Developing active poly(vinyl alcohol)-based membranes with encapsulated antimicrobial enzymes via electrospinning for food packaging

DEVELOPING ACTIVE POLY(VINYL ALCOHOL)-BASED MEMBRANES WITH ENCAPSULATED ANTIMICROBIAL ENZYMES VIA ELECTROSPINNING FOR FOOD PACKAGING M. Alonso-González1, A. Corral-González1, M. Félix2,*, A. Romero1, J. E. Martin- Alfonso 3 1Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012, Sevilla, Spain 2Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011, Sevilla, Spain. 3Department of Chemical Engineering and Materials Science, Campus de “El Carmen”, University of Huelva, Chemical Product and Process Technology Research Center (Pro2TecS) 21071 Huelva, Spain *MANUEL FELIX ANGEL Departamento de Ingeniería Química Escuela Politécnica Superior, Universidad de Sevilla, 41011, Sevilla (Spain) E-mail: [email protected] Phone: +34 954557179 Keywords: Enzyme; Electrospinning; Membrane; Polyvinyl alcohol; Polycaprolactone; Mechanical Properties 1. INTRODUCTION Traditionally, food packaging materials and methods have prevented food deterioration while providing safe and wholesome products. Current research focuses on active packaging, which, according to the European Commission, comprises packaging systems that interact with the food in such a way as to “deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food” [1]. In this sense, controlled release packaging (CRP) plays an active role in the food preservation process through the absorption or release of compounds such as antimicrobials and antioxidants at desirable rates [2]. For this purpose, active packaging materials interact with the internal gas environment and with the product surface with the aid of substances that remove (scavenge) or add (emit) gases inside the package, improving the food preservation and sensory properties while maintaining the quality and safety of the food [1]. Some examples are O2 scavengers and CO2 emitters, with the latter being increasingly attractive to food manufacturers due to their inhibitory activity against a wide range of aerobic bacteria and fungi, displaying a direct antimicrobial effect [3]. They are also frequently associated with Modified Atmosphere Packaging (MAP) systems, which can be incorporated into packaging materials by addition to the master batch via incorporation of active compounds in sachets and pads [4]. In this sense, food packaging can take the form of biodegradable and/or edible films [5], or metallic-based micro and nanocomposites [6]. However, polymers are the proper choice for this application due to their capacity to allow the passage of low molecular weight compounds [7]. Typically, packaging materials have been coated with antimicrobial polymeric films to inhibit spoilage and kill pathogenic microorganisms, although their low available surface area reduces their bioactivity. For this reason, fiber materials, such as electrospun fiber mats with submicron diameters (100-300 nm) and exceptionally high surface areas, are more suitable for this application. Electrospinning has been proved to be a suitable method for the fabrication of 3D and functional nanofiber mats through the immobilization/encapsulation of proper substances within the matrix [8–10]. Therefore, electrospun fibers with incorporated antimicrobial agents could kill bacteria and/or inhibit their growth on the surface of foods when used as a packaging material, thus helping to develop new and healthier food products. In addition, electrospun nanofibers offer some advantages over traditional fibers, including high surface area to volume ratio, tunable porosity and easy manipulation of chemical compositions and structures for desired properties and functionalities [11]. Fiber diameter and mat morphology are also easily tunable by varying process and material parameters, such as cospinning polymer, solution viscosity and conductivity, voltage, flow rate, nozzle-collector distance and collection method [12,13]. These characteristics make electrospun nanofibers very interesting for applications in different sectors, including biotechnology, drug delivery, wound healing, tissue engineering, environmental protection, energy harvest and storage [14]. The application determines the requirements demanded, such as the polymer or polymers and the solvent needed, depending on their characteristics. The most desired features in biotechnology are: biocompatibility, biodegradability with controlled degradation, antioxidability, antibacterial and antiviral properties, specific mechanical properties and high solubility in a particular solvent. Some polymers suit these characteristics and could be good candidates for enzyme encapsulation. Among them, PVOH is an easily electrospinnable, non-toxic, water-soluble (easily degradable by biological organisms) synthetic polymer with good thermal and chemical stability. These characteristics have been previously considered by Ping Shao et al. [15] for its use in food packaging applications and by Ardekani et al. [16] for the development of nanofiber membranes with potential applications in wound dressing. On the other hand, PCL shows high biocompatibility, good resistance to water, oil, solvents and chlorine and it is easily combinable with other polymers. In spite of the versatility and properties of electrospun nanofibers, further research is still needed for their widespread application in the food industry, since some challenges remain unsolved, such as maintaining the viability of the active agents in the functional electrospun fibers and the existing number of product development constraints concerning the inclusion of active components [17]. Among the different active agents that can be immobilized/encapsulated within the electrospun matrix, enzymes play a key role. Enzymes are responsible for bioreactions and are considered biocatalysts, exhibiting high specificity and thermodynamic efficiency. While traditional antibiotics are based on small molecules that fight pathogenic agents by preventing their functionality and reproduction, antibiotic enzymes work through two mechanisms: destroying a vital compound of the pathogenic agent or generating a compound that is toxic for it [18]. For example, glucose oxidase (GOX) reacts with D-glucose producing hydrogen peroxide (H2O2), which has a very strong oxidant character and, thus, antimicrobial capacity [19]. H2O2 reacts with sulfhydryl groups and double bonds in proteins and lipids, negatively affecting the cytoplasmic membrane and preventing cell proliferation. It is considered a natural antiseptic, since its metabolites are non-toxic for the environment, resulting in water and oxygen. The main disadvantage of employing enzymes is their instability under different temperature and pH conditions, which can lead to enzyme denaturalization and loss of catalytic activity. To retain the catalytic activity, enzymes can be immobilized, that is, confined in certain regions [20]. The present study addresses the immobilization of the GOX enzyme within a conventional polyvinyl alcohol (PVOH) membrane and within a multilayer membrane system (PCL/PVOH/PCL), synthetized by electrospinning. In more detail, the enzyme was encapsulated in a hydrophilic PVOH membrane which, at the same time, was protected by two hydrophobic polycaprolactone (PCL) membranes. The employed solvents were chloroform for PCL, and water for PVOH and the enzyme. The resulting systems were characterized via Fourier Transform Infrared Spectroscopy (FTIR), enzymatic kinetic tests and mechanical testing. To assess the morphology of the fibers, Scanning Electron Microscopy (SEM) and image analysis were used. Finally, to determine the antibacterial properties of the developed membranes, bacterial culture tests with Escherichia coli (E. coli) were carried out. 2. MATERIALS AND METHODS 2.1 Materials In this work, D-glucose anhydride along with hydrochloric acid (HCl) and sodium hydroxide (NaOH) (used to modify the pH of the solutions) were provided by PANREAC (Barcelona, Spain). β-D-glucose oxidase (GOX, 252,490 U/g) was used to catalyze the oxidation of D-glucose. This was purchased from Sigma-Aldrich (St. Luis, USA). PVOH (high molecular weight, 130,000 kDa) and chloroform (CHCl3) were also purchased from Sigma-Aldrich (St. Luis, USA). PCL (CapaTM FB100) was provided by CEPSA (Madrid, Spain). The buffer employed to establish the optimum pH conditions for the GOX enzyme was prepared using acetic acid (C2H4O2) provided by HONEYWELL (St. Luis, USA). 2.2 Preparation of solutions 2.2.1. Solutions for electrospinning Two solutions containing 7% PVOH in water and 10% PCL in chloroform were processed considering previous studies for the preparation of mats [21]. 2.2.1.1. PVOH solutions Solutions containing 7% PVOH in water were prepared to obtain PVOH membranes with and without the enzyme. The solutions required two hours under magnetic agitation on a heater at 50 ºC in order to be completely dissolved. To avoid solvent loss, the samples were stirred in closed containers, controlling the sample weight and adding water to counteract weight loss. 2.2.1.2. PVOH with encapsulated GOX solution PVOH membranes were synthetized with GOX at 10 U/mgmembrane. A PVOH solution with GOX enzyme was prepared assuming that the catalytic activity remained unaltered before and after the electrospinning process. The solutions were prepared right before the electrospinning procedure to avoid enzyme denaturalization. 2.2.1.3. PCL in chloroform solution A PCL solution (10%) was prepared. Due to the high volatility of chloroform, the weight loss was meticulously controlled, adding more solvent as needed. Since GOX is not soluble in this solution, the PCL membranes did not include the enzyme. 2.3. Membrane production The polymer solutions (PVOH and PCL) were introduced in a syringe to proceed with the electrospinning process. In this work, the electrospinning equipment was horizontally arranged to prevent the falling of solution drops from the syringe on the collector due to gravity. The operating conditions were stablished based on previous studies to obtain flawless nanofibers [21]. The parameters used for the manufacture of the both mats were the following: the needle-collector distance (11 cm), temperature (25±3 ºC), pump flow (0.4 ml/h), and voltage (12 kV). The mats were processed when the relative humidity was < 45%. To improve the properties of the synthetized membranes, the multilayer mats were studied by including GOX enzyme in the PVOH layer. The system consisted of a multilayer structure (PCL/PVOH/PCL), with each layer being independently electrospun after one another, coating the layer before them. 2.4. Membrane characterization 2.4.1. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectroscopy was used to determine the differences in composition between the various systems that could indicate the presence of the enzyme along with its possible denaturalization due to the electrospinning process. The FTIR spectrometer used to carry out the interaction between the sample and the IR radiation was a Bruker Hyperion 1000 (Massachusetts, USA). The obtained IR spectra show the transmittance of the sample against the wavelength from 4000 to 500 cm-1. 2.4.2. Measurement of GOX enzyme activity The enzyme activity unit for GOX is defined as “the required amount of GOX to catalyze the oxidation of 1 µmol of glucose to gluconolactone and H2O2 in 1 min at 25 ºC [22], due to the enzyme action mechanism. This flavoprotein catalyzes the oxidation of β-D-glucose to D-glucolactone and H2O2 using molecular oxygen as the electron acceptor. The analytical method to determine the activity of the enzyme in the different manufactured systems is based on its reaction mechanism. H2O2 is used to oxidize a chromogenic substrate in a secondary reaction, which causes the color of the solution to change, thus making it measurable by spectrophotometry. ABTS was the chromogenic agent used, and the followed method was proposed by Zenil et al. [23]. In this way, the samples were dispersed in 50 mM of pH 5.0 acetate buffer, with 0.1 M D-glucose solution acting as the substrate for the enzyme; 4 M HCl was used to stop the enzymatic reaction. 2.4.3. SEM and image analysis Scanning electron microscopy (SEM) is a very useful technique to obtain information regarding the morphology and composition of the samples. To study the surface morphology of the different systems, the samples were previously coated with gold (to make them conductive). After that, the samples were studied under a Zeiss EVO LS15 microscope (CITIUS) at an applied voltage of 10 kV. Two images were obtained for each sample with different magnifications (500x, 2000x and 10000x), which allowed selecting or rejecting the processed systems according to their morphology and the size of the obtained fibers. To analyze the SEM images of the different systems, the ImageJ free distribution analysis program was employed. In this study, the diameter of the fibers obtained by electrospinning was measured to evaluate the dispersion of the results and to obtain the size distribution. For each system, around 50 random measures were made in images with the same magnification. 2.4.4. Mechanical properties The samples were prepared from the 5x1 cm membranes with given thickness and they were fitted to the equipment using two grips: one fixed at the bottom and one at the top. The test was carried out on the different systems (PCL membranes, PVOH membranes with and without GOX and multilayer membranes with and without the enzyme) using different test conditions: dynamic and static mode. The equipment used was the DMA RSA3 from TA Instruments. All tests were carried out at room temperature (25±1 ºC) and according to standard ISO 570-2:2012, regarding the assessment of mechanical properties of polymeric samples [24]. system (pink) and the one containing the enzyme (in red), with the latter having a broad and heterogeneous distribution with higher values. Thus, it is assumed that the enzyme encapsulation in the PVOH polymeric matrix leads to a higher dispersion in the fiber diameter. Finally, the multilayer system containing the enzyme (blue) shows a more uniform size distribution and a fiber size between that of the PVOH system (with and without the enzyme) and that of the PCL system, that is, between their components. Thus, the combination of the PVOH membrane with enzyme encapsulation and the PCL membrane creates a synergic effect, resulting in improved size homogeneity when compared to the individual systems. In any case, the increase of fiber diameter does not involve a dramatic change in mat structure, with a fibrous structure remaining, which could have the same applications as before the addition of the enzyme. Furthermore, the high specific surface of the observed microstructures provides potentially higher bioactivities and, as a consequence, enable the obtained products to be employed for food conservation. In order to compare the morphology of the obtained fibers, Table 2 shows the mean fiber diameter for the studied membranes. This table evidences that all fiber diameters are of the order of hundreds of nanometers (from 214.7 to 388.5 nm), where the addition of GOX enzyme did not involve a dramatic increase in fiber diameter and about 2 microns, where a larger difference was found between PVOH and PCL fibers, with the latter being considerably thicker. By comparing the standard deviations, PVOH membrane fibers showed the highest fiber diameter homogeneity when compared to the PCL membrane fibers, which showed great deviation. In some previous research, Wang et al. [35] obtained a homogeneous fiber size distribution with similar average fiber diameter for electrospun PVOH fibers. In addition, Fernández-Pérez et al. [30] synthetized homogeneous PCL fibers with diameters around 200 nm, just like the ones obtained in this research. when using different processing parameters during the electrospinning process. For the conditions used in this research, PVOH exhibits a better behavior upon electrospinning than PCL. However, fiber size in the multilayer system was more similar to the one observed in the PVOH samples, which could be related to the fact that PVOH fiber morphology allows a higher membrane densification (higher number of PVOH fibers than PCL fibers in a given area), having a stronger influence on the average fiber size. It is also important to highlight the difference in fiber size, which can be observed in both the SEM images and Table 2, for the membranes obtained from the PVOH (7 wt.%) solution and the PVOH (7 wt.%) solution containing GOX enzyme (10 U/ml). Morphologically, small fiber widening can be observed in certain areas when including the enzyme, leading to a homogeneity loss compared to the reference systems (PVOH and multilayer system without the enzyme). Those deformations are shaped as a narrow ellipse. Regarding the multilayer system, it is not possible to observe the intermediate PVOH membrane, although it is assumed that the enzyme leads to the same deformations in all systems. These results are in line with those shown in Table 2, with the system with the lowest standard deviation being the PVOH membrane and the largest error corresponding to the PCL system. 3.4. Mechanical properties Mechanical properties are important parameters for determining and predicting characteristics, behaviors and usage of packaging materials. Apart from exhibiting good physical integrity, materials for this purpose should present certain resistance and good flexibility [36]. Dynamic tensile tests: Frequency sweeps Figure 5A and 5B show the results of the frequency sweep tensile tests obtained for the PVOH, PCL and multilayer (PCL/PVOH/PCL) membranes with and without the GOX enzyme (Fig. 5A and 5B, respectively). This test provides storage (E’) and loss (E’’) moduli as a function of the frequency. In all cases, the results show that the studied systems exhibit a mainly elastic behavior, since the elastic modulus (E’) is higher than the viscous modulus (E’’) (Figure 5A). In addition, it should be highlighted that the systems containing PVOH show higher viscoelastic moduli (E’ and E’’) than the membranes containing PCL, both individual and multilayer systems, although the latter improved only the properties of the PCL-based membranes. As can be observed in Figure 5B, the enzyme immobilization affected the mechanical properties of the individual PVOH and the multilayer system. This could be expected, since in the previous section (3.3) a change in the morphology of the fibers was induced by the enzyme immobilization. Thus, although the elastic modulus (E’) measurements seem to remain the same (a variation in the slope of the elastic modulus against frequency can be observed only for the multilayer system), the viscous modulus increases in all cases. This means that the systems containing the GOX enzyme exhibit a stronger viscous behavior and, as a consequence, the solid behavior of the systems decreases. This predominant solid behavior of these membranes is comparable with the rheological response of other bioplastic systems such as bioplastics from rapeseed, albumen, pea and soy proteins [37–39]. Tensile test to fracture Figure 5C shows the stress-strain curves obtained from the tensile tests of the studied samples. In all tests, the elastic limits that lead to plastic deformation, the break limits and the achieved strain can be observed. PVOH membranes exhibit a wider elastic range with higher slope (which corresponds to Young´s modulus) than the PCL and multilayer systems. The great toughness (area under the curve) of the PVOH sample compared to the rest of the membranes should be pointed out, as it reaches a greater plastic deformation. Furthermore, the maximum stress and strain at break decreased in the same order: PVOH membrane, PCL membrane and multilayer membrane. After enzyme incorporation, the mechanical properties of the PVOH membrane registered a strong decrease, reaching the values obtained for the PCL membranes, which according to results from Fig. 4, it could be related to the fiber dispersion observed for these systems [40]. This effect was not observed in the multilayer system with enzyme encapsulation, which can be related with the fact that the PCL layers do not contain the enzyme. This behavior has been previously observed for other systems where an active component has been included, such as the systems obtained by Fernández-Pérez et al. [30]. Table 3 shows the mean values from the tensile test. It can be observed that Young´s modulus showed the highest value in the individual PVOH membrane. However, once again, the PVOH membrane underwent a decrease in its properties with enzyme encapsulation, as happened in the previous tests (Figure 5A and 5B). Contrarily, the multilayer system showed the lowest maximum stress and strain at break values, even below the individual PCL system, although in this case enzyme incorporation did not lead to worse values. PCL membranes generally show an intermediate behavior between the PVOH and the multilayer systems. In any case, the mechanical properties of the material obtained are appropriate for use in the packaging industry, as they are of the same order of magnitude as other materials fabricated for the same purpose by El Fawal et al. [41]. 3.5. Antimicrobial activity test Figure 6 shows the results obtained for the E. coli in-vitro tests in the presence of the samples synthetized with GOX encapsulation, evaluating the antimicrobial activity of the systems. The first plate (Fig. 6A) did not contain E. coli bacteria in order to serve as a reference. The second and the third plates correspond to experiments with E. coli after 24 and 48 hours of growth (Fig. 6B and 6C, respectively). The plates containing bacteria (Fig. 6B and 6C) were incubated with the membranes including the GOX enzyme (samples 3 and 4) and without GOX enzyme (samples 1 and 2), indicating that the presence of encapsulated enzyme involves the inhibition of the growth of this microorganism after 24 and 48 hours. Immediately after 24 h of incubation (Fig. 6A), it was possible to visualize a halo around samples 3 and 4, in which E. coli growth was inhibited and, after 48 hours, the halo remained unchanged, with the unprotected areas showing greater bacterial growth. On the other hand, bacteria proliferated through the membranes without the enzyme (samples 1 and 2). These results suggest that the encapsulated membranes released the GOX enzyme, and that this enzyme was active after the electrospinning processing. Thus, the synthetized PVOH and the multilayer membranes showed bacteria inhibition capacity in the presence of glucose in a wide surface (wider than the membrane itself). In addition, this capacity prevailed for several days. 4. CONCLUSIONS The electrospinning technique is very useful and simple for processing polymer membranes aimed at the immobilization of enzymes. It is possible to immobilize enzymes using PVOH with a soluble matrix, allowing the release of the encapsulated enzyme. When the application requires a structurally stable system in aqueous medium, it would be suitable to use the PCL/PVOH/PCL multilayer membrane systems. SEM results indicate that PVOH fibers show a more reproducible behavior and greater homogeneity in fiber size than PCL membranes, whose fibers are acceptable but more heterogeneous. The morphology of the membranes result in a significant influence on the mechanical properties, as could be observed in the tensile tests results. The incorporation of the GOX enzyme drastically reduced the mechanical properties of the PVOH membrane. However, this does not occur in the multilayer system, since the PCL membranes present in the system contributes to maintaining the properties. In any case, the obtained membranes are suitable for use in service conditions. The results obtained from the bacterial culture indicate that both the PVOH membranes and the multilayer systems (PCL/PVOH/PCL) show inhibitory capacity for bacterial growth (i.e., antibacterial properties), due to the production of H2O2, which is a powerful antioxidant (corroborated by the kinetic measurements). To sum up, these results show that the membranes processed by electrospinning are suitable for the immobilization of enzymes, as they have good mechanical properties and antimicrobial activity, where the enzyme does not decrease its activity after the processing conditions. 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