Magnetically responsive chitosan-pectin films incorporating Fe3O4 nanoparticles with enhanced antimicrobial activity.

International Journal of Biological Macromolecules 227 (2023) 1070–1077 Available online 1 December 2022 0141-8130/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Magnetically responsive chitosan-pectin films incorporating Fe 3 O 4 nanoparticles with enhanced antimicrobial activity Iratxe Zarandona a , Daniela M. Correia b , Joana Moreira c , Carlos M. Costa c , d , e , Senentxu Lanceros-Mendez f , g , * , Pedro Guerrero a , f , h , ** , Koro de la Caba a , f a BIOMAT Research Group, University of the Basque Country (UPV/EHU), Escuela de Ingeniería de Gipuzkoa, Plaza de Europa 1, 20018 Donostia-San Sebasti´ an, Spain b Centre of Chemistry, University of Minho, 4710-053 Braga, Portugal c Physics Centre of Minho and Porto Universities (CF-UM-UP), University of Minho, 4710-057 Braga, Portugal d Institute of Science and Innovation for Bio-Sustainability (IB-S), University of Minho, 4710-053 Braga, Portugal e Laboratory of Physics for Materials and Emergent Technologies, LapMET, University of Minho, 4710-057 Braga, Portugal f BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain g Ikerbasque, Basque Foundation for Science, 48009 Bilbao, Spain h Proteinmat materials SL, Avenida de Tolosa 72, 20018 Donostia-San Sebasti´ an, Spain ARTICLE INFO Keywords: Chitosan-pectin films Fe 3 O 4 nanoparticles Antimicrobial capacity Magnetic properties ABSTRACT Chitosan-pectin films with iron oxide (Fe 3 O 4 ) magnetic nanoparticles were prepared by solution casting in order to produce biopolymer based magnetically active materials. Infrared (FTIR) spectra indicated physical interactions between the matrix and nanoparticles, corroborated by differential scanning calorimetry (DSC) results. In addition, thermal characterization suggested that the interactions between chitosan, pectin and the nanoparticles resulted in a less compact structure, influencing the film mechanical properties. Regarding vibratingsample magnetometry (VSM) and electrical analysis, chitosan-pectin films with Fe 3 O 4 nanoparticles showed ferrimagnetic behavior, with an increase of the dielectric constant as the nanoparticle concentration increased. Furthermore, films displayed enhanced antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) bacteria. Therefore, chitosan-pectin films with Fe 3 O 4 magnetic nanoparticles provide promising results for active and intelligent food packaging applications. 1. Introduction Food packaging plays an essential role in the food supply chain, guaranteeing food quality and safety from external factors, such as microorganisms, temperature, odors and light exposure, during food transport and storage, and supporting the prevention of food waste [1]. In this regard, packaging technologies are in continuous evolution seeking to improve the quality and freshness of food and prolonging its shelf life [2]. Additionally, with the goal of attenuate environmental issues and to implement circular economy models, sustainable materials are gaining relevance as potential candidates for food packaging [3]. Particularly, biopolymers could be a sustainable alternative for packaging materials due to their biodegradable character and non-toxicity [4–6]. Among the different biopolymers, and due to their specific properties, chitosan and pectin could be suitable materials for intelligent packaging films, since both polysaccharides are pH dependent. Under low pH conditions, positively charged chitosan and negatively charged pectin bind via ionic interactions forming a polyelectrolyte complex, resulting in the enhancement of the mechanical properties and hydrophilicity over chitosan and pectin matrices separately [7,8]. Few works have been reported on the use of chitosan-pectin matrix for food packaging. Among them, [9] prepared chitosan-pectin films with anthocyanin as a pH indicator device for intelligent food packaging, and [10] added Streptomyces coelicolor, which improved CO 2 barrier properties to the film. Regarding intelligent packaging, the aim is to monitors physical, chemical or biological status of food items by detectors and sensors, from the beginning of the food supply chain until it reaches the * Correspondence to: S. Lanceros-Mendez, BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/EHU Science Park, 48940 Leioa, Spain. ** Correspondence to: P. Guerrero, BIOMAT Research Group, University of the Basque Country (UPV/EHU), Escuela de Ingeniería de Gipuzkoa, Plaza de Europa 1, 20018 Donostia-San Sebasti´ an, Spain. E-mail addresses: [email protected] (S. Lanceros-Mendez), [email protected] (P. Guerrero). Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac https://doi.org/10.1016/j.ijbiomac.2022.11.286 Received 13 July 2022; Received in revised form 21 November 2022; Accepted 28 November 2022 International Journal of Biological Macromolecules 227 (2023) 1070–1077 1071 consumer, providing information about the food quality and internal environment conditions of the package [11,12]. Thus, intelligent packaging can provide information of the in situ conditions of the food quality and freshness, leading to a loss of dependency on food expiry dates. In this regard, nanoparticles of metal oxides have a great potential for applications in food industry because of their antibacterial capacity, non-toxicity, oxygen and ethylene scavenging capability, and thermal stability properties [13]. Indeed, metal oxides show sensing properties by the mechanism of adsorption and desorption with different gaseous compounds on the surface of the material, leading to changes in the electrical conductance [14]. In this regard, Fe 3 O 4 nanoparticles (NP) have been applied in a variety of including biomedicine, cosmetics and food preservation, due to their antimicrobial activity, magnetic response, biocompatibility and non-toxic properties [15,16]. For food applications, Fe 3 O 4 NPs have been used as sensors for detecting different compounds, such as heavy metals [17], caffeic acid (Department of Food Science and Technology, Ayatollah Amoli Branch, Islamic Azad University, Amol 46311-39631, Mazandaran, Iran; & Abdi, [18]), or foodborne spoilage bacteria [19]. In this context, the aim of the present work was to prepare chitosanpectin films with Fe 3 O 4 nanoparticles by solution casting, in order to assess the effect of Fe 3 O 4 NPs concentrations on the chitosan-pectin matrix, physicochemical, thermal, structural, magnetic, electric and antimicrobial properties. 2. Materials and methods 2.1. Materials Chitosan with a molecular weight of 375 kDa and a deacetylation degree ≥75 % was supplied by Sigma-Aldrich, Spain. High methoxylated pectin, with a molecular weight of 472 kDa and an esterification degree of 58 %, was kindly supplied by CEAMSA, Spain. Iron oxide (Fe 3 O 4 ) powder, with particle size of 50–100 nm and a purification degree of 97 %, was supplied by Nanostructured & Amorphous Materials, Inc., USA. Acetic acid solution (1 N), used as solvent, was supplied by Panreac, Spain. 2.2. Film preparation Chitosan-pectin films with Fe 3 O 4 nanoparticles were processed by solution casting. The polymers were dissolved separately. On the one hand, the required amount of chitosan was dissolved in 1 wt% acetic acid solution by stirring for 30 min. On the other hand, Fe 3 O 4 nanoparticles were dispersed in a 0.1 wt% aqueous solution of Triton 100-X by sonication for 3 h. Then, the required amount of pectin was added to the NP aqueous solution and stirred at 67 ◦C. Both solutions were mixed at 8000 rpm for 10 min (Ultraturrax UT25, IKA, Germany), and air bobbles were removed by vacuum. The mixture was placed in a petri dish and left to dry at room temperature. Mixture compositions incorporating Fe 3 O 4 NP concentrations between 0.1 and 10 wt% are shown in Table 1. 2.3. Film characterization 2.3.1. Fourier transform infrared (FTIR) spectroscopy An Alpha II spectrometer (Bruker, Madrid, Spain), with a Platinum ATR accessory, was used to collect FTIR spectra of chitosan-pectin films with Fe 3 O 4 nanoparticles. A total of 32 scans were performed with a resolution of 4 cm −1 in the wavelength between 4000 and 800 cm −1 . 2.3.2. Thermo-gravimetric analysis (TGA) A Mettler Toledo TGA/SDTA 851 thermo-balance was used to measure the thermal stability of the samples. Dynamic scans from 25 to 900 ◦C were carried out at a constant rate of 10 ◦C/min under nitrogen atmosphere to avoid thermo-oxidative reactions. 2.3.3. Differential scanning calorimetry (DSC) A Mettler Toledo DSC 822 was used to perform differential scanning calorimetry. Samples of around 3 mg were heated from −50 ◦C to 300 ◦C at a heating rate of 10 ◦C/min under nitrogen atmosphere to avoid oxidative reactions. 2.3.4. Scanning electron microscopy (SEM) Morphology was examined by using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 15 kV. Before analysis, fractured surfaces were coated with a gold layer by sputtering with a Polaron SC502 apparatus. Additionally, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX) was used to analyze the particle distribution within the samples with a Hitachi TM3000 Tabletop Microscope. 2.3.5. Mechanical properties Mechanical properties were measured with an Instron 5967 electromechanical testing system (Instron, Spain). According to ASTM D638-14 [20], tests were carried out with a load cell of 500 N and a crosshead rate of 1 mm/min. Films were cut into bone shaped samples of 4.75 mm ×22.25 mm. Five samples were measured for each system. Tensile strength (TS), elongation at break (EAB) and elastic modulus (E) were evaluated from the measurements. 2.3.6. Vibrating-sample magnetometry (VSM) The magnetic properties of the films were analyzed with a MicroSense EZ7 VSM from −1.8 to 1.8 T at room temperature. The hysteresis loops of the samples were measured and remanence (M r ), magnetization saturation (M s ) and coercive field (H c ) parameters were obtained. 2.3.7. Electrical characterization For the electric measurements, circular gold electrodes of 5 mm diameter were deposited by magnetron sputtering with a Polaron Coater SC502 onto both sides of each sample. The electrical conductivity of the films was measured through a Keithley 487 picoammeter/voltage source with a ±10 V voltage, and the volume conductivity of the films ( σ ) was calculated by: σ =d R.A(1) where R is the resistance of the film obtained from the slope of the I–V curves, d is thickness, and A is the electrode area. Dielectric measurements were performed using a Quadtech 1920 LCR precision meter. The capacitance (C) and the dielectric losses (tan δ) were obtained at room temperature in the frequency range from 20 Hz to 1 MHz with an applied voltage of 0.5 V. The real ( ε ´) part of the dielectric function was calculated as: ε ′=C⋅d ε 0⋅A(2) where C is the individual sample capacity, ε 0 is the permittivity of Table 1 Composition of chitosan-pectin films with different contents of Fe 3 O 4 nanoparticles. System designation Chitosan concentration (wt%) Pectin concentration (wt %) NP concentration (wt%) Control 50.00 50.00 0 0.1NP 49.95 49.95 0.1 0.5NP 49.75 49.75 0.5 1NP 49.50 49.50 1 5NP 47.50 47.50 5 10NP 45.00 45.00 10 I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1072 vacuum (8.85 ×10 −12 F⋅m −1 ), A is the electrode area and d is the film thickness. The real part of the conductivity of the dielectric material can be calculated from the dielectric measurements as follows: σ ′( ω ) = ε 0 ωε ′′( ω )(3) where ε 0 is the permittivity of free space, ω =2 π f is the angular frequency and ε ′′( ω ) = ε ′tan δ is the frequency dependent imaginary part of the dielectric permittivity. 2.3.8. Antimicrobial analysis To determine the inhibition capacity of films, two food pathogens were tested: a Gram negative E. coli K12 and a Gram-positive Staphylococcus epidermidis NCTC 11,047 purchased from American Type Culture Collection (LGC Standards S.L.U.) The bacterial pre-inoculum was prepared by using a single colony from the corresponding stock and resuspended in nutrient broth (NB). After incubating overnight at 37 ◦C and 200 rpm, the pre-inoculum was centrifuged (5000 rpm) and the pellet was resuspended in 0.9 % solution of NaCl at pH 6.5. The optical density, OD, of E. coli and S. epidermidis was measured at 600 nm and adjusted to 0.28 ±0.01 and 0.36 ±0.01 for E. coli and S. epidermidis, respectively. Samples were cut in circular pieces of 10 mm diameter, placed into falcons, and sterilized with ultraviolet light for 30 min on each side. Then, 2 mL of the final bacterial suspension was added and came into contact with the films for 2 h at 37 ◦C and 200 rpm. Falcons without any material were used as controls for bacterial growth. The viability of bacterial cells in suspension after contacting the material was evaluated using colony-forming units (CFUs) assay. Tenfold serial dilutions of the bacterial cultures of the falcons were carried out in 0.9 % NaCl aqueous solution. A volume of 10 μ L was placed on spread plates of NB and colony-forming units per milliliter (CFU ⋅ mL −1 ) count was carried out after incubating the plates at 37 ◦C for 24 h. Antimicrobial activity was determined by comparing viable bacteria of each system with that incubated without any film. The bacterial growth inhibition was calculated based on the following equation: Bacterial growth inhibition (%) = 100 −(CFU sample CFU control)×100% (4) 2.4. Statistical analysis With the purpose of determining the significant differences between measurements, analysis of variance (ANOVA) was carried out by means of SPSS software (SPSS Statictic 25.0). Tukey's multiple range test was used for multiple comparisons among different systems with a statistical significance at the p <0.05 level. 3. Results and discussion 3.1. FTIR analysis and thermal characterization In order to evaluate the interactions among the components of the films, FTIR analysis was carried out and the spectra are shown in Fig. 1a). The characteristic bands of chitosan and pectin were observed in control films: the band at 3247 cm −1 , associated to O – H bonds in both polymers and to N – H bonds in chitosan; the bands between 2925 and 2850 cm −1 , attributed to C – H stretching vibrations; the band at 1742 cm −1 , associated to C – – O in the ester bonds of pectin; the band at 1633 cm −1 , assigned to the stretching of C – – O bond in chitosan and to the asymmetric stretching of COO − in pectin; and the band between 1150 and 890 cm −1 , attributed to the C-O-C of the saccharide ring of chitosan and pectin. As shown in a previous study, the bands related to C – – O stretching of chitosan and to the ester bond of pectin showed displacements towards lower wavenumbers, indicating that the interactions between chitosan and pectin were physical [21]. Additionally, some bands displacements were observed when Fe 3 O 4 nanoparticles were added into the chitosan-pectin system. In particular, 4000 3500 3000 2500 2000 1500 1000 a) 10 NP 5 NP 1 NP 0.5 NP 0.1 NP .u.a/ecnattimsnarT Wavenumber / cm -1 Control 100 200 300 400 500 600 700 800 -0.008 -0.006 -0.004 -0.002 0.000 b) Cº% /GTD -1 Temperature / ºC Control Chitosan Pectin 100 200 300 400 500 600 700 800 -0.005 -0.004 -0.003 -0.002 -0.001 0.000 c) Cº%GTD -1 Temperature / ºC Control 0.1NP 0.5NP 1NP 5NP 10NP 0 50 100 150 200 250 300 g . W/wolFtaeH -1 Temperature / ºC 10NP 5NP 1NP 0.5NP 0.1NP Control odnE d) Fig. 1. a) FTIR spectra of chitosan-pectin films, b) DTG of neat chitosan, neat pectin and control film, c) DTG of chitosan-pectin films and d) DSC of chitosanpectin films. I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1073 O – H and N – H vibration bands shifted to higher wavenumbers, from 3247 cm −1 for control films up to 3263 cm −1 for 10NP, as the nanoparticle concentration increased. Moreover, the band associated to C – H stretching vibrations at 2853 cm −1 shifted to higher wavenumbers, becoming a shoulder of the band at 2923 cm −1 . All these band displacements indicated that Fe 3 O 4 nanoparticles interacted physically with chitosan-pectin matrix. TGA and DSC analyses were carried out in order to determine the thermal stability of the material. Concerning TGA, derivative thermogravimetric curves are presented in Fig. 1b and c. Regarding neat chitosan and pectin samples (Fig. 1b), thermal degradation was observed at 300 ◦C and 240 ◦C, respectively. For control films, 4 inflection points were presented. The first one was observed around 68 ◦C, related to the water evaporation due to moisture. The second inflection point, around 230 ◦C, was the greatest one and it was related to the thermal degradation of chitosan and pectin polymers. It should be noted that the thermal degradation of chitosan-pectin film happened at lower temperature than that of pure pectin and pure chitosan (Fig. 1b). This event could indicate that the ionic bonding between chitosan and pectin led to structure changes in the material [9]. The third inflection peak was a shoulder at 283 ◦C, related to the chitosan that was not bonded to pectin. Finally, a slight inflection point was observed at 447 ◦C, related to the decomposition of by-products. The addition of Fe 3 O 4 nanoparticles (Fig. 1c) caused the presence of a new inflection peak around 680 ◦C, which became more intense as the concentration of NP increased and can be attributed to the transition from Fe 3 O 4 to FeO [22]. The endothermic peaks for the DSC thermogram of chitosan-pectin systems with Fe 3 O 4 NP are shown in Fig. 1d. Two endothermic peaks were observed for control films: the first, at 94 ◦C, was attributed to the film moisture; the second, at 211 ◦C, was related to the entrapped water linked by hydrogen bonding with the polar groups of the biopolymers [23]. Regarding the films with nanoparticles, the same endothermic peaks were observed as for the control sample, although for the samples with higher concentration of nanoparticles, 5NP and 10NP, the peak at 94 ◦C for the control sample was shifted to lower temperatures, 82 ◦C and 76 ◦C, respectively. This displacement indicated that there were interactions between the matrix and the NP, as observed by FTIR. In addition, the shift to lower temperatures would indicate that the Fig. 2. SEM images of chitosan-pectin film cross-section: a) control, b) 0.1NP, c) 0.5NP, d) 1NP, e) 5NP, and f) 10NP. Yellow dashed circles indicate nanoparticle aggregations. I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1074 structure formed was less compact and, therefore, would require less energy to release the moisture. 3.2. Structure and mechanical properties The morphology of the films was analyzed by SEM and the crosssection images of the samples are shown in Fig. 2. Control films presented a homogeneous structure, indicating the compatibility between chitosan and pectin. When Fe 3 O 4 nanoparticles were added at low concentrations, the structure of the films remained homogeneous but, as NP concentration increased, especially for 5NP and 10NP samples, nanoparticle aggregations were observed. In particular, the nanoparticle clustering in 10NP films was bigger than in 5NP films. These results explain the temperature decrease observed by DSC analysis, since the nanoparticle aggregations led to a less compact polymer structure. The dispersion of iron on 1NP, 5NP and 10NP films surface was analyzed by SEM/EDX. As can be observed in Fig. 3, the Fe signal in blue showed that the nanoparticles were homogeneously dispersed through the surface of the films, though larger NP aggregates are shown for the samples with larger filler contents. Tensile strength (TS), elongation at break (EAB), and elastic modulus (E) of the films, shown in Table 2, were measured in order to assess the influence of the magnetic nanoparticles on the chitosan-pectin matrix. Regarding to control films, high values of TS were obtained, due to the strong intermolecular bonds between chitosan and pectin, which derived into a compact structure. These values are higher than those Fig. 3. SEM/EDX images of the surface of chitosan-pectin films with 1 % Fe 3 O 4 (1NP), 5 % Fe 3 O 4 (5NP) and 10 % Fe 3 O 4 (10NP). Table 2 Tensile strength (TS), elongation at break (EAB) and elastic modulus (E) of chitosan-pectin films without Fe 3 O 4 nanoparticles (Control) and with different contents of Fe 3 O 4 nanoparticles. Films TS (MPa) EAB (%) E (MPa) Control 47.0 ±1.1 a 6.0 ±0.5 a 2395 ±52 a 0.1NP 43.7 ±1.2 b 5.9 ±0.2 a 2397 ±18 a 0.5NP 43.9 ±1.2 b 6.0 ±0.6 a 2443 ±27 a 1NP 44.0 ±0.5 b 6.3 ±0.8 a 2417 ±40 a 5NP 40.7 ±0.8 c 7.0 ±0.5 a 2076 ±58 b 10NP 40.1 ±1.0 c 7.0 ±0.7 a 2053 ±46 b a-c: Two means followed by the same letter in the same column are not significantly (p >0.05) different according to the Tukey's multiple range test. -16000 -8000 0 8000 16000 -200 -100 0 100 200 g.ume/noitazitengaM -1 Applied field / Oe 0.1NP 0.5NP 1NP 5NP 10NP a) 0246810 0 50 100 150 200 M r M s H c NP content / % g.ume/noitazitengaM -1 30 35 40 45 50 55 60 Coercive field / Oe b) Fig. 4. a) Hysteresis loops and b) magnetic properties (Mr, remanence; magnetization saturation, Ms.; and Hc, coercive field) of chitosan-pectin films with different contents of Fe 3 O 4 nanoparticles. I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1075 found by other authors for chitosan-pectin films [24]. When the nanoparticles were added, no significant differences (p >0.05) were observed in EAB results. However, TS values decreased as the nanoparticles concentration increased, as well as E values for 5NP and 10NP, indicating that the NP acts as defective sites for mechanical properties, in particular for concentrations >5 %. These results indicate that Fe 3 O 4 nanoparticles affected the structure of the polymeric matrix, as observed in SEM images, hindering the interactions between chitosan and pectin chains [25]. 3.3. Magnetic properties Regarding magnetic properties, VSM analysis was carried out to obtain the hysteresis loops and calculate the remanence, the magnetization saturation, and the coercive field (Fig. 4). The systems exhibited a ferrimagnetic behavior, since the values of remanence and coercive field were unequal to zero [26]. As observed in Fig. 4b, the magnetic behavior of the systems was dependent of the nanoparticle concentration. As the nanoparticle concentration increased, M r values increased, from 0.23 emu/g (0.1NP) to 19.70 emu/g (10NP), as well as M s values from 2.38 emu/g (0.1NP) to 206.11 emu/g (10NP). However, coercive field increased as nanoparticle concentration increased up to 0.5 % (50 Oe) but started to decrease until 41 Oe (10NP) as nanoparticle concentration increased. The coercive field is dependent on the nanoparticle size: if the size increases, the coercivity increases until a critical particle size is reached, after which the coercivity decreases. Nevertheless, since the size of the nanoparticles in this study are the same for all samples, results may be related to the agglomeration of the nanoparticles, as described in previous works [27]. Fig. 5. a) Current-voltage (I-V) curves and b) electrical conductivity value of films as a function of Fe 3 O 4 content in the 1st regime (black) and in the 2nd regime (red). Fig. 6. a) Real part of dielectric constant ( ε ´), b) dielectric losses (tan δ), and c) a.c. conductivity ( σ ´) of chitosan-pectin films with different contents of Fe 3 O 4 nanoparticles. I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1076 3.4. Electric characterization Electrical conductivity of the films with Fe 3 O 4 nanoparticles was evaluated by performing I-V curves (Fig. 5a). It can be observed that the I-V curves depend on the Fe 3 O 4 content and does not present the straight-line dependence characteristics of Ohm's law. Instead, two regimes are observed at low (−10 V to −3 V) and high applied voltages (2 V to 10 V). These regimes are related to the water linked by hydrogen bonding of the polar groups and the movement of free ions by the application of the electric field. As can be observed in Fig. 5b, the electrical conductivity increases strongly as a function of Fe 3 O 4 content for both regimes. The electrical conductivity of control films and 10NP films was 3.2 ×10 −10 S⋅cm −1 and 9.6 ×10 −10 S⋅cm −1 , respectively in the 2nd regime. This behavior is due to the fact that the addition of Fe 3 O 4 nanoparticles increases the charge carriers and the conduction is assigned to the electron hopping. Additionally, the dielectric analysis was evaluated and results are shown in Fig. 6. The dielectric constant (Fig. 6a) and tan δ (Fig. 6b) depend on the frequency due to dipole relaxation. Regardless of frequency range, the dielectric constant and tan δ increases as a function of NP concentration due to polarization contributions derived from the addition of nanoparticles, mainly dominated by interfacial and spatial charge polarization [28]. Concerning a.c. conductivity (Fig. 6c), values increased with frequency, indicating the local contribution to the electrical conductivity. Regardless of frequency range, the addition of Fe 3 O 4 nanoparticles increased the a.c. conductivity due to the charge carrier hopping [29]. 3.5. Antimicrobial capacity All films showed antimicrobial activity against E. coli and S. epidermis as can be seen in the representative photographs presented in Fig. 7a, although different response was observed for both bacteria (Fig. 7b). In the case of S. epidermidis, 10 NP films reached 43 % of inhibition, while 98 % was reached for E. coli. The antimicrobial capacity of the films was driven by both chitosan and magnetic nanoparticles. The antimicrobial mechanism of chitosan was caused by the positive charge of the amino group of chitosan, which interacted with the negative charges of cell membranes, affecting the loss of protein and other intracellular components [30]. On the other hand, the antimicrobial effect of iron nanoparticles was related to the capacity of the nanoparticles to interact with the cell membrane and to penetrate inside the cells, causing membrane damage and inactivation of the bacteria [31]. Therefore, chitosan-pectin films with Fe 3 O 4 nanoparticles showed increased antibacterial activity due to the cooperative action of chitosan and Fe 3 O 4 nanoparticles. It is worth noting that Fe 3 O 4 nanoparticles improved antimicrobial capacity when compared to control films. Additionally, it should be noted the difference in the inhibitory capacity of the films with respect to the two bacteria under study. This difference might be due to the different cell walls of Gram-positive and Gram-negative bacteria. The cell wall of Gram-positive bacteria consists of an outer thick layer of peptidoglycan that acts as a layer of resistance against most inhibitory molecules, whereas the cell wall of Gram-negative bacteria has a thinner peptidoglycan layer [32]. 4. Conclusions The preparation of chitosan-pectin films with Fe 3 O 4 magnetic nanoparticles by solution casting led to homogeneous films. FTIR results indicated that the physical interactions between chitosan and pectin were not affected by the addition of the nanoparticles. However, the mechanical properties were influenced by Fe 3 O 4 nanoparticles, due to the aggregation of the nanoparticles, as shown by SEM. The incorporation of nanoparticles also influenced the electrical, magnetic and antimicrobial properties. As the nanoparticle concentration increased, the dielectric constant, the remanence and the magnetization saturation increased. For the antimicrobial properties, the addition of the nanoparticles increased the antimicrobial capacity of the films for pathogenic Escherichia coli (Gram-negative) and Straphylococcus epidermidis (Grampositive) bacteria. Therefore, Fe 3 O 4 nanoparticles incorporated into chitosan-pectin films forming solutions could extend food shelf-life, besides functioning as a potential sensor for food packaging due to their electric and magnetic properties. CRediT authorship contribution statement Iratxe Zarandona: Data curation, Formal analysis, Investigation, Writing – original draft. Daniela M. Correia: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Joana Moreira: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Carlos M. Costa: Conceptualization, Investigation, Methodology, Resources, Funding acquisition, Supervision, Validation, Writing – review & editing. Senentxu Lanceros-Mendez: Conceptualization, Investigation, Resources, Funding acquisition, Supervision, Validation, Writing – review & editing. Pedro Guerrero: Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – review & editing. Koro de la Caba: Conceptualization, Investigation, Resources, Funding acquisition, Supervision, Validation, Writing – review & editing. Fig. 7. a) Photographs of the antimicrobial activity against E. coli and S. epidermis for chitosan-pectin films with 10 wt% of Fe 3 O 4 nanoparticles, and b) inhibition capacity of chitosan-pectin films, without Fe 3 O 4 nanoparticles (Control) and with different contents of Fe 3 O 4 nanoparticles, against E. coli and S. epidermidis strains in solution after 2 h in contact with material. The results are the average of 3 independent assays. I. Zarandona et al. International Journal of Biological Macromolecules 227 (2023) 1070–1077 1077 Declaration of competing interest 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. Acknowledgments Grant PID2021-124294OB-C22 funded by MCI/AEI10.13039/ 501100011033 and by “ERDF A way of making Europe”. Biomat group thanks the Basque Government for funding (IT1658-22) and I.Z. thanks the Basque Government for her fellowship (22-2018-00078). This work was also supported by the Portuguese Foundation for Science and Technology (FCT) under strategic funding UIDB/04650/2020, UID/FIS/ 04650/2021, project PTDC/FIS-MAC/28157/2017, and Investigator FCT Contracts 2020.02915.CEECIND (D.M.C) and 2020.04028.CEECIND (C.M.C.) funded by national funds through FCT and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e Internacionalizaç˜ ao (POCI). 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