ion – Ti2+ and one highly biocompatible metal ion – Fe3+ (Stankic et al. 2016) in a 6 mixed metal oxide – Fe2TiO5 we hope for a cell friendly material, which is the first demand for future biodegradable active materials. We assessed the biocompatibility of Fe2TiO5 using a human colorectal immortalized Caco-2 cell line and a model microbiome bacterium Escherichia coli as well as on a panel of environmental and pathogenic bacteria. In addition, to evaluate Fe2TiO5 as a food-contact material, we investigated the migration from Fe2TiO5/alginate nanocomposites into food simulants and its antioxidant activity. A preservation study conducted on fresh whole and cut strawberries in open atmosphere was performed to further assess the antioxidant activity of Fe2TiO5/alginate nanocomposite films. This study may contribute to development of novel cell friendly bimetallic oxide NPs that could replace TiO2 NPs in active food packaging. 2. Materials and methods 2.1. Materials, reagents and solutions Iron(III) nonahydrate (Fe(NO3)39 H2O, ACS reagent, purity 98%), titanium isopropoxide (Ti(OCH)(CH3)2, purity 98%), oxalic acid (Puriss, purity 99%), citric acid monohydrate (ACS reagent, purity 99%), all from Sigma Aldrich (Darmstadt, Germany) were used for synthesis of the iron titanate (Fe2TiO5) nanoparticles. Alginic acid sodium salt (Sodium alginate) (Alfa Aesar, Thermo Fischer Scientific, Haverhill, Massachusetts, USA), calcium chloride (Lach-ner Chemicals, Neratovice, Czech Republic), glycerol (Galafarm, Belgrade, Serbia) and deionized water (Sigma Aldrich, Darmstadt, Germany) were used for alginate film preparation. Ethanol (Prolabo, Fontenay-sous-bois, France) and acetic acid (Prolabo, Fontenaysous-bois, France) were used to prepared food simulants. 2,2 Diphenyl-1-picrylhydrazyl (DPPH˙) (Sigma Aldrich, Saint Quentin Fallavier, France) and methanol (Prolabo, Fontenay- 7 sous-bois, France) were used in antioxidant tests. ZnO NPs of 50 nm average diameter (Sigma Aldrich, Saint Quentin Fallavier, France) were used in control toxicity tests. 2.2. Synthesis and characterisation of Fe2TiO5 nanoparticles Iron titanate nanoparticle synthesis was conducted following a modified sol-gel method (Vasiljevic et al. 2020a). Iron(III) nonahydrate, titanium isopropoxide, oxalic acid (chelating agent) and citric acid (surfactant) were mixed on a magnetic mixer maintaining the temperature between 80-90 oC until a gel was formed. The formed gel was heated to 300 oC until a powder formed, and then calcined in a chamber furnace at 750 oC for 3 h. Structural characteristics of the obtained powder were investigated by measuring X-ray diffraction (range 2 = 10-90o, step 0.05s, acquisition rate 1o/min, Rigaku Ultima IV diffractometer, Tokyo, Japan) and FT-IR spectrum (range 400-4000 cm-1, FT-IR Nicolet 6700 ATR device, Waltham, MA, USA). The optical band gap was determined by measuring and analysing UV-Vis diffuse reflectance spectrum (Shimadzu UV-2600 with an ISR2600 Plus integrating sphere attachment, Kyoto, Japan). The powder morphology was investigated by Field emission electron microscopy - FESEM (TESCAN MIRA3 XM, Brno, Czech Republic) and transmission electron microscopy - TEM (JEM-2100 200 kV, JEOL Ltd. Tokyo, Japan). The specific surface area and pore structure were determined from measured N2 adsorption-desorption isotherms (Micromeritics ASAP 2020, Norcross, GA, USA). Prior to measurement, the powder sample was degassed at 150 oC for 10 hours under reduced pressure. 2.3. Synthesis of alginate - Fe2TiO5 films Alginate film was prepared using the previously described method (Vizzini et al. 2020). Briefly, 1.167 g of sodium alginate was mixed with 50 mL of deionized water for 6 h, then 1.5 8 mL of calcium chloride aqueous solution (5% w/v) and 1.5 mL of glycerol (100%) were added and the mixture was mixed for another 10 minutes. The formed solution was poured into a Petri dish and evenly spread in order to obtain alginate film. Alginate/NPs packaging films were obtained using the solvent casting method. For this, 55 mg of Fe2TiO5 nanoparticles (NPs) were added to the solution prepared in the same way and poured into a Petri dish to obtain alginate - Fe2TiO5 films. Both plates were incubated at 50 oC for 12 h. The film was wetted with a solution of 5% calcium chloride for 10 minutes, washed and dried and sterilized before utilization. A scheme of the film preparation is shown in Fig. S1. The film structure was observed by FT-IR spectroscopy in the range 4000-400 cm-1, resolution 4 cm-1 (Perkin Elmer Spectrum Two, Waltham, MA, USA). 2.4. Bacterial strains The bacterial strains used in this study are listed in Table S1. Strains were grown in brain-heart-infusion (BHI) medium, Becton Dickinson (DB, Le Pont de Claix, France), with shaking at 37 °C or on BHI agar at 37 °C. As described previously, the revitalization procedure of all cultures stored at −80 °C was conducted at 37 °C overnight in BHI broth, with the exception of Campylobacter jejuni (Zanet et al. 2019). C. jejuni was firstly incubated in a Columbia blood agar base (Thermo Fisher Scientific Inc., Illkirch, France) supplemented with 5% v/v of sheep defibrinated blood (Thermo Ficher Scientific Inc.) for 48 h under microaerophilic condition. Pure colonies of C. jejuni were then isolated on BHI agar medium and cultivated under microaerophilic condition as explained previously (Vizzini et al. 2021). 2.5. Antibacterial activity 9 Antibacterial activity of Fe2TiO5 NPs was tested by the standard disk diffusion method and through following the kinetics of bacterial growth (Auger et al. 2018; Vasiljevic et al. 2020b) . The overnight-grown bacterial suspensions were standardized using the McFarland standard, and then spread onto the sterilized BHI agar dish. Whatman® filter paper circular disks of 5 mm diameter were placed on bacterial seeded plates and soaked with 15 µL of Fe2TiO5 NPs at different final concentrations (0.5, 1 and 5 mg/mL). Plates were incubated at 37 °C for 10–20 h, and the diameters of the inhibition zones were measured with a transparent ruler (Auger et al. 2018). The antibacterial kinetics of Fe2TiO5 NPs (0.1, 1, and 2 mg/mL) against Bacillus subtilis, Escherichia coli, Bacillus cytotoxicus and Salmonella enteritidis were investigated using a diluted bacterial culture in fresh BHI medium to initial optical density at 600 nm (OD600) of 0.1. Suspensions (200 µL each) were placed in 96 microplate wells and incubated at 37 °C. Bacterial growth was measured by following the OD600 overnight at 37 °C using the Infinite 200 PRO microplate reader (TECAN, Salzburg, Austria) as explained previously (Vasiljevic et al. 2020b). The absorbance of blanks containing the equivalent concentration of nanoparticles in BHI medium incubated under the same conditions was used to subtract the background absorption. A negative control was prepared replacing NPs with water. All data presented are the averages of at least triplicate measurements. 2.6. Ion migration Ion migration was tested in two food simulant solutions (3% v/v acetic acid – HAc and 95% v/v ethanol - EtOH) that simulated acidic and fatty foods according to the European regulation (EU 10/2011), by placing 0.01 g film pieces into a container with 40 ml of the 16 film, confirming the presence of NPs in the film. Bands in the higher wavenumber region originate from sodium alginate film (Dojcinovic et al. 2021). Bands in the region 800-100 cm-1 can be attributed to mannuronic acid residues, C1-H mannuronic acid deformation and CO stretching of uronic acids. The prominent band at  1032 is due to C-O stretching vibrations. COOsymmetric and asymmetric stretching vibrations are noted at 1400 and 1600 cm-1 originating from metal-carboxylate interactions, while the bands at 2910 can be assigned to the CH anomer. The prominent O-H stretching band is noted at 3292 cm-1 (Dojcinovic et al. 2021). The formation of such stretching bonds will have a significant effect on the physical and mechanical properties of packaging film. As shown in Fig. S1, the prepared Fe2TiO5/alginate film has a brown/reddish colour that is relatively uniform showing a relatively homogenous distribution of Fe2TiO5 nanoparticles in the alginate film. 3.3. ICP-OES measurement of ion release from Fe2TiO5/alginate films Migration or release of ions from metal or metal oxide nanoparticles from packaging films into the packaged food product is one of the parameters that may determinate the film biocompatibility (Perera et al. 2021). The ions release depends on a variety of factors that include nanoparticle (size, solubility, diffusivity within the polymer), composition and packaging characteristics (polymer structure, viscosity), environmental conditions (temperature or mechanical stress), and food conditions including its pH value and hydrophobicity. Ion release is usually determined in food simulants (Nikolic et al. 2021; Perera et al. 2021). In this work, we evaluated Ti and Fe ion release from Fe2TiO5/alginate films into 3% (w/v) aqueous acetic acid and 95% (v/v) aqueous ethanol, at 20 C for 10 days, sampling at day 1, 3, 5, 7 and 10. ICP-OES measurements performed on the prepared solutions showed that there was no Ti or Fe ion release into the food simulants, as all values determined were below the detection limit of the instrument 17 (in the range 0.001-0.032 mg/L). This is in accordance with previous analysis of Ti migration from other polymer matrixes such as chitosan (Enescu et al. 2020), or PET (Chen et al. 2019). In a recent review (Garcia et al. 2018) concluded that available research on nanoparticle migration, including Ti suggests that nanoparticles incorporated into polymers have a tendency to agglomerate and thus remain relatively firmly embedded in the polymer matrix. 3.4. Antioxidant effect Incorporation of antioxidants in food packaging is a way to prevent spoilage of oxidationsensitive food products. Antioxidant activity of Fe2TiO5 NPs was evaluated by the DPPH˙ free radical assay. This assay is based on DPPH˙ reduction to DPPH2 by accepting a hydrogen atom from the antioxidant molecule, which changes the solution purple colour to yellow with concomitant decrease in absorbance at 517 nm. The observed DPPH˙ radical scavenging activity of Fe2TiO5 NPs was dose dependent (Fig. 3a). Fig 3b displays the corresponding kinetics of antioxidant activity of NPs (as calculated from equation (1)), with the concentration EC50 of 0.16 mg/mL required to decrease the initial DPPH˙ concentration by 50% (0.66 µM, molecular weight of Fe2TiO5 is 239.46 g/mol). The calculated ARPµM was 1.5. Such strong scavenging activity of Fe2TiO5 NPs is similar to those previously reported for TiO2 NPs (0.1 mg/mL) of similar sizes (Ajmal et al. 2019). Interestingly, the hydrogen ions availability is probably high in aqueous solutions of two nanoparticles since we found that pH of 1 mg/mL Fe2TiO5 NPs and 1 mg/mL TiO2 was 2, and 4.5, respectively. Hematite (-Fe2O3) has also previously shown free radical scavenging activity (EC50 0.18 mg/mL) that has been attributed to electron transfer towards the free radical located at the nitrogen atom in DPPH (Bhattacharya et al. 2014; Dowlath et al. 2021). Thus, iron titanate (Fe2TiO5) that combines characteristics of both TiO2 and Fe2O3 NPs 18 shows strong antioxidant activity that was not controlled by metal ions release rate, but by the surface reactivity of NPs. Fig. 3. DPPH˙ assay showing antioxidant activity of Fe2TiO5 NPs with EC50 0.16 mg/mL (a) adsorption curves obtained with increasing concentrations of NPs. (b) EC50 plot. 3.5. Biocompatibility of Fe2TiO5 NPs In order to test the biocompatibility of Fe2TiO5 NPs, the human epithelial Caco-2 cells were incubated with different concentrations of the NPs (0.2 mg/ml - 2 mg/ml), stained with acridine orange and analysed by flow cytometry. Acridine orange fluorescence dye easily traverses the cell membrane and accumulates in lysosomes of live cells. During necrosis, when structural integrity of cell membranes is lost, lysosomes are ruptured and red fluorescence of acridine orange decreases. No significant decrease in acridine orange staining was observed in cells incubated with NPs compared to the control cells incubated with PBS (Fig. 4). This strongly suggests that Fe2TiO5 caused no cell damage. For comparison, 0.5 mg/ml ZnO NPs decreased fluorescent staining indicating that cells were necrotic. These findings suggest the biocompatibility of Fe2TiO5 NPs at concentrations ≤ 2 mg/mL. 19 Fig. 4. Acridine orange staining and flow cytometry representative plots (n = 3) of control Caco2 cells and Caco-2 cells incubated with different concentrations of Fe2TiO5 NPs and ZnO NPs for 24 h. For all plots, numbers indicate percentage of cells in relevant gate. Since cell membranes are negatively charged, cytotoxicity of metal oxides NPs is greatly influenced by their surface charges (Auger et al. 2019; Valgimigli et al. 2018). Positively charged NPs are usually more toxic to plasma membrane than negatively surface charged NPs (Auger et al. 2019; Fröhlich 2012). The zeta potential value of Fe2TiO5 NPs in deionized water was determined in our previous work showing a negative value (−21.6 ± 6.8) mV (Vasiljevic et al. 2020a). In contrast with Fe2TiO5 NPs analyzed in this work, and Fe oxide NPs, such as - Fe2O3 and Fe3O4 NPs (Stankic et al. 2016; Vihodceva et al. 2021), pure TiO2 NPs were shown to exhibit cytotoxicity, induce DNA damages and modify ATP-binding cassette (ABC) family xenobiotic efflux pumps in human Caco2 cells (Dorier et al. 2019; Gerloff et al. 2012). The toxic effect was highly dependent on the TiO2 particles’ size, morphology, crystal structure and amount of surface defects. 20 3.6. Antibacterial effect of Fe2TiO5 NPs We next sought to evaluate for potential cytotoxicity of Fe2TiO5 NPs towards E. coli, as a model of microbiota and environmental Gram-negative bacteria and B. subtilis, as a model of environmental Gram-positive bacteria. Different concentrations of Fe2TiO5 were studied by measuring growth kinetics of two bacteria in the BHI medium using a plate reader (Fig. 5). UV irradiation was performed before incubation. No antibacterial effect was observed for NPs concentrations ≤ 2 mg/mL. In case of B. subtilis a decrease of absorbance was observed after 7 h of incubation even in the absence of NPs probably due to the lower availability of oxygen in microplates (semi-anaerobiosis condition). In contrast, E. coli is a facultative anaerobic bacterium, able to grow in both aerobic and anaerobic environments. Fig. 5 Growth kinetics of E. coli and B. subtilis alone or in the presence of different concentrations of Fe2TiO5 nanoparticles in BHI. To confirm that Fe2TiO5 NPs show no toxicity towards bacterial cells, a panel of strains, listed in Table S1, was tested by following growing kinetics in the presence of NPs (Fig. S3) and by the disk diffusion method. Both tests indicated no toxicity of negatively surface charged 21 Fe2TiO5 NPs for concentration ≤ 5 mg/mL, i.e., concentrations much superior of those providing a strong antioxidant efficiency. Interestingly, TiO2 NPs of different sizes, morphology or crystal structures have been reported to exhibit marked antibacterial activity allowing efficient eradication of various bacterial strains (Stankic et al. 2016). Antibacterial activity has been reported for other metal oxides, such as ZnO, MgO and is strongly influenced by their composition, size, shape and crystal structure (Nikolic et al. 2021). Compared to TiO2, negatively charged hematite (-Fe2O3) NPs did not exhibit antimicrobial activity against E. coli or S. aureus in deionized water up to concentrations of 1mg/ml, while some antibacterial activity was noted for positively charged hematite NPs against E. coli, but not S. aureus (Vihodceva et al. 2021). Fe2TiO5 NPs produced here, also negatively charged in water (Vasiljevic et al. 2020a), showed higher biocompatibility towards both bacterial and human cells tested. 3.7 Preservation of fruit To verify the preservation effect of Fe2TiO5/alginate on a food sample sensitive to oxidation the preservation test was performed on cut and whole strawberries in open air conditions. Open air was used as it deteriorates fruit integrity (Vargas-Torrico et al. 2022). In the case of whole strawberries placed on pieces of alginate or Fe2TiO5/alginate composite film (Fig. 6A) there was no significant noticeable change on day 1, though on day 2 the ripening process started on all fruit samples, but most noticeably on the control sample (Fig. S4A). On day 3, visible deterioration was noticeable on the control sample, including evident textural damage, tissue degradation and visible growth of mould. The strawberry sample placed on alginate film also showed such damage, though tissue degradation was slightly less. The Fe2TiO5/alginate 22 composite film has acted as a barrier delaying the rotting process and reducing the tissue degradation rate. Cut strawberry spoils fast in open air conditions, and deterioration started on day 1 on the control sample, with slight curling of the edges (Fig. 6B, Fig. S4B). The cut strawberry samples covered with alginate or Fe2TiO5/alginate composite film showed no visible deterioration on day 1. This was more noticeable on day 2 when the fruit tissue started to brown on the control sample. On day 3 there was visible deterioration and fungal growth noticeable on the cut strawberry control sample (Fig. 6B, Fig. S4B), while the alginate film provided some protection in that tissue degradation had started and edge discoloration and deterioration was more noticeable, but was less than in the control sample. A Control Alginate film Fe2TiO5/algin. film Day 0 Day 3 B Control Alginate film Fe2TiO5/algin. film Day 0 Day 1 23 Day 2 Day 3 Fig. 6 Preservation study: (A) Conservation study of whole strawberries, (B) Conservation study of cut strawberries. 4. Conclusion We produced active alginate composite films by incorporating Fe2TiO5 NPs via the solvent-casting method to develop active biocompatible films. As synthetized, pure phase Fe2TiO5 NPs were mesoporous with an average particle size of 44 nm. The strong radical scavenging activity of Fe2TiO5 NPs observed in the DPPH tests suggests that its utilization in food packaging may prevent spoilage of oxidation sensitive food products. The ICP-OES measurements indicated no metal ion migration from alginate/Fe2TiO5 NPs to food simulants, which suggests the biocompatibility of the film. Moreover, nanoparticles alone showed no cytotoxicity as no inhibition of bacterial growth not human Caco-2 cells damage was observed in the presence of Fe2TiO5 NPs up to 5 mg/mL. This work is the first step in the evaluation of Fe2TiO5 NPs as a food-contact material. The protective antioxidant activity of this packaging film in a real food system was tested and confirmed in a preservation study of fresh whole and cut strawberries. Finally, the mesoporous structure of Fe2TiO5 nanocrystals suggests that they can be used as carriers to integrate other active compounds in the film, such as antibacterial peptides or essential oils, and to provide packaging films with multiple activities for prolonged shelf-life of various foods. 24 Credit author statement Francesco Rizzotto: Investigation and analysis of antioxidant, human cell line and antibacterial activity; Writing – original draft; Zorka Z. Vasiljevic: Synthesis and analysis of Fe2TiO5 nanoparticles and Fe2TiO5 embedded alginate films; Preservation test; UV/Vis measurement and analysis, Writing – original draft, Writing-review and editing, Visualization; Gordana Stanojevic, Ion migration measurement and analysis; Milena P. Dojcinovic, Investigation of Fe2TiO5 embedded alginate films, ion migration analysis; Writing-review and editing; Ivona JankovicCastvan, Nitrogen porosimetry measurement and analysis; Jelena D. Vujancevic, FESEM image measurement and analysis; Nenad B. Tadic, XRD measurement; G. O. Brankovic TEM measurement and analysis; Aurélie Magniez, flow cytometry measurements and analysis; Jasmina Vidic: Conceptualization, Supervision, Writing - Original draft, Writing-review and editing; Maria Vesna Nikolic: Conceptualization, Visualization, FT-IR measurements and analysis, Preservation test, Writing – Original draft, Writing-review and editing. Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors are grateful to Jugoslav Krstic (IHTM, Serbia) for valuable advice concerning ICP measurements, Goran Lakisic (INRAE, France) for valuable help with Caco2 cells. We are also grateful to Sandrine Auger (INRAE, France) for kindly gift of B. cytotoxicus and Florence Dubois-Brissonnet (AgroParisTech, France) for kindly gift of Salmonella strain. Z.Z.V., G.S., M.P.D., G.O.B. and M.V.N. are grateful for funding grant 451-03-9/2022-68/200053 and J.D.V. for the funding grant 451-03-9/2022-68/200175 from the Ministry for Education, Science and Technological Development of the Republic of Serbia. 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