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shell electrospun structures by using hydrophilic and hydrophobic polymers
Polymer Testing 93 (2021) 106937
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Antimicrobial food packaging system based on ethyl lauroyl arginate-loaded core/shell electrospun structures by using hydrophilic and hydrophobic polymers ˜o Vidal , Eliezer Vela ´squez , María Jos´e Galotto , Carol Lo ´pez de Dicastillo * Cristian Patin Packaging Innovation Center (LABEN), Department of Food Science and Technology, Technology Faculty, University of Santiago de Chile (USACH), CEDENNA (Center for the Development of Nanoscience and Nanotechnology), Santiago, 9170201, Chile
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrospinning Ethyl lauroyl arginate Core/shell fiber Antimicrobial Food packaging
Antimicrobial compounds can be encapsulated into core/shell structures through coaxial electrospinning in order to develop novel active food packaging materials. In this work, poly (vinyl alcohol)-(ethyl lauroyl arginate)/poly (lactic acid) core/shell fibers, (PVOH-LAE/PLA)f, were developed with the purpose to study the influence of combining a hydrophilic and a hydrophobic polymer on LAE release. LAE release studies were carried out in an aqueous and fatty food simulant, and data were analyzed through a phenomenological mass transfer model. Flatribbon fibers with a core/shell structure were observed by TEM. PLA crystallinity degree of coaxial fibers did not present significant differences with uniaxial PLA fibers, although thermal stability and the glass transition temperature decreased. The maximum concentrations of LAE released from (PVOH-LAE/PLA)f fibers to both simulants reached the MIC values of LAE against Listeria innocua, evidencing a higher affinity of LAE towards fatty simulant and a fast diffusion from fibers in the aqueous simulant.
1. Introduction Nowadays, technology has allowed the use of novel techniques as electrospinning for the development of new active food packaging ma terials. Unlike of traditional methods used to obtain packaging materials such as extrusion, casting or coating, the electrospinning is an efficient, simple, scalable and economic technology able to generate different micro- and nanostructures as fibers or spherical particles from a poly meric solution [1]. Fibers are produced when a voltage is applied to a polymeric solution obtaining a jet with a conical structure known as Taylor Cone’s and the solvent is evaporated in order to form ultrafine structures with a high aspect ratio [2,3]. Several polymers such as poly (lactic acid) (PLA) and poly (vinyl alcohol) (PVOH) have been used in the development of food packaging materials. PLA is a linear aliphatic thermoplastic polymer produced by the fermentation of the starch from natural resources such as potatoes, sugarcane, or corn, and it is “generally recognized as sure” (GRAS) by the Food and Drug Administration (FDA) [4,5]. On the other hand, PVOH is a hydrophilic, synthetic, semicrystalline, biocompatible and non-toxic polymer which is also widely used in food packaging [6,7]. Electrospinning technique can be used to obtain not only polymeric
fibers based on PLA or PVOH, but also their functionalization through the incorporation of active agents into these polymers. Several active compounds such as essential oils, natural extracts and antimicrobial or antioxidants substances have been incorporated into PLA or PVOH to produce active electrospun materials [8–10]. A compound with high antimicrobial activity that has been scarcely studied in the development of active fibers is the ethyl lauroyl arginate (LAE) [11]. LAE is a surfactant cationic derived from natural substances such as lauric acid, L-arginine and ethanol. It is a white powder with a melting temperature between 50 and 60 ◦ C and a degradation temper ature of 107 ◦ C, considered GRAS by FDA and authorized as a food preservative by European Food Safety Authority (EFSA) [12]. On the other hand, several studies have reported a burst release of the com pounds from active single electrospun fibers reducing its antimicrobial effectivity in food packaging applications [13,14]. For this reason, the development of core/shell structures through coaxial electrospinning to slowdown the release of encapsulated active compounds is an innova tive alternative to design packaging materials in order to extent food shelf life [15,16]. The traditional system of the coaxial electrospinning is composed of a voltage source, a stainless-steel coaxial needle, two in jection pumps and a flat or rotatory collector [17]. Thus, the use of this
* Corresponding author. E-mail address: [email protected] (C. L´ opez de Dicastillo). https://doi.org/10.1016/j.polymertesting.2020.106937 Received 19 August 2020; Received in revised form 14 October 2020; Accepted 26 October 2020 Available online 31 October 2020 0142-9418/© 2020 The Authors. Published by Elsevier Ltd. This is an open (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Polymer Testing 93 (2021) 106937
technique has principally allowed to combine different properties of the shell and core polymers into one structure in order to: (i) obtaining fi bers from non-electrospinnable materials, (ii) developing hollow fibers, (iii) protecting sensitive compounds to environment/processing condi tions, and (iv) modifying the release of the encapsulated compound [18–20]. In this work, antimicrobial polymeric fibers with a core/shell structure, whose inner and outer structures were composed by PVOH/ LAE and PLA, respectively, were developed through coaxial electro spinning with the aim of studying its morphological and thermal prop erties and release kinetics of the active compound.
for 24 h to obtain fresh early-stationary phase cells. 2.2. Antibacterial activity of LAE The antibacterial activity of LAE was tested against Gram (− ) E. coli and Gram (+) L. innocua bacteria by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentra tion (MBC) following the methodology of Muriel-Galet et al. (2012) [21]. A stock solution of LAE was previously prepared under ultrapure water. Subsequently, serial dilutions were done in sterile trypticase soy broth (TSB) contained in assay tubes. On the other hand, a loopful of each microorganism in stationary phase was transferred to TSB and incubated at 37 ◦ C until the exponential phase was reached (108 CFU/mL). A (1/10) triple dilution was done until a final concentration of 105 CFU/mL bacteria. Subsequently, 100 μL of this solution was added in the assay tubes that contained LAE and the samples were incubated at 37 ◦ C and 150 rpm for 24 h. After this period of contact between mi croorganisms and the antimicrobial compound, the turbidity of each tube as indicator of microbial growth was measured at 600 nm with a UV–vis spectrophotometer (Pharo 300 Spectroquant®, Germany) using TSB as blank. Subsequently, serial dilutions and spread plate method were applied to determine the cell concentrations. MIC was reported as the lowest LAE concentration that inhibited the growth of the micro organisms in TSB, while MBC was the lowest LAE concentration required to reduce the viability of the initial bacterial inoculum by a reduction ≥99.9%.
2. Material and methods 2.1. Polymers, chemicals and microorganisms Poly (lactic acid) 2003D (specific gravity 1.24) was obtained from Natureworks® (USA) and Gohsenol type AH-17 poly (vinyl alcohol) (saponification degree 97–98.5% and viscosity 25–30 mPa s) was ob tained from The Nippon Synthetic Chemical co. (Japan). Ethyl lauroyl arginate (LAE) was supplied by PRINAL (Chile). Chemical reagents such as chloroform (CLF), dimethylformamide (DMF), trifluoroacetic acid (TFA) and ethanol (EtOH) were obtained from Sigma Aldrich (Chile). Gram (+) Listeria innocua ATCC 33090 and Gram (− ) Escherichia coli ATCC 25922 bacteria were obtained from Biotechnology and Applied Microbiology Laboratory (Chile) and maintained in trypticase soy agar (TSA) at 4 ◦ C until their use. For the experimental assays, a loopful of each bacterial strain was transferred to TSA plate and incubated at 37 ◦ C
Fig. 1. Transmission electron microscopy (TEM) images of antimicrobial polymeric core/shell fibers (PVOH-LAE/PLA)f: (A–B) without imperfections; and (C–D) with imperfections. 2
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2.3. Electrospinning process of polymeric antimicrobial fibers (PVOHLAE/PLA)f
reverse phase Waters Symmetry® C18 (5 μm, 3.9 mm × 150 mm). The mobile phase consisted of a mixture acetonitrile and aqueous 0.1% TFA (50:50) at a flow rate of 1 mL/min [24]. In order to quantify LAE extracted, a calibration curve based on peak area against standard LAE concentrations solutions between 5 and 50 ppm was constructed.
Polymeric antimicrobial fibers (PVOH-LAE/PLA)f with core/shell structure were obtained through coaxial electrospinning system (Spraybase® power Supply Unit, Ireland). PVOH and PLA, polymers with different water affinity based on their composition and polarity, were used to obtain core and shell structures, respectively. PLA solution at 10% (w/v) under a solvent mixture of CLF:DMF (7:3) and an aqueous solution of PVOH at 10% (w/v) containing LAE at 2.5 wt% respect to the polymer were previously prepared. The concentration of LAE was selected according to results of the study presented in Supplemental File, Appendix 1, Both polymeric solutions were transferred to 5 mL syringes and connected through PTFE tubes to a coaxial device that contained two concentric stainless-steel needles of inner diameters 0.7 mm and 2.1 mm for the core and shell systems, respectively. Electrospinning device was set up with a distance of 14 cm between the tip of the coaxial needle and the collector, a voltage between 10 and 12 kW, and an internal and external flow rates of 0.5 and 1.8 mL/h for core and shell solutions, respectively. Uniaxial fibers PLAf, PVOHf and (PVOH-LAE)f were also obtained through electrospinning in order to evaluate and compare their thermal properties with the coaxial antimicrobial fibers (PVOH-LAE/ PLA)f. Processing parameters to obtain uniaxial fibers were: flow rate at 0.5 mL/h, single needle with 0.7 mm diameter, 14 cm distance and a voltage between 10 and 16 kW.
2.5. Study of the release kinetics of LAE from electrospun fibers with core/shell structure 2.5.1. Release assay procedure The release of LAE from (PVOH-LAE/PLA)f electrospun fibers to a fatty (EtOH 95%) and aqueous (EtOH 10%) food simulants was evalu ated according to EU Regulation N◦ October 2011 about materials and plastic objects to contact with food. Electrospun samples of 14 μm thickness and a weight of 0.014 g were placed in contact with the sim ulants with a ratio area/volume of 6 dm2/L at 40 ◦ C during 72 h in order to determine the active release kinetics. The released LAE was periodi cally analyzed through HPLC following the methodology earlier mentioned in section 2.4.3. 2.5.2. Determination of the LAE partition and diffusion coefficients The extent of a compound release is controlled by the phase equi librium and is given through the partition coefficient (K). The kinetics of the compound release process is defined by the diffusion coefficient (D) that measures the velocity of the particle diffusion in a polymeric matrix until the thermodynamic equilibrium is reached [25,26]. K and D co efficients of LAE were estimated based on the experimental results of this compound release from the electrospun fibers to food simulants. The partition coefficient, K, was calculated in the equilibrium as the ratio between LAE concentration in the electrospun fibers (Cf) and in the – Cf/Cs. simulant (Cs), K– The kinetics of the mass transport processes into the fibers were characterized through the Fick’s law, considering the limit conditions of the experiments such as: (i) presence of an equilibrium partition, (ii) a limited volume of solvent, and (iii) the coefficient D associated with the LAE transport into the fibers is independent of the time and position. Following these assumptions and the integration of Fick’s law, the mass ratio of released LAE into the solvent at time “t” and in the equilibrium ∞ ∑ S) was expressed through equation (2): m(t) = m(t)⋅(A⋅L⋅K+V =1− VS ⋅ciP ⋅A⋅L mfS n=1 ( ) 4⋅D⋅q2n ⋅t 2α(1+α) + exp − mtmSf = mt⋅(A⋅L⋅K + VS)VS⋅cPi⋅A⋅L = 1-n = 2 2 2 1+α+α q L
2.4. Characterization of active electrospun mats 2.4.1. Morphological analysis The internal morphology of (PVOH-LAE/PLA)f coaxial fibers was observed with an transmission electronic microscope (TEM) (Hitachi HT7700, Japan). The samples were collected on a carbon coated copper specimen grid during 10 s, and TEM micrographs of the coaxial fibers were obtained with different magnifications. 2.4.2. Thermal properties Thermal stability of the electrospun fibers was studied through thermogravimetric analysis (TGA) carried out in a Stare TGA/DSC sys tem (Mettler Toledo GC20, Switzerland). 5–6 mg of each sample were placed into alumina capsules and heated from 30 to 600 ◦ C at 10 ◦ C/min heating rate under nitrogen atmosphere. The effect of LAE incorporation and the coaxial processing on the thermal properties of both polymers were studied through differential scanning calorimetry (DSC) carried out in a Stare TGA/DSC system (Mettler Toledo GC20, Switzerland). 4–5 mg of each sample were placed into aluminum capsules and a thermal heating program from 0 to 250 ◦ C at 10 ◦ C/min heating rate was carried out under nitrogen atmosphere. Thermal parameters such as glass transition temperature (Tg), cold crystallization temperature (Tcc), melting temperature (Tm), and melting (ΔHm) and cold crystallization (ΔHcc) enthalpies were reported. Furthermore, the crystallinity of the PLA and PVOH polymers (Xc) was calculated through equation (1): Xc% = ΔHm-ΔHccΔHmo*W
n
1∞2α1+α1+α+α2qn2+exp-4⋅D⋅qn2⋅tL2mtmSf = mt⋅(A⋅L⋅K + VS) VS⋅cPi⋅A⋅L = 1-n = 1∞2α1+α1+α+α2qn2+exp-4⋅D⋅qn2⋅tL2 (2)
mtm∞ = 1-n = 1∞2α1+α1+α+α2qn2+exp-4Dqn2tL2
Where mt is the mass of LAE in the food simulant at t time, m⍰ is the mass of LAE in the food simulant in the equilibrium, L is the thickness of the film (cm), D is the diffusion coefficient (cm2/s), and t is the time (s). The α parameter is the mass ratio of the compound between liquid and polymer phases in the equilibrium, given by α = Vs/(K*A*L), where Vs is the volume of solution and A is the film area. Furthermore, qn represents the positive solutions of equation (3):
(1)
(3)
− tanqn = − αqn
Where ΔHm0 is the melting enthalpy of a wholly crystalline polymer (PLA = 93.1 J/g and PVOH = 138.6 J/g) [22,23], and W is the mass fraction of the polymer in the fiber.
The experimental data were fixed to equation (3), and the coefficient D was estimated using Solver of Excel 2016 and Sigmaplot v. 10.0 programs [27].
2.4.3. Quantification of LAE in the coaxial (PVOH-LAE/PLA)f fibers The effective concentration of LAE in the coaxial fibers was deter mined through an extraction process with ethanol and a later analysis through high-performance liquid chromatography (HPLC). 0.1 g of mat was put into a vial with 15 mL of EtOH and stirred at 60 ◦ C and 300 rpm for 3 h. Subsequently, liquid phase was filtered and injected (20 μL) in a HPLC chromatograph (Waters Alliance, USA) equipped with a column of
Table 1 Antibacterial activity of LAE expressed as MIC and MBC. Listeria innocua Escherichia coli
3
MIC (ppm)
MBC (ppm)
10 20
70 40
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2.6. Statistical analyses
diameters can be associated to differences in electrospinning process conditions such as: i) presence of Triton X-100 surfactant in the core solution, ii) a lower concentration of PLA in the shell, iii) use of higher voltage (19 kV), iv) lower flow rates in the core (0.1 mL/h) and shell (0.2 mL/h) solutions, v) different inner and outer needle diameters, and vi) use of EtOH:water mixture for PVOH solution. Likewise, da Silva et al. (2019) obtained diameters between 155 and 244 nm in core/shell nanofibers by using polymeric solutions of PLA at 20% (w/v) for the shell and PVOH at 15% (w/v) for the core with medical purposes. Authors have observed that the increase of the nanofibers diameters was principally produced by an increase in the flow rates of the shell solution from 0.5 to 1 mL/h and core solution from 0.06 to 1 mL/h. Besides, some micrographs showed also the presence of transverse fibers in the nanofibers [32].
Thermal properties were statistically analyzed by variance analysis (ANOVA) and Tukey’s Test, using InfoStat Program (2016) to detect differences between parameters with a confidence level of 95% (p < 0.05). The experimental design was random type for two replicates. 3. Results and discussion 3.1. Antibacterial activity of LAE Results of antibacterial activities of LAE against L. innocua and E. coli bacteria were reported as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), as Table 1 shows. Results revealed LAE exhibited high antibacterial activity against both micro organisms with MIC values of 10 and 20 ppm for L. innocua and E. coli, respectively. MIC values of LAE for both bacteria were lower than those values reported for other compounds such as essential oils and nano particles, demonstrating its effectiveness as antibacterial compound [28, 29]. On the other hand, a bactericidal effect was produced at 70 ppm for L. innocua and 40 ppm for E. coli. The great antibacterial activity of LAE is attributed to its behavior as cationic surfactant and its chemical structure able to react with the bacteria membrane. This reaction can produce the inhibition of the cell metabolic processes due to the increase of the permeability between cytoplasm and external layer of the Gram (− ) bacteria and between cell membrane and cytoplasm of Gram (+) bacteria [12,30].
3.3. Thermal properties Thermal parameters from the first and unique heating process are presented in Table 2. Uniaxial PLAf, PVOHf and (PVOH-LAE)f fibers were also developed in order to study the effect of LAE incorporation into PVOH thermal parameters, and the coaxial electrospinning process over PLA and PVOH thermal behavior. PLA fibers presented a Tg at approx. 73 ◦ C, a crystalline rearrangement at 91 ◦ C (Tcc) and a melting peak at 154 ◦ C (Tm). PVOH fibers showed a Tg and Tm at 49 and 223 ◦ C, respectively. The incorporation of LAE in PVOH fibers increased Tg value and significantly decreased Tm due to the chemical interactions between PVOH and LAE. These interactions possibly corresponded to hydrogen – O, OH) and the bonds between the functional groups of the polymer (C– – O, NH2 and NH) [30]. Unlike other works based on the surfactant (C– incorporation of active compounds into polymers, LAE did not present a plasticizer effect. The lower mobility of PVOH chains due to these in teractions significantly reduced crystallinity because the surfactant hindered the growth of polymeric crystals evidenced by a decrease in melting enthalpy and PVOH crystallinity degree of (PVOH-LAE)f [24]. On the other hand, Tg and Tcc temperatures of coaxial (PVOH-LAE/PLA)f fibers exhibited lower values than those of control uniaxial fibers PLAf. This fact was attributed to hydrogen bonds interactions between PLA and PVOH chains occurred in the contact zone between inner and outer structures [31]. Nonetheless, melting temperature, melting enthalpy and crystallinity degree of PLA were not significantly affected by coaxial electrospinning process. TGA thermogravimetric curves and their corresponding derivatives (DTGA) of LAE and fibers are shown in Fig. 2. PLAf initiated its degra dation at 280 ◦ C with a maximum degradation temperature at 353 ◦ C. PVOHf and PVOH-LAEf presented a weight loss below 100 ◦ C associated with adsorbed water due to the hydrophilicity of these components, and PVOH onset decomposition was registered at 250 ◦ C, in accordance with a previous work [33]. Subsequently, PVOHf evidenced two degradation processes with maximum values at 322 and 440 ◦ C, associated with the lateral groups separation and the degradation of backbone chain, respectively (Fig. 2B) [3,32]. LAE presented an onset decomposition temperature lower than polymers and degraded between 200 and 450 ◦ C, similarly to reported values in previous works [34]. The incor poration of LAE in PVOH produced a reduction of the polymeric thermal stability, accelerating its onset decomposition from 250 to 233 ◦ C, which can be attributed to the lower crystallinity of (PVOH-LAE)f respect to PVOHf (Table 2). The disruption of the crystal structure caused by LAE formed a network with lower stability due to competitive interactions PVOH-LAE and PVOH–PVOH chemical groups. Similar results have been reported for LAE incorporated into chito san/poly (ethylene oxide) fibers, where hydrogen bonds between poly mers and LAE were suggested by changes in FTIR bands at 3500-3100 – O) [11]. As Fig. 2B shows, the cm− 1 (-OH and –NH) and 1634 cm− 1 (C– degradation profile of (PVOH-LAE/PLA)f coaxial structures exhibited a similar behavior than PLAf probably due to the higher concentration of
3.2. Morphology of (PVOH-LAE/PLA)f electrospun fibers Coaxial electrospinning process resulted on the development of ho mogenous and bead-free (PVOH-LAE/PLA)f fibers, and TEM analysis confirmed their core/shell structure. Fig. 1A and B shows thick uniform coaxial fibers formed by a PVOH-LAE/PLA core/shell structure with diameters of 1.23 and 0.61 μm for the outer and inner layers, respec tively. The coaxial electrospinning process is more complex than uni axial system because it is dependent on the variables of both polymers, such as solution viscosity, conductivity, and flow rate. In some occasions during this process, some imperfections occurred and thin cross-fibers were scattered. As Fig. 1C and D shows, these thinner fibers exhibited inner and outer diameters of approx. 350 and 600 nm, respectively, and 15 nmtransversal fibers. Electrospun nanofibers based on PVOH and PLA with lower diameters have been previously obtained but focused on me chanical properties for biomedical applications. PVOH/PLA and PLA/ PVOH fibers without active compound were produced with inner and outer diameters of 35 nm and 165 nm, respectively [31]. Differences in Table 2 DSC parameters of the fibers. Fiber
Tg (◦ C)
Tcc (◦ C)
ΔHcc (J/g)
Tm (◦ C)
ΔHm (J/g)
PLAf
73.2 ± 0.7d 49.2 ± 0.3a 55.2 ± 0.6b 67.4 ± 0.8c
91.6 ± 0.5a –
12.6 ± 0.5b –
–
–
87.6 ± 2.5a
10.1 ± 1.0a
154.4 ± 0.4a 223.2 ± 1.0c 197.3 ± 0.3b 152.9 ± 0.2a
29.6 1.3a 68.6 0.2c 46.8 4.9b 23.2 0.1a
PVOHf (PVOHLAE)f (PVOHLAE/ PLA)f
Xc (%) ± ± ± ±
18.2 ± 1.9a 49.5 ± 0.2c 33.8 ± 3.6b 18.0 ± 1.5aa
Lowercase letters (a-d) indicate significant differences of DSC parameters be tween samples according to ANOVA analysis and Tukey’s test (p < 0.05). a Crystallinity was calculated respect to melting heat of 100% crystalline PLA, major component of the core/shell (PVOH-LAE/PLA)f fiber. 4
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Fig. 2. Curves (A) TGA and (B) DTGA of LAE and electrospun fibers.
this polymer in these fibers. Nevertheless, the maximum degradation occurred at lower temperature (330 ◦ C) than for PLAf because of the earlier degradation of PVOH and LAE [23].
ethanol into PLA matrix that produced a swelling of the polymeric matrix and the modification of its transport properties. LAE presents also a slightly higher solubility into ethanol than in water, therefore the release of this antimicrobial compound to the fatty simulant could be favored [35,36]. The maximum concentration of released LAE into the aqueous and fatty simulants were approx. 9.8 and 12.3 ppm, respec tively. These values evidenced that 29 and 36 wt% of total amount of encapsulated LAE into core/shell fibers was released towards the aqueous and fatty simulants, respectively. Interestingly, these values were similar to MIC value previously obtained for L. innocua (10 ppm), therefore the efficiency of these materials against this bacterium was confirmed. The diffusion coefficient, D, of the active compound into the poly meric matrix and the partition coefficient, K, that represents the extent of the compound release from the material to food are important pa rameters to design active food packaging materials [26]. Therefore, K and D coefficients of LAE-containing (PVOH-LAE/PLA)f fibers to 10 and
3.4. Study of the release kinetic of LAE from (PVOH-LAE/PLA)f The development of electrospun core/shell (PVOH-LAE/PLA)f fibers maintained a high encapsulation efficiency of this antimicrobial com pound during coaxial electrospinning process. The extraction process of these active fibers revealed an encapsulation efficiency value of 94% and the concentration of 0.51 wt% of LAE respect the total coaxial fiber. The release kinetic curves of LAE from (PVOH-LAE/PLA)f core/shell fibers to aqueous and fatty food simulants at 40 ◦ C are displayed in Fig. 3. LAE release curves indicated an exponential growth until an equilibrium during the first hours for both simulants, although the released active compound concentration into 95% EtOH was higher than in 10% EtOH. This behavior can be explained by the plasticizer effect of 5
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across them, from the PVOH core towards PLA shell [24,37]. Nonethe less, as it was earlier mentioned, total released LAE to the simulant in the equilibrium could be finally favored by a higher swelling grade of PLA caused by a higher concentration of ethanol in the fatty food simulant. Furthermore, several factors, such as structure and crystallinity of material, interaction between the polymer and active agent, and the type of polymer or active compound, can influence the particle speed diffusion into polymeric matrix and its posteriorly release towards the simulants [26,32,38]. Thus, D values of LAE into PLA obtained in this work were lower than values obtained in previous studies based on active PLA films and electrospun mats. A work developed by Torres et al. (2017) obtained D coefficients between 2.6 × 10− 11 and 1.7 × 10− 13 m2/s when PLA films loaded with thymol through carbon dioxide su percritical impregnation were put into 10% EtOH and 95% EtOH at 40 ◦ C for 40 h, respectively [39]. Likewise, D values between 1.9 and 2 ´pez de Dicastillo et al. (2017), when × 10− 13 m2/s were obtained by Lo the antioxidants compounds of PLA films extruded with merk´en were released into 10% EtOH and 95% EtOH at 40 ◦ C [40]. In both studies, the incorporation of the active compounds decreased the crystallinity of the materials generating more polymeric amorphous regions that could facilitate the fast diffusion of the compounds, and therefore, high D values. On the contrary, in this work, the crystallinity of PLA in the coaxial fibers was not altered by the incorporation of LAE (see Table 2), and thus, a slower diffusion occurred. On the other hand, D values are also influenced by the molecular weight of active compounds. Some works have evidenced that higher D coefficients have been obtained when compounds with lower weight ´pez de molecular than LAE were incorporated into electrospun fibers. Lo Dicastillo et al. (2018) obtained a D value of 1 × 10− 12 m2/s when PLA nanofibers loaded with cinnamaldehyde were put into 50% EtOH at 40 ◦ C [8]. Another study of PLA/PVOH coaxial fibers loaded with cur cumin exhibited D values between 5 and 5.5 × 10− 13 m2/s into 50% EtOH and 10% EtOH at 40 ◦ C, respectively [26]. Thereby, in spite of the good affinity and solubility of LAE towards both food simulants, the development of a coaxial electrospun structure has permitted to slow down the kinetic release of the active compound.
Fig. 3. Release kinetic curves of LAE from electrospun fibers to aqueous and fatty food simulants.
Fig. 4. Mathematical modulation of release kinetics of LAE from electrospun fibers to aqueous and fatty food simulants.
4. Conclusions Uniform antimicrobial fibers with core/shell structure were suc cessfully obtained through coaxial electrospinning technique. The maximum degradation of coaxial fibers (PVOH-LAE/PLA)f occurred at lower temperature than uniaxial PLAf probably due to the presence of PVOH and LAE whose thermal stabilities were lower. The incorporation of LAE in PVOHf decreased its glass transition temperature and crys tallinity possibly due to chemical interactions with this antimicrobial compound. The glass transition temperature of PLA in (PVOH-LAE/ PLA)f fiber decreased but its crystallinity degree was maintained. LAE exhibited a high affinity towards the fatty food simulant evidenced by the highest released LAE concentration, and therefore the lowest parti tion coefficient. Released LAE concentrations into both simulants reached MIC value for L. innocua. On the other hand, LAE diffusion into (PVOH-LAE/PLA)f fibers was faster in the aqueous simulant than in the fatty simulant due to the fast interaction of the water molecules with the hydrophilic inner structure of the coaxial fibers. The results suggested that the developed core/shell electrospun fibers containing LAE could be used in food packaging for maintaining or extending food shelf life. Furthermore, an increase of LAE concentration in the coaxial fibers could be achieved by modifying the concentrations of the core and shell polymeric solutions, and therefore, the development of antimicrobial materials against bacteria with higher MIC values could be attained.
95% EtOH as food simulants were studied through equations (2) and (3) and results were fitted in mathematical modelling (Fig. 4). Partition coefficients were estimated when released LAE from electrospun mate rials achieved the plateau in each kinetic profile. K values were 164.4 and 55.9 in 10% EtOH and 95% EtOH, respectively, indicating a large release extent on both food simulants, although a higher affinity of LAE for the fatty simulant. In addition to the good solubility of the antimi crobial compound in both simulants, the difference of K values can be attributed to the plasticizing effect of ethanol on PLA shell structure of the coaxial fibers. Likewise, this high affinity of LAE to both simulants was also evidenced when these partition coefficients were compared with K values of molecules with a hydrophobic behavior as curcumin. A release study of this polyphenol from coaxial PVOH/PLA fibers towards 10% and 50% EtOH exhibited K values between 349 and 10836 [26]. Fig. 4 reveals the experimental data greatly fitted with theoretical curves following Fick’s law. As Fig. 4 shows, the curves slope of released LAE kinetics were principally dependent on the type of food simulant. Although the extent of the LAE release was higher into fatty food sim ulant, the diffusion of this antimicrobial compound throughout (PVOHLAE/PLA)f fibers in the aqueous simulant was slightly faster (D = 3.9 × 10− 14 m2/s) than in the fatty simulant (D = 2.4 × 10− 14 m2/s). This could be attributed to the higher amount of water molecules in the aqueous simulant which are smaller than LAE and EtOH molecules, and have a higher affinity towards the hydrophilic polymer PVOH. There fore, water molecules could have penetrated faster into the inner structure of the coaxial fibers and accelerated the LAE release diffusion
CRediT authorship contribution statement ˜ o Vidal: Methodology, Validation, Formal analysis, Cristian Patin Investigation, Data curation, Writing - original draft. Eliezer 6
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´squez: Conceptualization, Data curation, Writing - review & edit Vela ´ Galotto: Resources, Supervision. Carol ing, Visualization. María Jose ´ pez de Dicastillo: Project administration, Funding acquisition, Lo Writing - review & editing, Supervision.
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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 The authors acknowledge the financial support of Agencia Nacional ´n y Desarrollo (ANID) (Chile) through the Fondecyt de Investigacio Regular Project N◦ 1200766, the Doctoral Scholarship CONICYTPFCHA/Doctorado Nacional/2019–21190316 and the “Programa de ´gicos de Exce Financiamiento Basal para Centros Científicos y Tecnolo lencia” (Project AFB180001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.polymertesting.2020.106937. References [1] A. Alehosseini, L.G. G´ omez-Mascaraque, M. Martínez-Sanz, A. L´ opez-Rubio, A. Ali, M.-S. Laura, G. G´ omez-Mascaraque, Marta, A. L´ opez-Rubio, Electrospun curcuminloaded protein nanofiber mats as active/bioactive coatings for food packaging applications, Food Hydrocolloids 87 (2019) 758–771, https://doi.org/10.1016/J. FOODHYD.2018.08.056. [2] L. Quiles-Carrillo, N. Montanes, J. Lagaron, R. Balart, S. Torres-Giner, L. QuilesCarrillo, N. Montanes, J.M. Lagaron, R. Balart, S. Torres-Giner, Bioactive multilayer polylactide films with controlled release capacity of gallic acid accomplished by incorporating electrospun nanostructured coatings and interlayers, Appl. Sci. 9 (2019) 533, https://doi.org/10.3390/app9030533. [3] C. L´ opez de Dicastillo, C. Pati˜ no, M.J. Galotto, J.L. Palma, D. Alburquenque, J. Escrig, Novel antimicrobial titanium dioxide nanotubes obtained through a combination of atomic layer deposition and electrospinning technologies, Nanomaterials 8 (2018), https://doi.org/10.3390/nano8020128. [4] I. Armentano, N. Bitinis, E. Fortunati, S. Mattioli, N. Rescignano, R. Verdejo, M. A. Lopez-Manchado, J.M. Kenny, Multifunctional nanostructured PLA materials for packaging and tissue engineering, Prog. Polym. Sci. 38 (2013) 1720–1747, https:// doi.org/10.1016/j.progpolymsci.2013.05.010. [5] E.M.B. Lima, A.M. Lima, A.P.S. Minguita, N.R. Rojas dos Santos, I.C.S. Pereira, T.T. M. Neves, L.F. da Costa Gonçalves, A.P.D. Moreira, A. Middea, R. Neumann, M.I. B. Tavares, R.N. Oliveira, Poly(lactic acid) biocomposites with mango waste and organo-montmorillonite for packaging, J. Appl. Polym. Sci. 136 (2019), https:// doi.org/10.1002/app.47512, 47512. [6] B. Wang, Z. Chen, J. Zhang, J. Cao, S. Wang, Q. Tian, M. Gao, Q. Xu, Fabrication of PVA/graphene oxide/TiO2 composite nanofibers through electrospinning and interface sol–gel reaction: effect of graphene oxide on PVA nanofibers and growth of TiO2, Colloids Surfaces A Physicochem. Eng. Asp. 457 (2014) 318–325, https:// doi.org/10.1016/j.colsurfa.2014.06.006. [7] A. L´ opez-C´ ordoba, G.R. Castro, S. Goyanes, A simple green route to obtain poly (vinyl alcohol) electrospun mats with improved water stability for use as potential carriers of drugs, Mater. Sci. Eng. C 69 (2016) 726–732, https://doi.org/10.1016/j. msec.2016.07.058. [8] C. L´ opez de Dicastillo, C. Villegas, L. Garrido, K. Roa, A. Torres, M. Galotto, A. Rojas, J. Romero, Modifying an active compound’s release kinetic using a supercritical impregnation process to incorporate an active agent into PLA electrospun mats, Polymers 10 (2018) 479, https://doi.org/10.3390/ polym10050479. [9] A. Moeini, A. Cimmino, M. Masi, A. Evidente, A. Van Reenen, The incorporation and release of ungeremine, an antifungal Amaryllidaceae alkaloid, in poly(lactic acid)/poly(ethylene glycol) nanofibers, J. Appl. Polym. Sci. (2020), https://doi. org/10.1002/app.49098, 49098. [10] T. Radusin, S. Torres-Giner, A. Stupar, I. Ristic, A. Miletic, A. Novakovic, J. M. Lagaron, Preparation, characterization and antimicrobial properties of electrospun polylactide films containing Allium ursinum L. extract, Food Packag. Shelf Life. 21 (2019), https://doi.org/10.1016/j.fpsl.2019.100357, 100357. [11] L. Deng, M. Taxipalati, A. Zhang, F. Que, H. Wei, F. Feng, H. Zhang, Electrospun chitosan/poly(ethylene oxide)/lauric arginate nanofibrous film with enhanced antimicrobial activity, J. Agric. Food Chem. 66 (2018) 6219–6226, https://doi. org/10.1021/acs.jafc.8b01493.
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Antimicrobial food packaging system based on ethyl lauroyl arginate-loaded core/shell electrospun structures by using hydrophilic and hydrophobic polymers ˜o Vidal , Eliezer Vela ´squez , María Jos´e Galotto , Carol Lo ´pez de Dicastillo * Cristian Patin Packaging Innovation Center (LABEN), Department of Food Science and Technology, Technology Faculty, University of Santiago de Chile (USACH), CEDENNA (Center for the Development of Nanoscience and Nanotechnology), Santiago, 9170201, Chile
A R T I C L E I N F O
A B S T R A C T
Keywords: Electrospinning Ethyl lauroyl arginate Core/shell fiber Antimicrobial Food packaging
Antimicrobial compounds can be encapsulated into core/shell structures through coaxial electrospinning in order to develop novel active food packaging materials. In this work, poly (vinyl alcohol)-(ethyl lauroyl arginate)/poly (lactic acid) core/shell fibers, (PVOH-LAE/PLA)f, were developed with the purpose to study the influence of combining a hydrophilic and a hydrophobic polymer on LAE release. LAE release studies were carried out in an aqueous and fatty food simulant, and data were analyzed through a phenomenological mass transfer model. Flatribbon fibers with a core/shell structure were observed by TEM. PLA crystallinity degree of coaxial fibers did not present significant differences with uniaxial PLA fibers, although thermal stability and the glass transition temperature decreased. The maximum concentrations of LAE released from (PVOH-LAE/PLA)f fibers to both simulants reached the MIC values of LAE against Listeria innocua, evidencing a higher affinity of LAE towards fatty simulant and a fast diffusion from fibers in the aqueous simulant.
1. Introduction Nowadays, technology has allowed the use of novel techniques as electrospinning for the development of new active food packaging ma terials. Unlike of traditional methods used to obtain packaging materials such as extrusion, casting or coating, the electrospinning is an efficient, simple, scalable and economic technology able to generate different micro- and nanostructures as fibers or spherical particles from a poly meric solution [1]. Fibers are produced when a voltage is applied to a polymeric solution obtaining a jet with a conical structure known as Taylor Cone’s and the solvent is evaporated in order to form ultrafine structures with a high aspect ratio [2,3]. Several polymers such as poly (lactic acid) (PLA) and poly (vinyl alcohol) (PVOH) have been used in the development of food packaging materials. PLA is a linear aliphatic thermoplastic polymer produced by the fermentation of the starch from natural resources such as potatoes, sugarcane, or corn, and it is “generally recognized as sure” (GRAS) by the Food and Drug Administration (FDA) [4,5]. On the other hand, PVOH is a hydrophilic, synthetic, semicrystalline, biocompatible and non-toxic polymer which is also widely used in food packaging [6,7]. Electrospinning technique can be used to obtain not only polymeric
fibers based on PLA or PVOH, but also their functionalization through the incorporation of active agents into these polymers. Several active compounds such as essential oils, natural extracts and antimicrobial or antioxidants substances have been incorporated into PLA or PVOH to produce active electrospun materials [8–10]. A compound with high antimicrobial activity that has been scarcely studied in the development of active fibers is the ethyl lauroyl arginate (LAE) [11]. LAE is a surfactant cationic derived from natural substances such as lauric acid, L-arginine and ethanol. It is a white powder with a melting temperature between 50 and 60 ◦ C and a degradation temper ature of 107 ◦ C, considered GRAS by FDA and authorized as a food preservative by European Food Safety Authority (EFSA) [12]. On the other hand, several studies have reported a burst release of the com pounds from active single electrospun fibers reducing its antimicrobial effectivity in food packaging applications [13,14]. For this reason, the development of core/shell structures through coaxial electrospinning to slowdown the release of encapsulated active compounds is an innova tive alternative to design packaging materials in order to extent food shelf life [15,16]. The traditional system of the coaxial electrospinning is composed of a voltage source, a stainless-steel coaxial needle, two in jection pumps and a flat or rotatory collector [17]. Thus, the use of this
* Corresponding author. E-mail address: [email protected] (C. L´ opez de Dicastillo). https://doi.org/10.1016/j.polymertesting.2020.106937 Received 19 August 2020; Received in revised form 14 October 2020; Accepted 26 October 2020 Available online 31 October 2020 0142-9418/© 2020 The Authors. Published by Elsevier Ltd. This is an open (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Polymer Testing 93 (2021) 106937
technique has principally allowed to combine different properties of the shell and core polymers into one structure in order to: (i) obtaining fi bers from non-electrospinnable materials, (ii) developing hollow fibers, (iii) protecting sensitive compounds to environment/processing condi tions, and (iv) modifying the release of the encapsulated compound [18–20]. In this work, antimicrobial polymeric fibers with a core/shell structure, whose inner and outer structures were composed by PVOH/ LAE and PLA, respectively, were developed through coaxial electro spinning with the aim of studying its morphological and thermal prop erties and release kinetics of the active compound.
for 24 h to obtain fresh early-stationary phase cells. 2.2. Antibacterial activity of LAE The antibacterial activity of LAE was tested against Gram (− ) E. coli and Gram (+) L. innocua bacteria by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentra tion (MBC) following the methodology of Muriel-Galet et al. (2012) [21]. A stock solution of LAE was previously prepared under ultrapure water. Subsequently, serial dilutions were done in sterile trypticase soy broth (TSB) contained in assay tubes. On the other hand, a loopful of each microorganism in stationary phase was transferred to TSB and incubated at 37 ◦ C until the exponential phase was reached (108 CFU/mL). A (1/10) triple dilution was done until a final concentration of 105 CFU/mL bacteria. Subsequently, 100 μL of this solution was added in the assay tubes that contained LAE and the samples were incubated at 37 ◦ C and 150 rpm for 24 h. After this period of contact between mi croorganisms and the antimicrobial compound, the turbidity of each tube as indicator of microbial growth was measured at 600 nm with a UV–vis spectrophotometer (Pharo 300 Spectroquant®, Germany) using TSB as blank. Subsequently, serial dilutions and spread plate method were applied to determine the cell concentrations. MIC was reported as the lowest LAE concentration that inhibited the growth of the micro organisms in TSB, while MBC was the lowest LAE concentration required to reduce the viability of the initial bacterial inoculum by a reduction ≥99.9%.
2. Material and methods 2.1. Polymers, chemicals and microorganisms Poly (lactic acid) 2003D (specific gravity 1.24) was obtained from Natureworks® (USA) and Gohsenol type AH-17 poly (vinyl alcohol) (saponification degree 97–98.5% and viscosity 25–30 mPa s) was ob tained from The Nippon Synthetic Chemical co. (Japan). Ethyl lauroyl arginate (LAE) was supplied by PRINAL (Chile). Chemical reagents such as chloroform (CLF), dimethylformamide (DMF), trifluoroacetic acid (TFA) and ethanol (EtOH) were obtained from Sigma Aldrich (Chile). Gram (+) Listeria innocua ATCC 33090 and Gram (− ) Escherichia coli ATCC 25922 bacteria were obtained from Biotechnology and Applied Microbiology Laboratory (Chile) and maintained in trypticase soy agar (TSA) at 4 ◦ C until their use. For the experimental assays, a loopful of each bacterial strain was transferred to TSA plate and incubated at 37 ◦ C
Fig. 1. Transmission electron microscopy (TEM) images of antimicrobial polymeric core/shell fibers (PVOH-LAE/PLA)f: (A–B) without imperfections; and (C–D) with imperfections. 2
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2.3. Electrospinning process of polymeric antimicrobial fibers (PVOHLAE/PLA)f
reverse phase Waters Symmetry® C18 (5 μm, 3.9 mm × 150 mm). The mobile phase consisted of a mixture acetonitrile and aqueous 0.1% TFA (50:50) at a flow rate of 1 mL/min [24]. In order to quantify LAE extracted, a calibration curve based on peak area against standard LAE concentrations solutions between 5 and 50 ppm was constructed.
Polymeric antimicrobial fibers (PVOH-LAE/PLA)f with core/shell structure were obtained through coaxial electrospinning system (Spraybase® power Supply Unit, Ireland). PVOH and PLA, polymers with different water affinity based on their composition and polarity, were used to obtain core and shell structures, respectively. PLA solution at 10% (w/v) under a solvent mixture of CLF:DMF (7:3) and an aqueous solution of PVOH at 10% (w/v) containing LAE at 2.5 wt% respect to the polymer were previously prepared. The concentration of LAE was selected according to results of the study presented in Supplemental File, Appendix 1, Both polymeric solutions were transferred to 5 mL syringes and connected through PTFE tubes to a coaxial device that contained two concentric stainless-steel needles of inner diameters 0.7 mm and 2.1 mm for the core and shell systems, respectively. Electrospinning device was set up with a distance of 14 cm between the tip of the coaxial needle and the collector, a voltage between 10 and 12 kW, and an internal and external flow rates of 0.5 and 1.8 mL/h for core and shell solutions, respectively. Uniaxial fibers PLAf, PVOHf and (PVOH-LAE)f were also obtained through electrospinning in order to evaluate and compare their thermal properties with the coaxial antimicrobial fibers (PVOH-LAE/ PLA)f. Processing parameters to obtain uniaxial fibers were: flow rate at 0.5 mL/h, single needle with 0.7 mm diameter, 14 cm distance and a voltage between 10 and 16 kW.
2.5. Study of the release kinetics of LAE from electrospun fibers with core/shell structure 2.5.1. Release assay procedure The release of LAE from (PVOH-LAE/PLA)f electrospun fibers to a fatty (EtOH 95%) and aqueous (EtOH 10%) food simulants was evalu ated according to EU Regulation N◦ October 2011 about materials and plastic objects to contact with food. Electrospun samples of 14 μm thickness and a weight of 0.014 g were placed in contact with the sim ulants with a ratio area/volume of 6 dm2/L at 40 ◦ C during 72 h in order to determine the active release kinetics. The released LAE was periodi cally analyzed through HPLC following the methodology earlier mentioned in section 2.4.3. 2.5.2. Determination of the LAE partition and diffusion coefficients The extent of a compound release is controlled by the phase equi librium and is given through the partition coefficient (K). The kinetics of the compound release process is defined by the diffusion coefficient (D) that measures the velocity of the particle diffusion in a polymeric matrix until the thermodynamic equilibrium is reached [25,26]. K and D co efficients of LAE were estimated based on the experimental results of this compound release from the electrospun fibers to food simulants. The partition coefficient, K, was calculated in the equilibrium as the ratio between LAE concentration in the electrospun fibers (Cf) and in the – Cf/Cs. simulant (Cs), K– The kinetics of the mass transport processes into the fibers were characterized through the Fick’s law, considering the limit conditions of the experiments such as: (i) presence of an equilibrium partition, (ii) a limited volume of solvent, and (iii) the coefficient D associated with the LAE transport into the fibers is independent of the time and position. Following these assumptions and the integration of Fick’s law, the mass ratio of released LAE into the solvent at time “t” and in the equilibrium ∞ ∑ S) was expressed through equation (2): m(t) = m(t)⋅(A⋅L⋅K+V =1− VS ⋅ciP ⋅A⋅L mfS n=1 ( ) 4⋅D⋅q2n ⋅t 2α(1+α) + exp − mtmSf = mt⋅(A⋅L⋅K + VS)VS⋅cPi⋅A⋅L = 1-n = 2 2 2 1+α+α q L
2.4. Characterization of active electrospun mats 2.4.1. Morphological analysis The internal morphology of (PVOH-LAE/PLA)f coaxial fibers was observed with an transmission electronic microscope (TEM) (Hitachi HT7700, Japan). The samples were collected on a carbon coated copper specimen grid during 10 s, and TEM micrographs of the coaxial fibers were obtained with different magnifications. 2.4.2. Thermal properties Thermal stability of the electrospun fibers was studied through thermogravimetric analysis (TGA) carried out in a Stare TGA/DSC sys tem (Mettler Toledo GC20, Switzerland). 5–6 mg of each sample were placed into alumina capsules and heated from 30 to 600 ◦ C at 10 ◦ C/min heating rate under nitrogen atmosphere. The effect of LAE incorporation and the coaxial processing on the thermal properties of both polymers were studied through differential scanning calorimetry (DSC) carried out in a Stare TGA/DSC system (Mettler Toledo GC20, Switzerland). 4–5 mg of each sample were placed into aluminum capsules and a thermal heating program from 0 to 250 ◦ C at 10 ◦ C/min heating rate was carried out under nitrogen atmosphere. Thermal parameters such as glass transition temperature (Tg), cold crystallization temperature (Tcc), melting temperature (Tm), and melting (ΔHm) and cold crystallization (ΔHcc) enthalpies were reported. Furthermore, the crystallinity of the PLA and PVOH polymers (Xc) was calculated through equation (1): Xc% = ΔHm-ΔHccΔHmo*W
n
1∞2α1+α1+α+α2qn2+exp-4⋅D⋅qn2⋅tL2mtmSf = mt⋅(A⋅L⋅K + VS) VS⋅cPi⋅A⋅L = 1-n = 1∞2α1+α1+α+α2qn2+exp-4⋅D⋅qn2⋅tL2 (2)
mtm∞ = 1-n = 1∞2α1+α1+α+α2qn2+exp-4Dqn2tL2
Where mt is the mass of LAE in the food simulant at t time, m⍰ is the mass of LAE in the food simulant in the equilibrium, L is the thickness of the film (cm), D is the diffusion coefficient (cm2/s), and t is the time (s). The α parameter is the mass ratio of the compound between liquid and polymer phases in the equilibrium, given by α = Vs/(K*A*L), where Vs is the volume of solution and A is the film area. Furthermore, qn represents the positive solutions of equation (3):
(1)
(3)
− tanqn = − αqn
Where ΔHm0 is the melting enthalpy of a wholly crystalline polymer (PLA = 93.1 J/g and PVOH = 138.6 J/g) [22,23], and W is the mass fraction of the polymer in the fiber.
The experimental data were fixed to equation (3), and the coefficient D was estimated using Solver of Excel 2016 and Sigmaplot v. 10.0 programs [27].
2.4.3. Quantification of LAE in the coaxial (PVOH-LAE/PLA)f fibers The effective concentration of LAE in the coaxial fibers was deter mined through an extraction process with ethanol and a later analysis through high-performance liquid chromatography (HPLC). 0.1 g of mat was put into a vial with 15 mL of EtOH and stirred at 60 ◦ C and 300 rpm for 3 h. Subsequently, liquid phase was filtered and injected (20 μL) in a HPLC chromatograph (Waters Alliance, USA) equipped with a column of
Table 1 Antibacterial activity of LAE expressed as MIC and MBC. Listeria innocua Escherichia coli
3
MIC (ppm)
MBC (ppm)
10 20
70 40
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2.6. Statistical analyses
diameters can be associated to differences in electrospinning process conditions such as: i) presence of Triton X-100 surfactant in the core solution, ii) a lower concentration of PLA in the shell, iii) use of higher voltage (19 kV), iv) lower flow rates in the core (0.1 mL/h) and shell (0.2 mL/h) solutions, v) different inner and outer needle diameters, and vi) use of EtOH:water mixture for PVOH solution. Likewise, da Silva et al. (2019) obtained diameters between 155 and 244 nm in core/shell nanofibers by using polymeric solutions of PLA at 20% (w/v) for the shell and PVOH at 15% (w/v) for the core with medical purposes. Authors have observed that the increase of the nanofibers diameters was principally produced by an increase in the flow rates of the shell solution from 0.5 to 1 mL/h and core solution from 0.06 to 1 mL/h. Besides, some micrographs showed also the presence of transverse fibers in the nanofibers [32].
Thermal properties were statistically analyzed by variance analysis (ANOVA) and Tukey’s Test, using InfoStat Program (2016) to detect differences between parameters with a confidence level of 95% (p < 0.05). The experimental design was random type for two replicates. 3. Results and discussion 3.1. Antibacterial activity of LAE Results of antibacterial activities of LAE against L. innocua and E. coli bacteria were reported as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), as Table 1 shows. Results revealed LAE exhibited high antibacterial activity against both micro organisms with MIC values of 10 and 20 ppm for L. innocua and E. coli, respectively. MIC values of LAE for both bacteria were lower than those values reported for other compounds such as essential oils and nano particles, demonstrating its effectiveness as antibacterial compound [28, 29]. On the other hand, a bactericidal effect was produced at 70 ppm for L. innocua and 40 ppm for E. coli. The great antibacterial activity of LAE is attributed to its behavior as cationic surfactant and its chemical structure able to react with the bacteria membrane. This reaction can produce the inhibition of the cell metabolic processes due to the increase of the permeability between cytoplasm and external layer of the Gram (− ) bacteria and between cell membrane and cytoplasm of Gram (+) bacteria [12,30].
3.3. Thermal properties Thermal parameters from the first and unique heating process are presented in Table 2. Uniaxial PLAf, PVOHf and (PVOH-LAE)f fibers were also developed in order to study the effect of LAE incorporation into PVOH thermal parameters, and the coaxial electrospinning process over PLA and PVOH thermal behavior. PLA fibers presented a Tg at approx. 73 ◦ C, a crystalline rearrangement at 91 ◦ C (Tcc) and a melting peak at 154 ◦ C (Tm). PVOH fibers showed a Tg and Tm at 49 and 223 ◦ C, respectively. The incorporation of LAE in PVOH fibers increased Tg value and significantly decreased Tm due to the chemical interactions between PVOH and LAE. These interactions possibly corresponded to hydrogen – O, OH) and the bonds between the functional groups of the polymer (C– – O, NH2 and NH) [30]. Unlike other works based on the surfactant (C– incorporation of active compounds into polymers, LAE did not present a plasticizer effect. The lower mobility of PVOH chains due to these in teractions significantly reduced crystallinity because the surfactant hindered the growth of polymeric crystals evidenced by a decrease in melting enthalpy and PVOH crystallinity degree of (PVOH-LAE)f [24]. On the other hand, Tg and Tcc temperatures of coaxial (PVOH-LAE/PLA)f fibers exhibited lower values than those of control uniaxial fibers PLAf. This fact was attributed to hydrogen bonds interactions between PLA and PVOH chains occurred in the contact zone between inner and outer structures [31]. Nonetheless, melting temperature, melting enthalpy and crystallinity degree of PLA were not significantly affected by coaxial electrospinning process. TGA thermogravimetric curves and their corresponding derivatives (DTGA) of LAE and fibers are shown in Fig. 2. PLAf initiated its degra dation at 280 ◦ C with a maximum degradation temperature at 353 ◦ C. PVOHf and PVOH-LAEf presented a weight loss below 100 ◦ C associated with adsorbed water due to the hydrophilicity of these components, and PVOH onset decomposition was registered at 250 ◦ C, in accordance with a previous work [33]. Subsequently, PVOHf evidenced two degradation processes with maximum values at 322 and 440 ◦ C, associated with the lateral groups separation and the degradation of backbone chain, respectively (Fig. 2B) [3,32]. LAE presented an onset decomposition temperature lower than polymers and degraded between 200 and 450 ◦ C, similarly to reported values in previous works [34]. The incor poration of LAE in PVOH produced a reduction of the polymeric thermal stability, accelerating its onset decomposition from 250 to 233 ◦ C, which can be attributed to the lower crystallinity of (PVOH-LAE)f respect to PVOHf (Table 2). The disruption of the crystal structure caused by LAE formed a network with lower stability due to competitive interactions PVOH-LAE and PVOH–PVOH chemical groups. Similar results have been reported for LAE incorporated into chito san/poly (ethylene oxide) fibers, where hydrogen bonds between poly mers and LAE were suggested by changes in FTIR bands at 3500-3100 – O) [11]. As Fig. 2B shows, the cm− 1 (-OH and –NH) and 1634 cm− 1 (C– degradation profile of (PVOH-LAE/PLA)f coaxial structures exhibited a similar behavior than PLAf probably due to the higher concentration of
3.2. Morphology of (PVOH-LAE/PLA)f electrospun fibers Coaxial electrospinning process resulted on the development of ho mogenous and bead-free (PVOH-LAE/PLA)f fibers, and TEM analysis confirmed their core/shell structure. Fig. 1A and B shows thick uniform coaxial fibers formed by a PVOH-LAE/PLA core/shell structure with diameters of 1.23 and 0.61 μm for the outer and inner layers, respec tively. The coaxial electrospinning process is more complex than uni axial system because it is dependent on the variables of both polymers, such as solution viscosity, conductivity, and flow rate. In some occasions during this process, some imperfections occurred and thin cross-fibers were scattered. As Fig. 1C and D shows, these thinner fibers exhibited inner and outer diameters of approx. 350 and 600 nm, respectively, and 15 nmtransversal fibers. Electrospun nanofibers based on PVOH and PLA with lower diameters have been previously obtained but focused on me chanical properties for biomedical applications. PVOH/PLA and PLA/ PVOH fibers without active compound were produced with inner and outer diameters of 35 nm and 165 nm, respectively [31]. Differences in Table 2 DSC parameters of the fibers. Fiber
Tg (◦ C)
Tcc (◦ C)
ΔHcc (J/g)
Tm (◦ C)
ΔHm (J/g)
PLAf
73.2 ± 0.7d 49.2 ± 0.3a 55.2 ± 0.6b 67.4 ± 0.8c
91.6 ± 0.5a –
12.6 ± 0.5b –
–
–
87.6 ± 2.5a
10.1 ± 1.0a
154.4 ± 0.4a 223.2 ± 1.0c 197.3 ± 0.3b 152.9 ± 0.2a
29.6 1.3a 68.6 0.2c 46.8 4.9b 23.2 0.1a
PVOHf (PVOHLAE)f (PVOHLAE/ PLA)f
Xc (%) ± ± ± ±
18.2 ± 1.9a 49.5 ± 0.2c 33.8 ± 3.6b 18.0 ± 1.5aa
Lowercase letters (a-d) indicate significant differences of DSC parameters be tween samples according to ANOVA analysis and Tukey’s test (p < 0.05). a Crystallinity was calculated respect to melting heat of 100% crystalline PLA, major component of the core/shell (PVOH-LAE/PLA)f fiber. 4
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Fig. 2. Curves (A) TGA and (B) DTGA of LAE and electrospun fibers.
this polymer in these fibers. Nevertheless, the maximum degradation occurred at lower temperature (330 ◦ C) than for PLAf because of the earlier degradation of PVOH and LAE [23].
ethanol into PLA matrix that produced a swelling of the polymeric matrix and the modification of its transport properties. LAE presents also a slightly higher solubility into ethanol than in water, therefore the release of this antimicrobial compound to the fatty simulant could be favored [35,36]. The maximum concentration of released LAE into the aqueous and fatty simulants were approx. 9.8 and 12.3 ppm, respec tively. These values evidenced that 29 and 36 wt% of total amount of encapsulated LAE into core/shell fibers was released towards the aqueous and fatty simulants, respectively. Interestingly, these values were similar to MIC value previously obtained for L. innocua (10 ppm), therefore the efficiency of these materials against this bacterium was confirmed. The diffusion coefficient, D, of the active compound into the poly meric matrix and the partition coefficient, K, that represents the extent of the compound release from the material to food are important pa rameters to design active food packaging materials [26]. Therefore, K and D coefficients of LAE-containing (PVOH-LAE/PLA)f fibers to 10 and
3.4. Study of the release kinetic of LAE from (PVOH-LAE/PLA)f The development of electrospun core/shell (PVOH-LAE/PLA)f fibers maintained a high encapsulation efficiency of this antimicrobial com pound during coaxial electrospinning process. The extraction process of these active fibers revealed an encapsulation efficiency value of 94% and the concentration of 0.51 wt% of LAE respect the total coaxial fiber. The release kinetic curves of LAE from (PVOH-LAE/PLA)f core/shell fibers to aqueous and fatty food simulants at 40 ◦ C are displayed in Fig. 3. LAE release curves indicated an exponential growth until an equilibrium during the first hours for both simulants, although the released active compound concentration into 95% EtOH was higher than in 10% EtOH. This behavior can be explained by the plasticizer effect of 5
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across them, from the PVOH core towards PLA shell [24,37]. Nonethe less, as it was earlier mentioned, total released LAE to the simulant in the equilibrium could be finally favored by a higher swelling grade of PLA caused by a higher concentration of ethanol in the fatty food simulant. Furthermore, several factors, such as structure and crystallinity of material, interaction between the polymer and active agent, and the type of polymer or active compound, can influence the particle speed diffusion into polymeric matrix and its posteriorly release towards the simulants [26,32,38]. Thus, D values of LAE into PLA obtained in this work were lower than values obtained in previous studies based on active PLA films and electrospun mats. A work developed by Torres et al. (2017) obtained D coefficients between 2.6 × 10− 11 and 1.7 × 10− 13 m2/s when PLA films loaded with thymol through carbon dioxide su percritical impregnation were put into 10% EtOH and 95% EtOH at 40 ◦ C for 40 h, respectively [39]. Likewise, D values between 1.9 and 2 ´pez de Dicastillo et al. (2017), when × 10− 13 m2/s were obtained by Lo the antioxidants compounds of PLA films extruded with merk´en were released into 10% EtOH and 95% EtOH at 40 ◦ C [40]. In both studies, the incorporation of the active compounds decreased the crystallinity of the materials generating more polymeric amorphous regions that could facilitate the fast diffusion of the compounds, and therefore, high D values. On the contrary, in this work, the crystallinity of PLA in the coaxial fibers was not altered by the incorporation of LAE (see Table 2), and thus, a slower diffusion occurred. On the other hand, D values are also influenced by the molecular weight of active compounds. Some works have evidenced that higher D coefficients have been obtained when compounds with lower weight ´pez de molecular than LAE were incorporated into electrospun fibers. Lo Dicastillo et al. (2018) obtained a D value of 1 × 10− 12 m2/s when PLA nanofibers loaded with cinnamaldehyde were put into 50% EtOH at 40 ◦ C [8]. Another study of PLA/PVOH coaxial fibers loaded with cur cumin exhibited D values between 5 and 5.5 × 10− 13 m2/s into 50% EtOH and 10% EtOH at 40 ◦ C, respectively [26]. Thereby, in spite of the good affinity and solubility of LAE towards both food simulants, the development of a coaxial electrospun structure has permitted to slow down the kinetic release of the active compound.
Fig. 3. Release kinetic curves of LAE from electrospun fibers to aqueous and fatty food simulants.
Fig. 4. Mathematical modulation of release kinetics of LAE from electrospun fibers to aqueous and fatty food simulants.
4. Conclusions Uniform antimicrobial fibers with core/shell structure were suc cessfully obtained through coaxial electrospinning technique. The maximum degradation of coaxial fibers (PVOH-LAE/PLA)f occurred at lower temperature than uniaxial PLAf probably due to the presence of PVOH and LAE whose thermal stabilities were lower. The incorporation of LAE in PVOHf decreased its glass transition temperature and crys tallinity possibly due to chemical interactions with this antimicrobial compound. The glass transition temperature of PLA in (PVOH-LAE/ PLA)f fiber decreased but its crystallinity degree was maintained. LAE exhibited a high affinity towards the fatty food simulant evidenced by the highest released LAE concentration, and therefore the lowest parti tion coefficient. Released LAE concentrations into both simulants reached MIC value for L. innocua. On the other hand, LAE diffusion into (PVOH-LAE/PLA)f fibers was faster in the aqueous simulant than in the fatty simulant due to the fast interaction of the water molecules with the hydrophilic inner structure of the coaxial fibers. The results suggested that the developed core/shell electrospun fibers containing LAE could be used in food packaging for maintaining or extending food shelf life. Furthermore, an increase of LAE concentration in the coaxial fibers could be achieved by modifying the concentrations of the core and shell polymeric solutions, and therefore, the development of antimicrobial materials against bacteria with higher MIC values could be attained.
95% EtOH as food simulants were studied through equations (2) and (3) and results were fitted in mathematical modelling (Fig. 4). Partition coefficients were estimated when released LAE from electrospun mate rials achieved the plateau in each kinetic profile. K values were 164.4 and 55.9 in 10% EtOH and 95% EtOH, respectively, indicating a large release extent on both food simulants, although a higher affinity of LAE for the fatty simulant. In addition to the good solubility of the antimi crobial compound in both simulants, the difference of K values can be attributed to the plasticizing effect of ethanol on PLA shell structure of the coaxial fibers. Likewise, this high affinity of LAE to both simulants was also evidenced when these partition coefficients were compared with K values of molecules with a hydrophobic behavior as curcumin. A release study of this polyphenol from coaxial PVOH/PLA fibers towards 10% and 50% EtOH exhibited K values between 349 and 10836 [26]. Fig. 4 reveals the experimental data greatly fitted with theoretical curves following Fick’s law. As Fig. 4 shows, the curves slope of released LAE kinetics were principally dependent on the type of food simulant. Although the extent of the LAE release was higher into fatty food sim ulant, the diffusion of this antimicrobial compound throughout (PVOHLAE/PLA)f fibers in the aqueous simulant was slightly faster (D = 3.9 × 10− 14 m2/s) than in the fatty simulant (D = 2.4 × 10− 14 m2/s). This could be attributed to the higher amount of water molecules in the aqueous simulant which are smaller than LAE and EtOH molecules, and have a higher affinity towards the hydrophilic polymer PVOH. There fore, water molecules could have penetrated faster into the inner structure of the coaxial fibers and accelerated the LAE release diffusion
CRediT authorship contribution statement ˜ o Vidal: Methodology, Validation, Formal analysis, Cristian Patin Investigation, Data curation, Writing - original draft. Eliezer 6
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´squez: Conceptualization, Data curation, Writing - review & edit Vela ´ Galotto: Resources, Supervision. Carol ing, Visualization. María Jose ´ pez de Dicastillo: Project administration, Funding acquisition, Lo Writing - review & editing, Supervision.
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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 The authors acknowledge the financial support of Agencia Nacional ´n y Desarrollo (ANID) (Chile) through the Fondecyt de Investigacio Regular Project N◦ 1200766, the Doctoral Scholarship CONICYTPFCHA/Doctorado Nacional/2019–21190316 and the “Programa de ´gicos de Exce Financiamiento Basal para Centros Científicos y Tecnolo lencia” (Project AFB180001). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.polymertesting.2020.106937. References [1] A. Alehosseini, L.G. G´ omez-Mascaraque, M. Martínez-Sanz, A. L´ opez-Rubio, A. Ali, M.-S. Laura, G. G´ omez-Mascaraque, Marta, A. L´ opez-Rubio, Electrospun curcuminloaded protein nanofiber mats as active/bioactive coatings for food packaging applications, Food Hydrocolloids 87 (2019) 758–771, https://doi.org/10.1016/J. FOODHYD.2018.08.056. [2] L. Quiles-Carrillo, N. Montanes, J. Lagaron, R. Balart, S. Torres-Giner, L. QuilesCarrillo, N. Montanes, J.M. Lagaron, R. Balart, S. Torres-Giner, Bioactive multilayer polylactide films with controlled release capacity of gallic acid accomplished by incorporating electrospun nanostructured coatings and interlayers, Appl. Sci. 9 (2019) 533, https://doi.org/10.3390/app9030533. [3] C. L´ opez de Dicastillo, C. Pati˜ no, M.J. Galotto, J.L. Palma, D. Alburquenque, J. Escrig, Novel antimicrobial titanium dioxide nanotubes obtained through a combination of atomic layer deposition and electrospinning technologies, Nanomaterials 8 (2018), https://doi.org/10.3390/nano8020128. [4] I. Armentano, N. Bitinis, E. Fortunati, S. Mattioli, N. Rescignano, R. Verdejo, M. A. Lopez-Manchado, J.M. Kenny, Multifunctional nanostructured PLA materials for packaging and tissue engineering, Prog. Polym. Sci. 38 (2013) 1720–1747, https:// doi.org/10.1016/j.progpolymsci.2013.05.010. [5] E.M.B. Lima, A.M. Lima, A.P.S. Minguita, N.R. Rojas dos Santos, I.C.S. Pereira, T.T. M. Neves, L.F. da Costa Gonçalves, A.P.D. Moreira, A. Middea, R. Neumann, M.I. B. Tavares, R.N. Oliveira, Poly(lactic acid) biocomposites with mango waste and organo-montmorillonite for packaging, J. Appl. Polym. Sci. 136 (2019), https:// doi.org/10.1002/app.47512, 47512. [6] B. Wang, Z. Chen, J. Zhang, J. Cao, S. Wang, Q. Tian, M. Gao, Q. Xu, Fabrication of PVA/graphene oxide/TiO2 composite nanofibers through electrospinning and interface sol–gel reaction: effect of graphene oxide on PVA nanofibers and growth of TiO2, Colloids Surfaces A Physicochem. Eng. Asp. 457 (2014) 318–325, https:// doi.org/10.1016/j.colsurfa.2014.06.006. [7] A. L´ opez-C´ ordoba, G.R. Castro, S. Goyanes, A simple green route to obtain poly (vinyl alcohol) electrospun mats with improved water stability for use as potential carriers of drugs, Mater. Sci. Eng. C 69 (2016) 726–732, https://doi.org/10.1016/j. msec.2016.07.058. [8] C. L´ opez de Dicastillo, C. Villegas, L. Garrido, K. Roa, A. Torres, M. Galotto, A. Rojas, J. Romero, Modifying an active compound’s release kinetic using a supercritical impregnation process to incorporate an active agent into PLA electrospun mats, Polymers 10 (2018) 479, https://doi.org/10.3390/ polym10050479. [9] A. Moeini, A. Cimmino, M. Masi, A. Evidente, A. Van Reenen, The incorporation and release of ungeremine, an antifungal Amaryllidaceae alkaloid, in poly(lactic acid)/poly(ethylene glycol) nanofibers, J. Appl. Polym. Sci. (2020), https://doi. org/10.1002/app.49098, 49098. [10] T. Radusin, S. Torres-Giner, A. Stupar, I. Ristic, A. Miletic, A. Novakovic, J. M. Lagaron, Preparation, characterization and antimicrobial properties of electrospun polylactide films containing Allium ursinum L. extract, Food Packag. Shelf Life. 21 (2019), https://doi.org/10.1016/j.fpsl.2019.100357, 100357. [11] L. Deng, M. Taxipalati, A. Zhang, F. Que, H. Wei, F. Feng, H. Zhang, Electrospun chitosan/poly(ethylene oxide)/lauric arginate nanofibrous film with enhanced antimicrobial activity, J. Agric. Food Chem. 66 (2018) 6219–6226, https://doi. org/10.1021/acs.jafc.8b01493.
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