1. Introduction
Pectin is one of the most abundant polysaccharides in nature, which is found in the middle lamella of cell wall, primary cell walls, and plasma membrane of plants [
1,
2]. It is commercially produced by the industrial waste of apple pomace, citrus peel, and sugar beet pulp [
3]. Pectin is mainly composed of linear chains of α-1,4 linked
d-galacturonic acid units but also different types of side chains may exist in its chemical structure such as those containing rhamnose, xylose, galactose, and arabinose [
2,
4]. Depending on the side chains, pectin domains are named differently such as homogalacturonan (HGA), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II), and xylogalacturonan (XGA) [
5,
6]. HGA units are habitually referred as ‘smooth’ regions of pectin and it comprises galacturonic acid groups whereas other units are called ‘hairy’ regions [
4,
7].
Pectin can be used as gelling, thickening, and stabilizing agent in the food and pharmaceutical industry. Moreover, the polyelectrolyte nature, biodegradability, biocompatibility, and water solubility of pectin also open up new uses including coatings or edible films for food packaging applications [
8,
9]. Nevertheless, the intrinsically high hydrophilicity and low mechanical strength of pectin-based films compared to conventional ones such as those made of high-density polyethylene (HDPE) and polypropylene (PP) currently limit the application of this carbohydrate [
10,
11,
12]. In this context, to enhance the mechanical integrity and also reduce brittleness, plasticizers are habitually added to the pectin formulations to form the films [
13]. The addition of different kinds of plasticizers, for instance glycerol [
14,
15,
16], sorbitol [
13,
17,
18], polyethylene glycol (PEG) [
13,
19] or xylitol [
20], to polysaccharide film-forming solutions during the casting process can improve both film formation and flexibility by enhancing the motion of polymer chains and reducing their intra- and intermolecular forces [
21,
22]. Additionally, water dissolution of pectin can be decreased by in situ cross-linking with divalent metal ions including Ca
2+, Zn
2+ or Mg
2+ [
23,
24]. Furthermore, the mechanical, thermal, and barrier properties of the pectin-based films can also be enhanced by blending with other biopolymers such as chitosan [
25,
26], cellulose, and its derivatives [
27,
28] or the addition of inorganics such as nanoclays [
11].
In the polymer literature, neat pectin films are technically not feasible to be produced by conventional melt technologies and they have been so far obtained by the solvent casting method using large contents of plasticizers [
24,
25,
29,
30]. In this regard, electrospinning is a novel technique that provides manufacturing of ultrathin fibers with diameters extending from several nanometers to a few micrometers [
31]. Electrospun nanofibers may offer many functional advantages such as superior mechanical properties, large surface-to-mass ratio, tailored fiber morphology, and the capability of encapsulation and subsequent release of active and bioactive principles [
32,
33,
34,
35,
36]. However, aqueous solutions of neat pectin cannot be electrospun due to the limited viscoelasticity of pectin and its insufficient chain entanglements [
37,
38,
39]. As a result, electrospun pectin nanofibers have been only obtained by blending with different synthetic polymers such as polyethylene oxide (PEO) [
39,
40,
41,
42,
43,
44,
45], polyvinyl alcohol (PVOH) [
46,
47,
48], and pullulan [
38]. In addition, ternary blends of alginate/pectin/PEO [
49] and chitosan/pectin/PVOH [
50] have been recently successfully electrospun for biomedical purposes.
The few studies reporting the development of electrospun pectin-based nanofibers have been mainly focused on the areas of antibacterial surfaces [
48], tissue engineering [
40,
41,
42,
50], drug delivery [
44], and encapsulation [
38,
49], whereas their utilization for food packaging applications remains unexplored due to the inherent discontinuity and porous structure of the nanofibers mats. Interestingly, electrospun mats can be subjected to a thermal post-treatment above the glass transition (T
g) and below the melting temperature (T
m) of the polymer, also termed annealing, in order to remove or minimize their porosity and produce continuous and homogenous films [
51,
52,
53,
54,
55]. Until now, this technology has been successfully applied to different polyester-type biopolymers with different potential applications in the food packaging field. For instance, electrospun poly(3-hydroxybutyrate) (PHB) films showed better optical properties, similar barrier performance, and higher elongation at break and toughness in comparison with equivalent films obtained by compression molding [
51]. Electrospun films of PHB, PVOH, and also polylactide (PLA) were also developed by electrospinning and originally applied as coating materials on a paper-based packaging material to develop multilayers with improved barrier properties against water and limonene vapors [
52]. In another study carried out by Cherpinski et al. [
53], a similar strategy was followed to coat cellulose nanopapers by PHB and poly(3-hydroxybutyrate-
co-3-hydroxyvalerate) (PHBV) electrospun layers. Similarly, electrospun ultrathin fibers of bio-waste derived PHBV were subjected to annealing by Melendez-Rodriguez et al. [
54] to successfully produce continuous biopolymer films with similar barrier performance than petroleum-based polyethylene terephthalate (PET) films. Other recent studies have been focused on the incorporation of antimicrobial or antioxidant ingredients in the electrospun fibers, which can be thereafter integrated as active layers in packaging structures. For example, Figueroa-Lopez et al. [
56] prepared electrospun active films of PHBV with antimicrobial and antioxidant properties by the incorporation into the fibers of different essential oils (EOs) and natural extracts (NEs). Also, Quiles-Carrillo et al. [
57] recently developed multilayer bioactive films with controlled release capacity of the natural antioxidant gallic acid (GA) by the incorporation of electrospun PLA interlayers into cast-extruded PLA films. Lastly, Radusin et al. [
58] recently prepared antimicrobial PLA films containing
Allium ursinum L. extract by electrospinning.
The aim of this study was to obtain, for the first time, electrospun pectin-based films as potential candidates for food packaging applications. First, various water-based pectin solutions containing different amounts of PEO and/or in combination with the addition of two different types of plasticizers were processed by electrospinning to determine the best system to produce a film. Thereafter, the selected electrospun mats were characterized and the most promising fibers were selected and subjected to annealing to produce pectin-based films. The morphology, chemical, and thermal properties of the films were reported. The optimal electrospun film was, finally, applied as an interlayer in a multilayer structure based on PHBV and the barrier properties of the resultant multilayers were analyzed and compared to an equivalent multilayer of a cast-film pectin interlayer.
2. Materials and Methods
2.1. Materials
Low methyl esterified amidated pectin was kindly received from AROMSA Inc. (Gebze, Turkey). The product (GENU pectin, LM-104 AS-FS, degree of esterification 27%, degree of amidation 20%) was produced and delivered in powder form by CP Kelco (Copenhagen, Denmark). PEO with molecular weight (Mw) of 2000 kDa, that is, PEO2000, was obtained in powder form as SENTRYTM POLYOXTM WSR N80-LEO NF grade by The Dow Chemical Company (Midland, MI, USA). PEG with Mw of 900 kDa, that is, PEG900, was provided by Honeywell Fluka Chemicals Company (Bucharest, Romania). Bacterial aliphatic copolyester PHBV was ENMAT™ Y1000P, produced by Tianan Biologic Materials (Ningbo, China) and distributed by NaturePlast (Ifs, France). The product was delivered as off-white pellets packaged in plastics bags. The biopolymer resin presents a true density of 1.23 g/cm3 and the pellets a bulk density of 0.74 g/cm3, as determined by ISO 1183 and ISO 60, respectively. Sorbitan monolaurate was obtained from Sigma-Aldrich S.A. (Madrid, Spain) as Span® 20. According to the manufacturer, its fatty acid composition was lauric acid (C12:0) ≥ 44%; balance primarily myristic (C14:0), palmitic (C16:0), and linolenic (C18:3) acids. Calcium chloride, dichloromethane, 2,2,2-trifluoroethanol (TFE), ≥99% purity, and glycerol, ≥99.5% purity, were all also purchased from Sigma-Aldrich S.A. (Madrid, Spain).
2.2. Preparation of Solutions
The total concentration of solids in distilled water to prepare the fiber-forming solution for electrospinning was set at 10 wt%. For this, pectin was first dissolved in water at 70 °C for 3 h and the solution was gently stirred overnight at room temperature. Then, PEO
2000 with or without a plasticizer, that is, glycerol or PEG
900, was added to the pectin solution and it was further stirred for 24 h. In all cases, Span
® 20 was added as a surfactant to the pectin solutions at 2 wt% with respect to the total solid weight content of the solution.
Table 1 summarizes the compositions of the water-based solutions prepared for electrospinning. For the electrospinning of the PHBV layers, the copolyester resin was dissolved at 10% (w/v) in TFE at room conditions during 24 h.
2.3. Characterization of Solutions
Prior to electrospinning, all the prepared pectin solutions were characterized in terms of their viscosity, surface tension, and conductivity. Solution viscosity was determined by a VISCO BASIC Plus L rotational viscosity meter equipped with a low-viscosity adapter (LCP) from Fungilab S.A. (San Feliu de Llobregat, Spain). Conductivity was measured in a conductivity meter XS Con6 from Lab-box (Barcelona, Spain). Surface tension was determined in an EasyDyne tensiometer K20 model Krüss GmbH (Hamburg, Germany) following the Wilhelmy plate method. All measurements were carried out at room temperature in triplicate.
2.4. Electrospinning
An electrospinning machine Fluidnatek
® LE500 (Bioinicia S.L., Valencia, Spain) placed in a closed chamber and connected to an environmental control unit was used in the lab mode. Fibers were collected in vertical mode on a rectangular plate collector covered with aluminum foil. The most suitable conditions for the electrospinning of pectin were determined during the process. The flow-rate of the pectin-base solutions was set at the highest value possible in order to attain the maximum yield. Flow-rate was optimal at 3 mL/h since higher values led to some droplets on the collector. The tip-to-collector distance was also adjusted by decreasing it until the fibers were formed, being optimal at 25 cm. Finally, the applied voltage was smoothly increased up to the point a stable jet was obtained. The most optimal values of voltage ranged from 16 to 20 kV. For the electrospinning of PHBV, the voltage was set at 10 kV, the tip-to-collector distance was 15 cm, and the flow-rate was 6 mL/h. These values were selected based on our previous study [
54]. All the experiments were conducted at 25 °C and 30% relative humidity (RH).
2.5. Washing and Drying
The resultant electrospun pectin-based fibers were washed by soaking the mats into dichloromethane for 60 s. Dichloromethane was chosen since pectin is not soluble in this solvent [
39], but it could facilitate fiber coalescence by reducing the porosity of the electrospun mats. The washed mats were then placed in a Vaciotem-TV (P. Selecta, Barcelona, Spain) vacuum drying oven connected to a Vacuubrand vacuum pump at 27 °C and 100 mmHg pressure for 18 h in order to remove the organic solvent.
2.6. Films Preparation
The washed and dried fibers were then subjected to annealing in a 4122-model press from Carver, Inc. (Wabash, IN, USA). A set of experiments were conducted to select the optimal temperature, time, and load to produce homogenous and transparent films. To this end, the electrospun mats were post-treated in the temperature range of 50–240 °C and the pressure range of 6–30 kN for times ranging from 5 s to 120 s. A pectin film was also prepared by casting as a control material. To this end, 2 g of pectin powder was dissolved in 100 mL of distilled water and then 0.92 g of glycerol was added. After 24 h of mixing, 10 mL of solution was poured into polystyrene (PS) petri dishes and left at room conditions, that is, 25 °C and 40%, for 3 days.
The multilayer films were prepared by placing either the solvent-casted or electrospun pectin-based films as an interlayer between two electrospun layers of PHBV. This was accomplished by electrospinning PHBV fibers on one side of the previously prepared pectin films. The resultant coated films were turned down and coated on the other side. The two side coated films were then placed in the press and annealed at 160 °C for 10 s, without pressure, based on our previous research [
55]. These annealing conditions were selected since the film-forming process is controlled by the external layers, which are habitually thicker. Control films made of two electrospun layers of PHBV without pectin were also prepared in the same conditions.
2.7. Characterization of the Electrospun Fibers and Films
2.7.1. Thickness and Conditioning
Prior to testing, the thickness of the electrospun mats and films was measured using a digital micrometer series S00014 (Mitutoyo Corporation, Kawasaki, Japan) with ±0.001 mm accuracy. Measurements were performed at five random positions and values were averaged. The samples were stored in a desiccator at 25 °C and 0% RH for 24 h before characterization.
2.7.2. Morphology
The morphologies of the electrospun fibers and the top views and cross-sections of the pectin-based films were investigated by a scanning electron microscope (SEM, Hitachi S-4800, Tokyo, Japan). The samples were cryo-fractures using nitrogen liquid. Prior to analysis, all the samples were coated with a gold/palladium alloy for 2 min by a Polaron sputter coater (Quarum Technologies, Kent, UK). A 5 kV voltage was applied during SEM analysis. Fiber diameters and layer thicknesses were determined by the software ImageJ, Java v.1.52a from the measurement of, at least, 50 fibers.
2.7.3. Thermal Analysis
The thermal properties of PEO2000 and pectin powders as well as the electrospun pectin-based fibers obtained from S3 and from S5 to S10 were determined by differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA). Thermal transitions were determined in a DSC-7 analyzer from PerkinElmer, Inc. (Waltham, MA, USA), equipped with a cooling accessory Intracooler 2 from PerkinElmer, Inc. Approximately 3 mg of sample was placed into the aluminum pan, while an empty pan was used as reference. Calibration was previously conducted using an indium sample. The samples were first heated from −70 °C to 160 °C, then cooled back to −70 °C, and then heated again to 300 °C. The heating and cooling rates were set at 10 °C/min. The experiments were conducted under nitrogen atmosphere and all DSC tests were performed in triplicate.
To ascertain their thermal stability, TGA was performed under nitrogen atmosphere in a Thermobalance TG-STDA Mettler Toledo model TGA/STDA851e/LF/1600 analyzer (Greifensee, Switzerland). TGA curves were obtained after conditioning the samples in the sensor for 5 min at 30 °C. The samples were heated from 25 °C to 700 °C at a heating rate of 10 °C/min. All TGA tests were also carried out in triplicate.
2.7.4. Fourier Transform Infrared Spectroscopy
Fourier transform infrared spectroscopy (FTIR) spectra of the raw materials, that is, the pectin powder, glycerol, and PEO2000, and also the electrospun fibers and film obtained from S7 solution were obtained from an average of 20 scans by a Bruker Tensor 37 (Rheinstetten, Germany) spectrometer connected with a Golden Gate of Specac, Ltd. (Orpington, UK) attenuated total reflection (ATR) accessory. ATR-FTIR was performed in order to analyze the existence of any chemical interactions between the components. The scans were collected in the wavelength values from 4000 cm−1 to 600 cm−1 at a resolution of 4 cm−1.
2.7.5. Color Measurements
The color of the pectin-based interlayers and the resultant multilayers in PHBV was carried out in a benchtop spectrophotometer Konica Minolta CM-5, from Hunter Associates Laboratory, Inc. (Reston, VA, USA). The Commission Internationale de l’Eclairage (CIE) standard illuminant D65 was used to assess the CIE Lab color space coordinates L*a*b* using an observer angle of 10°. L* represents the luminance (black to white), a* indicates the change between green and red, and b* represents the change from blue to yellow. The colorimeter was calibrated with a white standard tile and a mirror device for the black (no light reflection). The L*a*b* coordinate values were obtained on five different samples and the color difference (ΔE*) was calculated following Equation (1).
where ∆E*, ∆a*, and ∆b* corresponded to the differences between the color parameters of the multilayer samples and the values of the PHBV/PHBV multilayer. Color change was evaluated as follows: Unnoticeable (ΔE*
ab < 1), only an experienced observer can notice the difference (ΔE*
ab ≥ 1 and <2), an unexperienced observer notices the difference (ΔE*
ab ≥ 2 and < 3.5), clear noticeable difference (ΔE*
ab ≥ 3.5 and < 5), and the observer notices different colors (ΔE*
ab ≥ 5) [
59].
2.7.6. Permeability Tests
Water Vapor Permeance
The water vapor permeance of the multilayer films was measured according to the ASTM 2011 gravimetric method. In order to conduct this test, 5 mL of distilled water was put inside a Payne permeability cup (Inner diameter = 3.5 cm) (Elcometer Sprl, Belgium). The films were located in the cups so that on one side they were exposed to 100% RH, avoiding direct contact with water. Then, the cups were locked with silicon rings and kept in a conditioned desiccator (25 °C and 0% RH). The control samples were cups with aluminum films to estimate solvent loss through the sealing. The cups were weighed regularly for every 24 h using an analytical balance having an accuracy of ±0.0001 g, until the values reached a plateau. Water vapor permeation rate corresponded to the slope value of the steady state line of time versus weight loss per unit area and the weight loss was calculated as the total loss minus the loss through the sealing. Water permeance was obtained by correcting the water vapor permeation rate for the permeant partial pressure. Tests were conducted in triplicate.
Limonene Vapor Permeance
Limonene vapor permeance of the multilayer films was determined as similar as described above for water vapor. For this 5 mL of d-limonene was placed inside the Payne permeability cups and the cups containing the films were placed at controlled room conditions of 25 °C and 40% RH. The limonene vapor permeation rates were estimated from the steady-state permeation slopes and the weight loss was calculated as the total cell loss minus the loss through the sealing. Limonene permeance was obtained by correcting the limonene vapor permeation rate for the permeant partial pressure. Tests were conducted in triplicate.
2.8. Statistical Analysis
All data were analyzed statistically by SPSS Statistics 17.0 (IBM, Chicago, IL, USA). Tukey’s HSD test was used to determine the significant differences among samples (p < 0.05). Different superscripts show statistically different results. Unlike groups of letters were used to express each group of properties.