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PEO blend and phenolic compounds with antibacterial activity
Accepted Manuscript Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity
Suelen Goettems Kuntzler, Jorge Alberto Vieira Costa, Michele Greque de Morais PII: DOI: Reference:
S0141-8130(18)31649-0 doi:10.1016/j.ijbiomac.2018.05.224 BIOMAC 9829
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9 April 2018 15 May 2018 30 May 2018
Please cite this article as: Suelen Goettems Kuntzler, Jorge Alberto Vieira Costa, Michele Greque de Morais , Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity. Biomac (2017), doi:10.1016/ j.ijbiomac.2018.05.224
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ACCEPTED MANUSCRIPT 1 Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity Suelen Goettems Kuntzlera, Jorge Alberto Vieira Costab and Michele Greque de Moraisa*
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Laboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering,
Brazil. Phone: +55-53-32336908, Fax: +55-53-32338745
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering,
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b
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Federal University of Rio Grande (FURG), P.O. Box 474, 96203-900, Rio Grande, RS -
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Federal University of Rio Grande (FURG), P.O. Box 474, 96203-900, Rio Grande, RS – Brazil. Phone: +55-53-32336908, Fax: +55-53-32338745
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*
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Corresponding author: [email protected]
Abstract
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Electrospun nanofibers can be formed with chitosan as the polymers found in biological
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sources have antibacterial ability. The objective of this work was to evaluate whether chitosan/polyethylene oxide (PEO) blend nanofibers containing microalgal phenolic
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compounds exhibit antibacterial activity. Nanofibers produced with a 3% chitosan/2% PEO blend containing 1% phenolic compounds had an average diameter of 214 ± 37 nm, which
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resulted in a high temperature of maximum degradation, an important parameter for food packaging. The potential antibacterial activity of this nanofibers was confirmed by their inhibition of Staphylococcus aureus ATCC 25923 (6.4 ± 1.1 mm) and Escherichia coli ATCC 25972 (5.5 ± 0.4 mm). The polymeric nanofibers produced from chitosan and containing phenolic compounds have properties that therefore allow their application as active packaging. In addition, chitosan is an excellent polymer for packaging as it presents biodegradability, biocompatibility and, non-toxicity.
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Keywords: Active packaging; Electrospinning; Microalga. 1 Introduction Microbial growth accelerates changes in the aroma, color and texture of food, resulting in reduced shelf life and an increased risk of diseases transmitted by contaminated
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products. An alternative that can reduce or inhibit the growth of bacteria in food is the use of
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active packaging that in addition to being an inert barrier to the external environment,
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incorporates additives that improve food preservation [1].
Electrospun nanofibers are nanotechnology materials that have important
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properties for food applications because of their nanometric diameter, which optimizes mechanical properties such as elasticity, strength, porosity and surface area in relation to
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volume [2]. Active packaging includes the use of natural antimicrobial polymers and compounds for the purpose of developing materials that are biodegradable and reducing the
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use of synthetic preservatives [3].
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Chitosan is an amino polysaccharide obtained from chitin, an abundant byproduct of seafood processing, through a deacetylation reaction with an alkali [4]. The final structure
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of chitosan has one primary amine and two free hydroxyl groups for each monomer. Chitosan has good film-forming ability and, because of its high versatility, this polymer can be applied
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in food preservation [5]. Alibadi et al. [6] prepared a nanofiber membrane with chitosan and polyethylene oxide by electrospinning, investigating its application in the removal of metals in aqueous solution. This natural polymer has biological properties as an antibacterial [7] and antifungal [8] in addition to exhibiting gas barrier characteristics and an aroma in dry conditions. Goy, Morais, Assis [9] evaluated the antibacterial activity of chitosan that had undergone the quaternization process and in gel form. The authors verified that chitosan has
ACCEPTED MANUSCRIPT 3 an effect against Staphylococcus aureus and Escherichia coli. These characteristics make chitosan an interesting choice for nanofiber production by electrospinning for various applications. Some natural compounds, such as phenolic compounds of microalgal origin, can be incorporated into nanofibers to add antimicrobial properties. The phenolic compounds in
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Spirulina microalgae are organic acids such as caffeic, gallic, salicylic and trans-cinnamic acids [10], which act individually or synergistically and may exhibit various biological
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activities as antioxidants [11], antibacterials [12] and antifungals [13]. The objective of this
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work was to evaluate whether chitosan/PEO blend nanofibers containing microalgal phenolic
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compounds exhibit antibacterial activity. 2 Methodology
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2.1 Obtaining microalgal biomass of Spirulina sp. LEB 18 and extraction of its phenolic compounds
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In this study, the biomass of Spirulina sp. LEB 18 was dried at 50 °C for 4 h in a
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tray dryer, ground in a ball mill for 2 h, and sieved with a 37 μm stainless steel sieve (Granutest, Brasil) [14].
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The extraction of the phenolic compounds was performed according to Souza et al. [15]. Supernatant containing the phenolic compounds used for microbiological tests was
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concentrated by a rotary vacuum evaporator (Quimis, 344B2, Brazil) at 60 °C to remove methanol, and the remaining sample was then resuspended in sterilized distilled water [13]. The phenolic extract used in the polymer solution was placed in a rotavaporator (Quimis 344B2, Brazil) at 60 ° C to remove methanol, and the remaining sample was resuspended in 90% (v v-1) acetic acid solution. 2.2 Development and characterization of nanofibers 2.2.1 Preparation of polymer solutions for electrospinning
ACCEPTED MANUSCRIPT 4 Solutions with polymer blends containing 2 and 3% (w v-1) chitosan (60-120,000 g mol-1) and 2% (w v-1) polyethylene oxide (PEO) (900,000 g mol-1) (Sigma-Aldrich®, USA) were prepared with 90% (v v-1) acetic acid solution. In the solution that formed uniform nanofibers, the phenolic compounds extracted from the biomass were incorporated at a 1% (w v-1) concentration. All of the solutions were homogenized using a magnetic stirrer (Fisatom,
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Brazil) for 16 h. 2.2.2 Development of nanofibers
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The electrospinning equipment consists of a positive displacement pump (Model
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KDS 100, KD Scientific, United States) and a source of high-voltage, direct-current power
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(Model ET 5000 CC, Electric Test Serta, Brazil). The polymer solutions were injected through capillaries with a diameter of 0.45, 0.55, 0.70 and 0.80 mm. The fixed parameters
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were an electric potential of 20 kV, a distance of the capillary to the collector of 100 mm and a feed rate of 300 μL h-1, according to Vrieze et al. [16]. All tests were conducted at 22 °C
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with the relative humidity maintained at 60±1%.
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2.2.3 Shape and diameter of the nanofibers Images and 30 measurements of the diameters of the nanofibers with and without
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phenolic compounds were obtained using a scanning electron microscope (SEM) (JEOL JSM6610 LV, Japan). Before the analyses, the samples were fixed in a metallic holder with carbon
[17].
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tape and coated with gold using a sputtering diode (Denton Vacuum CAR001-0038, USA)
2.2.4 Viscosity of the polymer solutions The viscosity of polymer solutions was determined from 0.5 mL of each sample using a rheometer (Brookfield DV-III Ultra Programmable Rheometer, USA). 2.2.5 Porosity of the nanofibers
ACCEPTED MANUSCRIPT 5 The porosity was calculated from Equation 1 [18], with ρ and ρ0 (g cm-3) representing the densities of the nanofibers and polymer film, respectively, which were determined according to their dimensions and masses. Porosity (%) = 1 100 0
(1)
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The film samples were obtained by a casting technique using polymer solution distributed longitudinally in petri plates, followed by evaporation of the solvent [19].
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2.2.6 Endothermic and exothermic transitions
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To determine the melting temperature of the nanofibers, phenolic compounds and polymers, differential scanning calorimetry (DSC) (Shimadzu DSC-60, Japan) was used.
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Approximately 4 mg of each sample was placed in an aluminum capsule, which was sealed with a lid; the samples were tested under a nitrogen atmosphere with a flow rate of 50 mL
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min-1. The analyses were carried out at room temperature and heated at a rate of 10 °C min-1 until the samples reached 400 °C. The melting temperature was determined from the melting
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peak shown in the DSC curve according to ASTM (American Society for Testing and Materials) D7426-08 [20], and the degree of crystallinity (Xc) was determined according to Equation 2.
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Hm 100 Xc (%) 0 Hm
(2)
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where ΔH0m represents the melting enthalpy for 100% crystalline PEO (118 J g-1)
and ΔHm indicates the melting enthalpy calculated from DSC thermograms [21]. 2.2.7 Thermal degradation and residual solvent content The analyses were conducted on nanofibers, phenolic compounds and polymers at temperatures from room temperature to 500 °C in an inert atmosphere consisting of nitrogen with a flow rate of 30 mL min-1 and with a 10 °C min-1 heating rate, using 2 to 6 mg of
ACCEPTED MANUSCRIPT 6 sample. A thermogravimetric analyzer (TGA) (Shimadzu DTG-60, Japan) was used according to the ASTM D3850-12 [22] method. 2.2.8 Structural characterization of nanofibers, phenolic compounds and polymers Fourier transform infrared spectroscopy (FTIR) was performed on a Thermo Scientific-Nicolet 6700 (Shimadzu, Japan), and the sample spectra were analyzed at
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wavenumbers from 0 to 4000 cm-1. Nanofibers samples, phenolic compounds and the polymers, all in solid form, were homogenized and prepared with potassium bromide (KBr).
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2.2.9 Contact angle of the nanofibers
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A drop of water was inserted into the surface of the nanofibers, and the image was
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then obtained by microscope (Digital Blue). The Surftens 3.0 software was used to make five measurements of each image using five different points arranged around the drop. The
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maximum standard deviation of the water angle contact was ± 3% [23]. 2.2.10 X-ray diffraction (XRD) of nanofibers and polymers
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This analysis was performed on a D8 Advance X-ray diffractometer (Bruker,
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United States) equipped with a copper tube. The radiation was produced at a wavelength of 0.154 nm and at 40 kV and 40 mA. The diffractograms were obtained at room temperature
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(25 °C) at a 2θ angle between 10 and 90° at a step rate of 0.05° sec-1. The nanofibers samples were prepared as films having a diameter of approximately 50 mm and the polymers were
8.0.
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analyzed in granular state. The diffractograms were analyzed using the software Origin Pro
2.3 Evaluation of antibacterial activity 2.3.1 Disc diffusion method The determination of the antibacterial activity of the phenolic compounds and phenolic compounds in nanofibers was performed against the microorganisms Escherichia coli ATCC 25972 and Staphylococcus aureus ATCC 25923 with the disc diffusion test
ACCEPTED MANUSCRIPT 7 according to the M7 - A6 standard established by National Committee for Clinical Laboratory Standards [24]. Bacterial cultures were prepared on Mueller-Hinton agar for E. coli ATCC 25972 and plate count agar for S. aureus ATCC 25923. An isolated colony was removed from each culture and utilized for a bacterial suspension that was prepared in saline solution (0.85% w v-1) in order to obtain an optical turbidity comparable to a McFarland standard solution of
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0.5 measured in a spectrophotometer (Shimadzu, UV Mini 1240, Japan) at wavelength 600 nm (equivalent to λ = 0.1). This approach resulted in a suspension containing 1 to 2 x 108
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CFU mL-1 bacteria.
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The bacterial suspension was added to petri plates containing the corresponding agar for each microorganism. Qualitative filter paper discs with 7 mm diameters containing
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different amounts (20, 60, 100, 160, 200, 240, 280, 320 and 380 μL) of the phenolic extract
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were added to the petri plates, and all tests were performed in triplicate. Discs produced with approximately 4 mm the nanofibers with and without the phenolic compounds were added to
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the plates. All tests were performed in triplicate. The samples were stored for 24 h in a
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bacteriological incubator (AmericanLab 101/100, Brazil) at 35±1 °C. After this period, growth inhibition was quantified by the diameter of the sample inhibition zones [24]. The
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percent inhibition of the phenolic compounds was calculated relative to control inhibition zone, (chloramphenicol 30 μg) which was considered 100% inhibition of growth.
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2.3.2 Macrodilution method with colony count The determination of the antibacterial activity of the phenolic compounds was
performed according to section 2.3.1. Test tubes with different concentrations (200, 100, 50, 10, 5 and 3 μg mL-1 for S. aureus ATCC 25923 and 500, 400, 300, 200, 100, 50, 10, 5 and 3 μg mL-1 for E. coli ATCC 25972) of the phenolic compounds were prepared with 2 mL of nutrient broth and 1 mL of the bacterial suspensions. The positive control tube (without the phenolic extract) was prepared with 2 mL of nutrient broth and 1 mL of bacterial suspension,
ACCEPTED MANUSCRIPT 8 and the negative control tube (without the bacterial suspension) was prepared with 2 mL of nutrient broth and 3 μg mL-1 phenolic compound. The samples were stored for 24 h in a bacteriological incubator (AmericanLab 101/100, Brazil) at 35±1 °C. After this period, plating was performed with serial dilutions (100, 10-1, 10-2 and 10-3) using 1 mL of sample, which was added to petri plates with the appropriate agar for each bacterium. The plates were
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then stored for 24 h in a bacteriological incubator (AmericanLab 101/100, Brasil) at 35±1 °C. The tests were conducted in duplicate.
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2.4 Statistical analysis
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Statistical analysis was performed using a confidence level of 95% for the related
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data of inhibition zones and diameters of the nanofibers. The results were expressed as the mean and standard deviation, were evaluated by an analysis of variance (ANOVA) and
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compared by the Tukey test. 3 Results and Discussion
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formation of nanofibers
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3.1 Influence of capillary diameters and the concentration of the polymer solution on the
The average diameters of nanofibers (Table 1) verify that, statistically, experiment
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5 presented nanofibers with small diameters (262 ± 32 nm) produced with 3% chitosan, 2% PEO, and 0.45 mm capillary. Table 1 shows that addition of more chitosan produced
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nanofibers of smaller diameters, which is a favorable result for the present study since it favors the antibacterial properties of this polymer for application in foods, in addition to producing nanofibers with greater contact area. TABLE 1 After determination of the conditions for the nanofibers production, phenolic compounds were added to a concentration of 1% to the polymer solution of 3% chitosan/2% PEO (Fig. 1), and they statistically (p<0.05) reduced the average diameter of the nanofibers.
ACCEPTED MANUSCRIPT 9 FIG. 1 Polymer solutions of 2% chitosan/2% PEO, 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds exhibited viscosities of 1.7, 2.6, and 3.4 Pa.s, respectively. The increases in chitosan concentration in solutions of chitosan and PEO blends increased the viscosity, which increased the entanglement of the chains between polymeric
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structures. This entanglement overcomes the surface tension at the tip of the capillary and results in uniform nanofibers with smaller diameters [25]. The same viscosity increase was
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increased concentrations of the solution components.
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observed when the phenolic compounds are added to the polymer blend because of the
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3.2 Wettability and porosity of chitosan/PEO nanofibers
The nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic
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compounds possess hydrophilic character because they exhibit contact angles less than 90°, i.e., 41.3 ± 2.9 and 26.9 ± 3.1°, respectively (Fig. 2). The hydrophilicity of nanofibers is
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related to the polymers and phenolic compounds that are soluble in solution aqueous at room
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temperature. In addition, the hydrophilicity of the nanofibers assists the phenolic compounds to interact with the product and to exert its antibacterial function.
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FIG. 2
The porosity analysis of polymeric nanofibers resulted in values of 97 ± 1.8 and
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90 ± 1% for nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds, respectively. In addition, high porosity allows for greater transfer between the content of the interstices (phenolic compounds) and the product to which it is applied, and yet the compounds remain protected by the polymer. 3.3 Thermal properties The thermogram of a chitosan polymer sample showed displacement in the endothermic curve at 84.8 °C, corresponding to dehydration, and an exothermic peak in the
ACCEPTED MANUSCRIPT 10 curve at 306 °C, possibly related to the crystallization of the material (Table 2). Kriegel et al. [26] found a melting temperature of 82.23 ± 3.50 °C for commercial chitosan. The thermograms of nanofibers of 3% chitosan/2% PEO showed two endothermic and exothermic displacements. These displacements generally indicate the glass transition temperature of the material in the cases of amorphous and semi-crystalline materials. This
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temperature suggests that the macromolecules acquire a greater degree of freedom, passing from the vitreous state to the elastomeric state and increasing the degree of mobility in the
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polymer chains without structural changes.
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The lower melting temperature and enthalpy in nanofibers than in polymers and
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phenolic compounds may be due to interactions of the PEO and chitosan chains. According to Peponi, García, Kenny [27] and Aou, Hsu [28], the electrospinning orientate the
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macromolecular chains in the longitudinal direction from chemicals reactions between polymers that produce the nanofibers. Thus, the degradation temperature of the material is
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increased due to a strong interaction between polymers.
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TABLE 2
Like the melting temperature and enthalpy, the crystallinity of the nanofibers was
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lower than that of the PEO polymer. This difference may be associated with the electrically forced orientation of the polymer chains during electrospinning, transforming them into
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oriented tangled structures, which are also present in solidified nanofibers [29]. The thermal stability of the nanofibers with phenolic compounds had different
temperatures maximum of degradation than the polymers in their granular form. During degradation, the largest mass losses (65.4 and 55.1%) occurred for nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds, respectively. For the polymers chitosan and PEO and the phenolic compounds, the loss of mass corresponded to 34.1, 89.9 and 34.0% of the initial masses, respectively.
ACCEPTED MANUSCRIPT 11 All curves thus showed that no visible changes in this temperature range. Due to these results, it can be concluded that there was the complete elimination of the solvent during the formation of nanofibers via electrospinning. 3.4 Analysis of the chemical composition and structure of nanofibers The interactions of the chemical species in the nanofibers and the incorporation of
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the phenolic compounds were analyzed by FTIR spectra. The characteristic absorption bands of chitosan were observed at 1100 cm-1, showing the antisymmetric elongation of the C-O-C
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bridge, and at 1060 and 1029 cm-1, which represent vibrations involving C-O stretching (Fig.
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3). These peaks are characteristic of chitosan polysaccharides [30, 31]. The chitosan amide I and II bands at 1650 and 1565 cm-1, respectively, were exhibited by the sample.
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The 1467 cm-1 band was attributed to C-H flexion, and the 1358 and 1340 cm-1
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bands were assigned to CH deformation of the methyl group. The OH absorption bands can be neglected because the PEO used in the experimental setup has a relatively high molecular
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mass (900 kDa) [32]. The chitosan/PEO nanofibers show a peak at 2885 cm-1 whose intensity
FIG. 3
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is lower than that of the CH2 component of the PEO polymer.
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The peak at around 2351 cm−1 corresponds to the C-H stretch of the phenolic compounds. It is observed that this peak has higher intensity in the spectrum of the phenolic
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compounds and spectrum of the 3% chitosan/2% PEO nanofibers with 1% phenolic compounds, confirming the incorporation of the compounds into the nanofibers. An analysis of the infrared spectra of the 3% chitosan/2% PEO nanofibers
indicated that these consist of the chemical components present in the chitosan and PEO powder polymers but at lower concentrations. This result may be related to the electrospinning process, which may have altered the structures of the polymer molecules for the formation of nanofibers. However, the addition of the phenolic compounds to the 3%
ACCEPTED MANUSCRIPT 12 chitosan/2% PEO solution led to a reduction in the intensity of the polymer bands, while the characteristic peaks of the phenolic compounds increased in intensity. X-ray diffraction analysis was used to study the changes in the crystalline structure of the nanofibers that occurred as a consequence of electrospinning and the addition of phenolic compounds (Fig. 4). A curve of the phenolic compounds was unobtainable since
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the equipment requires solid material and the compounds were liquids. The diffractogram showed that the powdered PEO polymer has a crystalline
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structure, which is represented by the strongly reflected peaks at 23.20 and 19.03°; however,
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the chitosan did not show peaks in the angle range analyzed. These PEO peaks were also
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analyzed by Zhang et al. [33]. After PEO was mixed with the chitosan, the reflection of both PEO peaks decreased with a flat pattern (Fig. 4 (a, b)). The diffractogram of the nanofiber
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samples exhibited amorphous halos of low intensity.
The results found by the XRD experiments may be related to the electrospinning
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since the PEO crystals were oriented through the application of tension during the technique
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to form the nanofibers [34]. In addition, amorphous halos detected in nanofibers may be due to the presence of chitosan with a semicrystalline structure. The crystallinity results calculated
than the PEO.
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through the enthalpy (Table 2) also confirmed that the nanofibers have a lower crystallinity
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FIG. 4
3.5 Antibacterial properties of phenolic compounds The sizes of the inhibition zones increased as the amount of phenolic extract
added on the discs increased, until the concentration reached the inhibition limit, at which point the zones were not significantly different (p<0.05) (Table 3). The large inhibition zones of the phenolic compounds of Spirulina sp. LEB 18 were 11.0 ± ≤0.1 and 15.7 ± 0.6 mm for S. aureus ATCC 25923 and E. coli ATCC 25972,
ACCEPTED MANUSCRIPT 13 respectively, corresponding to percentages of inhibition of 68.7 and 72.7%. Ouerghemmi et al. [35] extracted phenolic compounds from the leaves and flowers of the Ruta chalepensis plant, and these compounds generated inhibition zones of 12.3 ± 1.5 and 15.7 ± 0.9 mm for S. aureus and E. coli, respectively. Moubayed et al. [36] obtained phenolic extracts of Sargassum latifolium algae; these extracts were tested with E. coli and S. aureus and
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generated inhibition zones of 7 and 10 mm, respectively. TABLE 3
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The phenolic compounds acted to inhibit 100% growth of S. aureus ATCC 25923
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at a concentration of 200 μg mL-1, whereas 100% growth inhibition of E. coli ATCC 25972
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was possible with 500 μg mL-1.
Authors have published results on the antifungal activity of phenolic compounds
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of Spirulina platensis [11, 13, 37], but little information is available about the antibacterial activity of phenols in this microalgae [12]. The phenolic compounds and concentrations have
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shown that gram-negative bacteria (E. coli) are more resistant than gram-positive (S. aureus).
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This result may have occurred due to the different membrane compositions of gram-negative and gram-positive bacteria. The gram-negative cell wall has an additional component, the
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outer membrane, which corresponds to a second lipid bilayer that adheres firmly to the peptidoglycan layer, giving greater stiffness. The outer face of the membrane is composed of
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lipopolysaccharides, which makes it more lipophilic in relation to exogenous substances [38]. 3.6 Antibacterial properties of nanofibers The nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic
compounds inhibited the growth of S. aureus ATCC 25923, forming zones of 6.0 ± 0.7 and 6.4 ± 1.1 mm, respectively. The bacteria E. coli ATCC 25972 showed resistance to 3% chitosan/2% PEO nanofibers and sensitivity to nanofibers containing the phenolic
ACCEPTED MANUSCRIPT 14 compounds, which generated a zone of 5.5 ± 0.4 mm. The addition of phenolic compounds to the nanofibers enhanced the inhibition of S. aureus ATCC 25923 and E. coli ATCC 25972. The present study thus confirms that chitosan in the form of nanofibers exhibits antibacterial activity against S. aureus ATCC 25923. Sedghi et al. [39] developed chitosan nanofibers and incorporated curcumin as a bioactive compound. The authors verified that
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nanofibers presented antibacterial activity against S. aureus and S. epidermidis. Goy, Morais, Assis [9] developed chitosan gel at two different concentrations and evaluated their activity
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against E. coli and S. aureus using the diffusion disc method. The authors observed that the
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gel showed a zone of inhibition for both bacterias after 24 h of incubation.
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The antibacterial activity of chitosan can be explained by two main mechanisms. The first mechanism proposes that positively charged chitosan can interact with negatively
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charged groups on the cell surface and, as a consequence, change its permeability. This prevents essential materials from entering the cells and/or losing fundamental solutes out of
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the cell. The second mechanism involves chitosan binding with cellular DNA, which would
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lead to the inhibition of the synthesis of microbial RNA. The antimicrobial property of chitosan may also result from a combination of both mechanisms [40, 41].
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Phenolic compounds were incorporated into the nanofibers and did not lose their activity after electrospinning since the growth of E. coli ATCC 25972 was inhibited only by
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nanofibers containing the phenolic compounds. When the concentration of the phenolic compounds was increased, the nanofibers exhibited greater antibacterial activity and preserved products more. In addition, incorporating the phenolic compounds into the nanofibers is important because the absence of polymer protection allows a loss of their activity when they directly contact the surfaces of the products. 4 Conclusion
ACCEPTED MANUSCRIPT 15 Electrospinning method produced long and continuous nanofibers containing 3% chitosan/2% PEO/1% phenolic compounds were 214 ± 37 nm in diameter. These nanofibers presented degradation temperature higher in relation of the phenolic compounds. Chitosan/PEO nanofibers exhibited antibacterial activity against S. aureus ATCC 25923 (6.0 ± 0.7 mm) and, chitosan/PEO/phenolic compounds nanofiber presented 6.4 ± 1.1 mm and 5.5
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± 0.4 mm for S. aureus ATCC 25923 and E. coli ATCC 25972, respectively. Thus, the nanofibers developed in this study are promising for the incorporation and distribution
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uniform of bioactive compounds, as well as being able to maintain the antibacterial activity of
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the microalgal phenolic compounds.
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Acknowledgements
The authors would like to thank the CNPq (Project 310490/2014-6) (National Council of
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Technological and Scientific Development), the MCTIC (Project 01200.005005/2014-49) (Ministry of Science Technology, Innovation and Communication), CAPES (Coordination for
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the Improvement of Higher Education Personnel) and the CEME-SUL (Center of Electronic
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Microscopy of the South) for their support of this study. References
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ACCEPTED MANUSCRIPT 17 [14] M.G. Morais, E.M. Radmann, M.R. Andrade, G.G. Teixeira, L.R.F. Brusch, J.A.V. Costa, Pilot scale semicontinuous production of Spirulina biomass in Southern Brazil, Aquacult. 294, (2009) 60-64. [15] M.M. Souza, M.S. Oliveira, M. Rocha, E.B. Furlong, Antifungal activity evaluation in phenolic extracts from onion, rice bran, and Chlorella phyrenoidosa, Food Sci Technol. 3
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ACCEPTED MANUSCRIPT 18 [23] V. Fombuena, J. Balart, T. Boronat, L.S. Nácher, D.G. Sanoguera, Improving mechanical performance of thermoplastic adhesion joints by atmospheric plasma, Mater. Des. 47 (2013) 49–56. [24] NCCLS (National Committee for Clinical Laboratory Standards), Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 6th Ed., M7-A6
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[27] L. Peponi, A.M. García, J.M. Kenny, Electrospinning of PLA, in: A. Jiménez, M.
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Properties, Additives and Applications. Londres: The Royal Society of Chemistry, 2015. [28] K. Aou, S.L. Hsu, Trichroic vibrational analysis on the α-form of poly(lactic acid)
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ACCEPTED MANUSCRIPT 19 [32] C. Sawatari, T. Kondo, Interchain hydrogen bonds in blend films of poly(vinyl alcohol) and its derivatives with poly (ethylene oxide), Macromol. 32 (1999) 1949–1955. [33] J.F. Zhang, D.Z. Yang, F. Xu, Z.P. Zhang, R.X. Yin, J. Nie, Electrospun core-shell structure nanofibers from homogeneous solution of poly(ethylene oxide)/chitosan, Macromol. 42 (2009) 5278–5284.
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ACCEPTED MANUSCRIPT 20 [41] Y.-C. Chung, C.-Y. Chen, Antibacterial characteristics and activity of acid-soluble
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Figure captions Figure 1 - Nanofibers developed from 3% chitosan/2% PEO (a) and 3% chitosan/2% PEO and 1% phenolic compounds (b) Figure 2 - Water contact angle measurements of the electrospun nanofibers.
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Figure 3 - FTIR spectra of the electrospun nanofibers of 35 chitosan/2% PEO/1% phenolic
(d) and chitosan (e) polymers in the granular state.
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compounds (a) and 3% chitosan/2% PEO (b), phenolic compounds solid and pure (c), PEO
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Figure 4 - XRD patterns of the electrospun nanofibers of 3% chitosan/2% PEO/1% phenolic
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compounds (a) and 3% chitosan/2% PEO (b), PEO (d) and chitosan (e) polymers in the
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granular state.
ACCEPTED MANUSCRIPT 22 Table 1 - Mean diameters of the nanofibers (Dm, nm) according to variation of the concentration of chitosan/PEO (Ch/PEO % v w-1) and the capillary diameter (Dc, mm). Experiment Dc Ch/PEO Dm
1 0.45 2/2 393 ± 31abc
2 0.55 2/2 430 ± 36bcd
4 0.80 2/2 476 ± 45d
5 0.45 3/2 262 ± 32e
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6 0.55 3/2 360 ± 57a
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Experiment Dc Ch/PEO Dm
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Nanofibers
3 0.70 2/2 383 ± 30abc
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Nanofibers
7 0.70 3/2 377 ± 112ab
8 0.80 3/2 434 ± 71cd
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Experiment Dc Ch/PEO Dm
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Nanofibers
Identical lowercase letters indicate that there is no significant difference (p <0.05) between
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the average diameters.
ACCEPTED MANUSCRIPT 23 Table 2 - Melting temperature (Tm); enthalpy (ΔH); crystallinity (Xc); initial temperature of degradation (Tid); final temperature of degradation (Tfd) and temperature maximum of
Tf (°C)
ΔH (J g-1)
Xc (%)
Tid (°C)
Tfd (°C)
Tmd (°C)
Chitosan granules
84.8
136.4
-
284.9
320.4
301.0
PEO granules
72.3
111.0
59.0
371.8
406.9
384.8
Phenolic compounds
126.5
174.5
-
119.9
170.3
140.6
62.3
32.7
17.4
264.5
351.6
297.5
60.2
43.6
23.2
310.4
370.4
323.0
Ch/PEO
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Nanofibers
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-: sample not analyzed.
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Ch/PEO/phenolic compounds
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Samples
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degradation (Tmd) of the samples.
ACCEPTED MANUSCRIPT 24 Table 3 - Inhibition zones of phenolic compounds (PC) of Spirulina sp. LEB 18. PC concentration (μL)
Inhibition zone (mm)
Inhibition (%)
Inhibition zone (mm)
E. coli ATCC 25972
20
7.0 ± ≤0.1c
43.7
8.0 ± ≤0.1a
37.0
60
8.0 ± ≤0.1cd
50.0
8.0 ± 1.0a
37.0
100
9.3 ± 0.6bd
58.1
8.5 ± 1.3a
39.3
160
11.0 ± ≤0.1a
68.7
10.8 ± 0.3af
50.0
200
9.7 ± 0.6ab
60.6
12.0 ± 1.0bc
55.5
62.5
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S. aureus ATCC 25923
Inhibition (%)
d
72.7
13.7 ± 0.3bcd
63.4
240
10.0 ± ≤0.1
280
10.7 ± 0.6ab
66.9
320
10.7 ± 0.6ab
66.9
12.0 ± 1.0bc
55.5
380
11.0 ± 1.0a
68.7
14.7 ± 2.3cd
68.1
16.0 ± ≤0.1
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ab
21.6 ± 0.9
100.0
Chloramphenicol (30 µg/disk)
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15.7 ± 0.6
100.0
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Identical lowercase letters indicate that there is no significant difference (p <0.05) between
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the inhibition zone dimensions of the concentrations tested.
ACCEPTED MANUSCRIPT 25 Highlights 1. Phenolic compounds of Spirulina sp. LEB 18 have antibacterial activity 2. Chitosan and polyoxyethylene oxide formed nanofibers with a diameter of 262 ± 32 nm 3. Chitosan and polyoxyethylene oxide nanofibers exhibited antibacterial activity
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4. Phenolic compound-incorporated nanofibers have potential applications in packaging
Figure 1
Figure 2
Figure 3
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Suelen Goettems Kuntzler, Jorge Alberto Vieira Costa, Michele Greque de Morais PII: DOI: Reference:
S0141-8130(18)31649-0 doi:10.1016/j.ijbiomac.2018.05.224 BIOMAC 9829
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
9 April 2018 15 May 2018 30 May 2018
Please cite this article as: Suelen Goettems Kuntzler, Jorge Alberto Vieira Costa, Michele Greque de Morais , Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity. Biomac (2017), doi:10.1016/ j.ijbiomac.2018.05.224
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ACCEPTED MANUSCRIPT 1 Development of electrospun nanofibers containing chitosan/PEO blend and phenolic compounds with antibacterial activity Suelen Goettems Kuntzlera, Jorge Alberto Vieira Costab and Michele Greque de Moraisa*
a
Laboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering,
Brazil. Phone: +55-53-32336908, Fax: +55-53-32338745
Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering,
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b
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Federal University of Rio Grande (FURG), P.O. Box 474, 96203-900, Rio Grande, RS -
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Federal University of Rio Grande (FURG), P.O. Box 474, 96203-900, Rio Grande, RS – Brazil. Phone: +55-53-32336908, Fax: +55-53-32338745
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*
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Corresponding author: [email protected]
Abstract
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Electrospun nanofibers can be formed with chitosan as the polymers found in biological
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sources have antibacterial ability. The objective of this work was to evaluate whether chitosan/polyethylene oxide (PEO) blend nanofibers containing microalgal phenolic
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compounds exhibit antibacterial activity. Nanofibers produced with a 3% chitosan/2% PEO blend containing 1% phenolic compounds had an average diameter of 214 ± 37 nm, which
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resulted in a high temperature of maximum degradation, an important parameter for food packaging. The potential antibacterial activity of this nanofibers was confirmed by their inhibition of Staphylococcus aureus ATCC 25923 (6.4 ± 1.1 mm) and Escherichia coli ATCC 25972 (5.5 ± 0.4 mm). The polymeric nanofibers produced from chitosan and containing phenolic compounds have properties that therefore allow their application as active packaging. In addition, chitosan is an excellent polymer for packaging as it presents biodegradability, biocompatibility and, non-toxicity.
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Keywords: Active packaging; Electrospinning; Microalga. 1 Introduction Microbial growth accelerates changes in the aroma, color and texture of food, resulting in reduced shelf life and an increased risk of diseases transmitted by contaminated
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products. An alternative that can reduce or inhibit the growth of bacteria in food is the use of
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active packaging that in addition to being an inert barrier to the external environment,
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incorporates additives that improve food preservation [1].
Electrospun nanofibers are nanotechnology materials that have important
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properties for food applications because of their nanometric diameter, which optimizes mechanical properties such as elasticity, strength, porosity and surface area in relation to
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volume [2]. Active packaging includes the use of natural antimicrobial polymers and compounds for the purpose of developing materials that are biodegradable and reducing the
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use of synthetic preservatives [3].
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Chitosan is an amino polysaccharide obtained from chitin, an abundant byproduct of seafood processing, through a deacetylation reaction with an alkali [4]. The final structure
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of chitosan has one primary amine and two free hydroxyl groups for each monomer. Chitosan has good film-forming ability and, because of its high versatility, this polymer can be applied
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in food preservation [5]. Alibadi et al. [6] prepared a nanofiber membrane with chitosan and polyethylene oxide by electrospinning, investigating its application in the removal of metals in aqueous solution. This natural polymer has biological properties as an antibacterial [7] and antifungal [8] in addition to exhibiting gas barrier characteristics and an aroma in dry conditions. Goy, Morais, Assis [9] evaluated the antibacterial activity of chitosan that had undergone the quaternization process and in gel form. The authors verified that chitosan has
ACCEPTED MANUSCRIPT 3 an effect against Staphylococcus aureus and Escherichia coli. These characteristics make chitosan an interesting choice for nanofiber production by electrospinning for various applications. Some natural compounds, such as phenolic compounds of microalgal origin, can be incorporated into nanofibers to add antimicrobial properties. The phenolic compounds in
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Spirulina microalgae are organic acids such as caffeic, gallic, salicylic and trans-cinnamic acids [10], which act individually or synergistically and may exhibit various biological
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activities as antioxidants [11], antibacterials [12] and antifungals [13]. The objective of this
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work was to evaluate whether chitosan/PEO blend nanofibers containing microalgal phenolic
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compounds exhibit antibacterial activity. 2 Methodology
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2.1 Obtaining microalgal biomass of Spirulina sp. LEB 18 and extraction of its phenolic compounds
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In this study, the biomass of Spirulina sp. LEB 18 was dried at 50 °C for 4 h in a
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tray dryer, ground in a ball mill for 2 h, and sieved with a 37 μm stainless steel sieve (Granutest, Brasil) [14].
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The extraction of the phenolic compounds was performed according to Souza et al. [15]. Supernatant containing the phenolic compounds used for microbiological tests was
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concentrated by a rotary vacuum evaporator (Quimis, 344B2, Brazil) at 60 °C to remove methanol, and the remaining sample was then resuspended in sterilized distilled water [13]. The phenolic extract used in the polymer solution was placed in a rotavaporator (Quimis 344B2, Brazil) at 60 ° C to remove methanol, and the remaining sample was resuspended in 90% (v v-1) acetic acid solution. 2.2 Development and characterization of nanofibers 2.2.1 Preparation of polymer solutions for electrospinning
ACCEPTED MANUSCRIPT 4 Solutions with polymer blends containing 2 and 3% (w v-1) chitosan (60-120,000 g mol-1) and 2% (w v-1) polyethylene oxide (PEO) (900,000 g mol-1) (Sigma-Aldrich®, USA) were prepared with 90% (v v-1) acetic acid solution. In the solution that formed uniform nanofibers, the phenolic compounds extracted from the biomass were incorporated at a 1% (w v-1) concentration. All of the solutions were homogenized using a magnetic stirrer (Fisatom,
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Brazil) for 16 h. 2.2.2 Development of nanofibers
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The electrospinning equipment consists of a positive displacement pump (Model
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KDS 100, KD Scientific, United States) and a source of high-voltage, direct-current power
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(Model ET 5000 CC, Electric Test Serta, Brazil). The polymer solutions were injected through capillaries with a diameter of 0.45, 0.55, 0.70 and 0.80 mm. The fixed parameters
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were an electric potential of 20 kV, a distance of the capillary to the collector of 100 mm and a feed rate of 300 μL h-1, according to Vrieze et al. [16]. All tests were conducted at 22 °C
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with the relative humidity maintained at 60±1%.
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2.2.3 Shape and diameter of the nanofibers Images and 30 measurements of the diameters of the nanofibers with and without
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phenolic compounds were obtained using a scanning electron microscope (SEM) (JEOL JSM6610 LV, Japan). Before the analyses, the samples were fixed in a metallic holder with carbon
[17].
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tape and coated with gold using a sputtering diode (Denton Vacuum CAR001-0038, USA)
2.2.4 Viscosity of the polymer solutions The viscosity of polymer solutions was determined from 0.5 mL of each sample using a rheometer (Brookfield DV-III Ultra Programmable Rheometer, USA). 2.2.5 Porosity of the nanofibers
ACCEPTED MANUSCRIPT 5 The porosity was calculated from Equation 1 [18], with ρ and ρ0 (g cm-3) representing the densities of the nanofibers and polymer film, respectively, which were determined according to their dimensions and masses. Porosity (%) = 1 100 0
(1)
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The film samples were obtained by a casting technique using polymer solution distributed longitudinally in petri plates, followed by evaporation of the solvent [19].
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2.2.6 Endothermic and exothermic transitions
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To determine the melting temperature of the nanofibers, phenolic compounds and polymers, differential scanning calorimetry (DSC) (Shimadzu DSC-60, Japan) was used.
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Approximately 4 mg of each sample was placed in an aluminum capsule, which was sealed with a lid; the samples were tested under a nitrogen atmosphere with a flow rate of 50 mL
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min-1. The analyses were carried out at room temperature and heated at a rate of 10 °C min-1 until the samples reached 400 °C. The melting temperature was determined from the melting
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peak shown in the DSC curve according to ASTM (American Society for Testing and Materials) D7426-08 [20], and the degree of crystallinity (Xc) was determined according to Equation 2.
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Hm 100 Xc (%) 0 Hm
(2)
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where ΔH0m represents the melting enthalpy for 100% crystalline PEO (118 J g-1)
and ΔHm indicates the melting enthalpy calculated from DSC thermograms [21]. 2.2.7 Thermal degradation and residual solvent content The analyses were conducted on nanofibers, phenolic compounds and polymers at temperatures from room temperature to 500 °C in an inert atmosphere consisting of nitrogen with a flow rate of 30 mL min-1 and with a 10 °C min-1 heating rate, using 2 to 6 mg of
ACCEPTED MANUSCRIPT 6 sample. A thermogravimetric analyzer (TGA) (Shimadzu DTG-60, Japan) was used according to the ASTM D3850-12 [22] method. 2.2.8 Structural characterization of nanofibers, phenolic compounds and polymers Fourier transform infrared spectroscopy (FTIR) was performed on a Thermo Scientific-Nicolet 6700 (Shimadzu, Japan), and the sample spectra were analyzed at
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wavenumbers from 0 to 4000 cm-1. Nanofibers samples, phenolic compounds and the polymers, all in solid form, were homogenized and prepared with potassium bromide (KBr).
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2.2.9 Contact angle of the nanofibers
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A drop of water was inserted into the surface of the nanofibers, and the image was
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then obtained by microscope (Digital Blue). The Surftens 3.0 software was used to make five measurements of each image using five different points arranged around the drop. The
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maximum standard deviation of the water angle contact was ± 3% [23]. 2.2.10 X-ray diffraction (XRD) of nanofibers and polymers
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This analysis was performed on a D8 Advance X-ray diffractometer (Bruker,
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United States) equipped with a copper tube. The radiation was produced at a wavelength of 0.154 nm and at 40 kV and 40 mA. The diffractograms were obtained at room temperature
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(25 °C) at a 2θ angle between 10 and 90° at a step rate of 0.05° sec-1. The nanofibers samples were prepared as films having a diameter of approximately 50 mm and the polymers were
8.0.
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analyzed in granular state. The diffractograms were analyzed using the software Origin Pro
2.3 Evaluation of antibacterial activity 2.3.1 Disc diffusion method The determination of the antibacterial activity of the phenolic compounds and phenolic compounds in nanofibers was performed against the microorganisms Escherichia coli ATCC 25972 and Staphylococcus aureus ATCC 25923 with the disc diffusion test
ACCEPTED MANUSCRIPT 7 according to the M7 - A6 standard established by National Committee for Clinical Laboratory Standards [24]. Bacterial cultures were prepared on Mueller-Hinton agar for E. coli ATCC 25972 and plate count agar for S. aureus ATCC 25923. An isolated colony was removed from each culture and utilized for a bacterial suspension that was prepared in saline solution (0.85% w v-1) in order to obtain an optical turbidity comparable to a McFarland standard solution of
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0.5 measured in a spectrophotometer (Shimadzu, UV Mini 1240, Japan) at wavelength 600 nm (equivalent to λ = 0.1). This approach resulted in a suspension containing 1 to 2 x 108
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CFU mL-1 bacteria.
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The bacterial suspension was added to petri plates containing the corresponding agar for each microorganism. Qualitative filter paper discs with 7 mm diameters containing
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different amounts (20, 60, 100, 160, 200, 240, 280, 320 and 380 μL) of the phenolic extract
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were added to the petri plates, and all tests were performed in triplicate. Discs produced with approximately 4 mm the nanofibers with and without the phenolic compounds were added to
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the plates. All tests were performed in triplicate. The samples were stored for 24 h in a
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bacteriological incubator (AmericanLab 101/100, Brazil) at 35±1 °C. After this period, growth inhibition was quantified by the diameter of the sample inhibition zones [24]. The
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percent inhibition of the phenolic compounds was calculated relative to control inhibition zone, (chloramphenicol 30 μg) which was considered 100% inhibition of growth.
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2.3.2 Macrodilution method with colony count The determination of the antibacterial activity of the phenolic compounds was
performed according to section 2.3.1. Test tubes with different concentrations (200, 100, 50, 10, 5 and 3 μg mL-1 for S. aureus ATCC 25923 and 500, 400, 300, 200, 100, 50, 10, 5 and 3 μg mL-1 for E. coli ATCC 25972) of the phenolic compounds were prepared with 2 mL of nutrient broth and 1 mL of the bacterial suspensions. The positive control tube (without the phenolic extract) was prepared with 2 mL of nutrient broth and 1 mL of bacterial suspension,
ACCEPTED MANUSCRIPT 8 and the negative control tube (without the bacterial suspension) was prepared with 2 mL of nutrient broth and 3 μg mL-1 phenolic compound. The samples were stored for 24 h in a bacteriological incubator (AmericanLab 101/100, Brazil) at 35±1 °C. After this period, plating was performed with serial dilutions (100, 10-1, 10-2 and 10-3) using 1 mL of sample, which was added to petri plates with the appropriate agar for each bacterium. The plates were
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then stored for 24 h in a bacteriological incubator (AmericanLab 101/100, Brasil) at 35±1 °C. The tests were conducted in duplicate.
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2.4 Statistical analysis
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Statistical analysis was performed using a confidence level of 95% for the related
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data of inhibition zones and diameters of the nanofibers. The results were expressed as the mean and standard deviation, were evaluated by an analysis of variance (ANOVA) and
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compared by the Tukey test. 3 Results and Discussion
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formation of nanofibers
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3.1 Influence of capillary diameters and the concentration of the polymer solution on the
The average diameters of nanofibers (Table 1) verify that, statistically, experiment
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5 presented nanofibers with small diameters (262 ± 32 nm) produced with 3% chitosan, 2% PEO, and 0.45 mm capillary. Table 1 shows that addition of more chitosan produced
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nanofibers of smaller diameters, which is a favorable result for the present study since it favors the antibacterial properties of this polymer for application in foods, in addition to producing nanofibers with greater contact area. TABLE 1 After determination of the conditions for the nanofibers production, phenolic compounds were added to a concentration of 1% to the polymer solution of 3% chitosan/2% PEO (Fig. 1), and they statistically (p<0.05) reduced the average diameter of the nanofibers.
ACCEPTED MANUSCRIPT 9 FIG. 1 Polymer solutions of 2% chitosan/2% PEO, 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds exhibited viscosities of 1.7, 2.6, and 3.4 Pa.s, respectively. The increases in chitosan concentration in solutions of chitosan and PEO blends increased the viscosity, which increased the entanglement of the chains between polymeric
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structures. This entanglement overcomes the surface tension at the tip of the capillary and results in uniform nanofibers with smaller diameters [25]. The same viscosity increase was
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increased concentrations of the solution components.
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observed when the phenolic compounds are added to the polymer blend because of the
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3.2 Wettability and porosity of chitosan/PEO nanofibers
The nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic
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compounds possess hydrophilic character because they exhibit contact angles less than 90°, i.e., 41.3 ± 2.9 and 26.9 ± 3.1°, respectively (Fig. 2). The hydrophilicity of nanofibers is
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related to the polymers and phenolic compounds that are soluble in solution aqueous at room
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temperature. In addition, the hydrophilicity of the nanofibers assists the phenolic compounds to interact with the product and to exert its antibacterial function.
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FIG. 2
The porosity analysis of polymeric nanofibers resulted in values of 97 ± 1.8 and
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90 ± 1% for nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds, respectively. In addition, high porosity allows for greater transfer between the content of the interstices (phenolic compounds) and the product to which it is applied, and yet the compounds remain protected by the polymer. 3.3 Thermal properties The thermogram of a chitosan polymer sample showed displacement in the endothermic curve at 84.8 °C, corresponding to dehydration, and an exothermic peak in the
ACCEPTED MANUSCRIPT 10 curve at 306 °C, possibly related to the crystallization of the material (Table 2). Kriegel et al. [26] found a melting temperature of 82.23 ± 3.50 °C for commercial chitosan. The thermograms of nanofibers of 3% chitosan/2% PEO showed two endothermic and exothermic displacements. These displacements generally indicate the glass transition temperature of the material in the cases of amorphous and semi-crystalline materials. This
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temperature suggests that the macromolecules acquire a greater degree of freedom, passing from the vitreous state to the elastomeric state and increasing the degree of mobility in the
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polymer chains without structural changes.
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The lower melting temperature and enthalpy in nanofibers than in polymers and
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phenolic compounds may be due to interactions of the PEO and chitosan chains. According to Peponi, García, Kenny [27] and Aou, Hsu [28], the electrospinning orientate the
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macromolecular chains in the longitudinal direction from chemicals reactions between polymers that produce the nanofibers. Thus, the degradation temperature of the material is
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increased due to a strong interaction between polymers.
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TABLE 2
Like the melting temperature and enthalpy, the crystallinity of the nanofibers was
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lower than that of the PEO polymer. This difference may be associated with the electrically forced orientation of the polymer chains during electrospinning, transforming them into
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oriented tangled structures, which are also present in solidified nanofibers [29]. The thermal stability of the nanofibers with phenolic compounds had different
temperatures maximum of degradation than the polymers in their granular form. During degradation, the largest mass losses (65.4 and 55.1%) occurred for nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic compounds, respectively. For the polymers chitosan and PEO and the phenolic compounds, the loss of mass corresponded to 34.1, 89.9 and 34.0% of the initial masses, respectively.
ACCEPTED MANUSCRIPT 11 All curves thus showed that no visible changes in this temperature range. Due to these results, it can be concluded that there was the complete elimination of the solvent during the formation of nanofibers via electrospinning. 3.4 Analysis of the chemical composition and structure of nanofibers The interactions of the chemical species in the nanofibers and the incorporation of
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the phenolic compounds were analyzed by FTIR spectra. The characteristic absorption bands of chitosan were observed at 1100 cm-1, showing the antisymmetric elongation of the C-O-C
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bridge, and at 1060 and 1029 cm-1, which represent vibrations involving C-O stretching (Fig.
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3). These peaks are characteristic of chitosan polysaccharides [30, 31]. The chitosan amide I and II bands at 1650 and 1565 cm-1, respectively, were exhibited by the sample.
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The 1467 cm-1 band was attributed to C-H flexion, and the 1358 and 1340 cm-1
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bands were assigned to CH deformation of the methyl group. The OH absorption bands can be neglected because the PEO used in the experimental setup has a relatively high molecular
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mass (900 kDa) [32]. The chitosan/PEO nanofibers show a peak at 2885 cm-1 whose intensity
FIG. 3
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is lower than that of the CH2 component of the PEO polymer.
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The peak at around 2351 cm−1 corresponds to the C-H stretch of the phenolic compounds. It is observed that this peak has higher intensity in the spectrum of the phenolic
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compounds and spectrum of the 3% chitosan/2% PEO nanofibers with 1% phenolic compounds, confirming the incorporation of the compounds into the nanofibers. An analysis of the infrared spectra of the 3% chitosan/2% PEO nanofibers
indicated that these consist of the chemical components present in the chitosan and PEO powder polymers but at lower concentrations. This result may be related to the electrospinning process, which may have altered the structures of the polymer molecules for the formation of nanofibers. However, the addition of the phenolic compounds to the 3%
ACCEPTED MANUSCRIPT 12 chitosan/2% PEO solution led to a reduction in the intensity of the polymer bands, while the characteristic peaks of the phenolic compounds increased in intensity. X-ray diffraction analysis was used to study the changes in the crystalline structure of the nanofibers that occurred as a consequence of electrospinning and the addition of phenolic compounds (Fig. 4). A curve of the phenolic compounds was unobtainable since
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the equipment requires solid material and the compounds were liquids. The diffractogram showed that the powdered PEO polymer has a crystalline
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structure, which is represented by the strongly reflected peaks at 23.20 and 19.03°; however,
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the chitosan did not show peaks in the angle range analyzed. These PEO peaks were also
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analyzed by Zhang et al. [33]. After PEO was mixed with the chitosan, the reflection of both PEO peaks decreased with a flat pattern (Fig. 4 (a, b)). The diffractogram of the nanofiber
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samples exhibited amorphous halos of low intensity.
The results found by the XRD experiments may be related to the electrospinning
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since the PEO crystals were oriented through the application of tension during the technique
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to form the nanofibers [34]. In addition, amorphous halos detected in nanofibers may be due to the presence of chitosan with a semicrystalline structure. The crystallinity results calculated
than the PEO.
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through the enthalpy (Table 2) also confirmed that the nanofibers have a lower crystallinity
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FIG. 4
3.5 Antibacterial properties of phenolic compounds The sizes of the inhibition zones increased as the amount of phenolic extract
added on the discs increased, until the concentration reached the inhibition limit, at which point the zones were not significantly different (p<0.05) (Table 3). The large inhibition zones of the phenolic compounds of Spirulina sp. LEB 18 were 11.0 ± ≤0.1 and 15.7 ± 0.6 mm for S. aureus ATCC 25923 and E. coli ATCC 25972,
ACCEPTED MANUSCRIPT 13 respectively, corresponding to percentages of inhibition of 68.7 and 72.7%. Ouerghemmi et al. [35] extracted phenolic compounds from the leaves and flowers of the Ruta chalepensis plant, and these compounds generated inhibition zones of 12.3 ± 1.5 and 15.7 ± 0.9 mm for S. aureus and E. coli, respectively. Moubayed et al. [36] obtained phenolic extracts of Sargassum latifolium algae; these extracts were tested with E. coli and S. aureus and
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generated inhibition zones of 7 and 10 mm, respectively. TABLE 3
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The phenolic compounds acted to inhibit 100% growth of S. aureus ATCC 25923
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at a concentration of 200 μg mL-1, whereas 100% growth inhibition of E. coli ATCC 25972
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was possible with 500 μg mL-1.
Authors have published results on the antifungal activity of phenolic compounds
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of Spirulina platensis [11, 13, 37], but little information is available about the antibacterial activity of phenols in this microalgae [12]. The phenolic compounds and concentrations have
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shown that gram-negative bacteria (E. coli) are more resistant than gram-positive (S. aureus).
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This result may have occurred due to the different membrane compositions of gram-negative and gram-positive bacteria. The gram-negative cell wall has an additional component, the
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outer membrane, which corresponds to a second lipid bilayer that adheres firmly to the peptidoglycan layer, giving greater stiffness. The outer face of the membrane is composed of
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lipopolysaccharides, which makes it more lipophilic in relation to exogenous substances [38]. 3.6 Antibacterial properties of nanofibers The nanofibers of 3% chitosan/2% PEO and 3% chitosan/2% PEO/1% phenolic
compounds inhibited the growth of S. aureus ATCC 25923, forming zones of 6.0 ± 0.7 and 6.4 ± 1.1 mm, respectively. The bacteria E. coli ATCC 25972 showed resistance to 3% chitosan/2% PEO nanofibers and sensitivity to nanofibers containing the phenolic
ACCEPTED MANUSCRIPT 14 compounds, which generated a zone of 5.5 ± 0.4 mm. The addition of phenolic compounds to the nanofibers enhanced the inhibition of S. aureus ATCC 25923 and E. coli ATCC 25972. The present study thus confirms that chitosan in the form of nanofibers exhibits antibacterial activity against S. aureus ATCC 25923. Sedghi et al. [39] developed chitosan nanofibers and incorporated curcumin as a bioactive compound. The authors verified that
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nanofibers presented antibacterial activity against S. aureus and S. epidermidis. Goy, Morais, Assis [9] developed chitosan gel at two different concentrations and evaluated their activity
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against E. coli and S. aureus using the diffusion disc method. The authors observed that the
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gel showed a zone of inhibition for both bacterias after 24 h of incubation.
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The antibacterial activity of chitosan can be explained by two main mechanisms. The first mechanism proposes that positively charged chitosan can interact with negatively
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charged groups on the cell surface and, as a consequence, change its permeability. This prevents essential materials from entering the cells and/or losing fundamental solutes out of
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the cell. The second mechanism involves chitosan binding with cellular DNA, which would
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lead to the inhibition of the synthesis of microbial RNA. The antimicrobial property of chitosan may also result from a combination of both mechanisms [40, 41].
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Phenolic compounds were incorporated into the nanofibers and did not lose their activity after electrospinning since the growth of E. coli ATCC 25972 was inhibited only by
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nanofibers containing the phenolic compounds. When the concentration of the phenolic compounds was increased, the nanofibers exhibited greater antibacterial activity and preserved products more. In addition, incorporating the phenolic compounds into the nanofibers is important because the absence of polymer protection allows a loss of their activity when they directly contact the surfaces of the products. 4 Conclusion
ACCEPTED MANUSCRIPT 15 Electrospinning method produced long and continuous nanofibers containing 3% chitosan/2% PEO/1% phenolic compounds were 214 ± 37 nm in diameter. These nanofibers presented degradation temperature higher in relation of the phenolic compounds. Chitosan/PEO nanofibers exhibited antibacterial activity against S. aureus ATCC 25923 (6.0 ± 0.7 mm) and, chitosan/PEO/phenolic compounds nanofiber presented 6.4 ± 1.1 mm and 5.5
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± 0.4 mm for S. aureus ATCC 25923 and E. coli ATCC 25972, respectively. Thus, the nanofibers developed in this study are promising for the incorporation and distribution
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uniform of bioactive compounds, as well as being able to maintain the antibacterial activity of
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the microalgal phenolic compounds.
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Acknowledgements
The authors would like to thank the CNPq (Project 310490/2014-6) (National Council of
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Technological and Scientific Development), the MCTIC (Project 01200.005005/2014-49) (Ministry of Science Technology, Innovation and Communication), CAPES (Coordination for
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the Improvement of Higher Education Personnel) and the CEME-SUL (Center of Electronic
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Microscopy of the South) for their support of this study. References
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[27] L. Peponi, A.M. García, J.M. Kenny, Electrospinning of PLA, in: A. Jiménez, M.
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Peltzer, R. Ruseckaite (Eds), Poly (lactic acid) Science and Technology: Processing,
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Properties, Additives and Applications. Londres: The Royal Society of Chemistry, 2015. [28] K. Aou, S.L. Hsu, Trichroic vibrational analysis on the α-form of poly(lactic acid)
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ACCEPTED MANUSCRIPT 19 [32] C. Sawatari, T. Kondo, Interchain hydrogen bonds in blend films of poly(vinyl alcohol) and its derivatives with poly (ethylene oxide), Macromol. 32 (1999) 1949–1955. [33] J.F. Zhang, D.Z. Yang, F. Xu, Z.P. Zhang, R.X. Yin, J. Nie, Electrospun core-shell structure nanofibers from homogeneous solution of poly(ethylene oxide)/chitosan, Macromol. 42 (2009) 5278–5284.
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[36] N.M.S. Moubayed, H.J. Houri, M.M. Khulaifi, D.A. Farraj, Antimicrobial, antioxidant properties and chemical composition of seaweeds collected from Saudi Arabia (Red Sea and
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ACCEPTED MANUSCRIPT 20 [41] Y.-C. Chung, C.-Y. Chen, Antibacterial characteristics and activity of acid-soluble
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ACCEPTED MANUSCRIPT 21
Figure captions Figure 1 - Nanofibers developed from 3% chitosan/2% PEO (a) and 3% chitosan/2% PEO and 1% phenolic compounds (b) Figure 2 - Water contact angle measurements of the electrospun nanofibers.
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Figure 3 - FTIR spectra of the electrospun nanofibers of 35 chitosan/2% PEO/1% phenolic
(d) and chitosan (e) polymers in the granular state.
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compounds (a) and 3% chitosan/2% PEO (b), phenolic compounds solid and pure (c), PEO
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Figure 4 - XRD patterns of the electrospun nanofibers of 3% chitosan/2% PEO/1% phenolic
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compounds (a) and 3% chitosan/2% PEO (b), PEO (d) and chitosan (e) polymers in the
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granular state.
ACCEPTED MANUSCRIPT 22 Table 1 - Mean diameters of the nanofibers (Dm, nm) according to variation of the concentration of chitosan/PEO (Ch/PEO % v w-1) and the capillary diameter (Dc, mm). Experiment Dc Ch/PEO Dm
1 0.45 2/2 393 ± 31abc
2 0.55 2/2 430 ± 36bcd
4 0.80 2/2 476 ± 45d
5 0.45 3/2 262 ± 32e
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6 0.55 3/2 360 ± 57a
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Experiment Dc Ch/PEO Dm
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Nanofibers
3 0.70 2/2 383 ± 30abc
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Nanofibers
7 0.70 3/2 377 ± 112ab
8 0.80 3/2 434 ± 71cd
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Experiment Dc Ch/PEO Dm
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Nanofibers
Identical lowercase letters indicate that there is no significant difference (p <0.05) between
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the average diameters.
ACCEPTED MANUSCRIPT 23 Table 2 - Melting temperature (Tm); enthalpy (ΔH); crystallinity (Xc); initial temperature of degradation (Tid); final temperature of degradation (Tfd) and temperature maximum of
Tf (°C)
ΔH (J g-1)
Xc (%)
Tid (°C)
Tfd (°C)
Tmd (°C)
Chitosan granules
84.8
136.4
-
284.9
320.4
301.0
PEO granules
72.3
111.0
59.0
371.8
406.9
384.8
Phenolic compounds
126.5
174.5
-
119.9
170.3
140.6
62.3
32.7
17.4
264.5
351.6
297.5
60.2
43.6
23.2
310.4
370.4
323.0
Ch/PEO
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Nanofibers
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-: sample not analyzed.
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Ch/PEO/phenolic compounds
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Samples
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degradation (Tmd) of the samples.
ACCEPTED MANUSCRIPT 24 Table 3 - Inhibition zones of phenolic compounds (PC) of Spirulina sp. LEB 18. PC concentration (μL)
Inhibition zone (mm)
Inhibition (%)
Inhibition zone (mm)
E. coli ATCC 25972
20
7.0 ± ≤0.1c
43.7
8.0 ± ≤0.1a
37.0
60
8.0 ± ≤0.1cd
50.0
8.0 ± 1.0a
37.0
100
9.3 ± 0.6bd
58.1
8.5 ± 1.3a
39.3
160
11.0 ± ≤0.1a
68.7
10.8 ± 0.3af
50.0
200
9.7 ± 0.6ab
60.6
12.0 ± 1.0bc
55.5
62.5
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S. aureus ATCC 25923
Inhibition (%)
d
72.7
13.7 ± 0.3bcd
63.4
240
10.0 ± ≤0.1
280
10.7 ± 0.6ab
66.9
320
10.7 ± 0.6ab
66.9
12.0 ± 1.0bc
55.5
380
11.0 ± 1.0a
68.7
14.7 ± 2.3cd
68.1
16.0 ± ≤0.1
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ab
21.6 ± 0.9
100.0
Chloramphenicol (30 µg/disk)
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15.7 ± 0.6
100.0
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Identical lowercase letters indicate that there is no significant difference (p <0.05) between
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the inhibition zone dimensions of the concentrations tested.
ACCEPTED MANUSCRIPT 25 Highlights 1. Phenolic compounds of Spirulina sp. LEB 18 have antibacterial activity 2. Chitosan and polyoxyethylene oxide formed nanofibers with a diameter of 262 ± 32 nm 3. Chitosan and polyoxyethylene oxide nanofibers exhibited antibacterial activity
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4. Phenolic compound-incorporated nanofibers have potential applications in packaging
Figure 1
Figure 2
Figure 3
Figure 4