- Email: [email protected]
Food-grade gliadin microstructures obtained by electrohydrodynamic processing
Accepted Manuscript Food-grade gliadin microstructures electrohydrodynamic processing
obtained
Niloufar Sharif, Mohammad-Taghi Golmakani, Niakousari, Behrouz Ghorani, Amparo Lopez-Rubio
by
Mehrdad
PII: DOI: Reference:
S0963-9969(18)30814-7 doi:10.1016/j.foodres.2018.10.027 FRIN 7997
To appear in:
Food Research International
Received date: Revised date: Accepted date:
6 May 2018 27 September 2018 7 October 2018
Please cite this article as: Niloufar Sharif, Mohammad-Taghi Golmakani, Mehrdad Niakousari, Behrouz Ghorani, Amparo Lopez-Rubio , Food-grade gliadin microstructures obtained by electrohydrodynamic processing. Frin (2018), doi:10.1016/ j.foodres.2018.10.027
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ACCEPTED MANUSCRIPT Food-Grade
Gliadin
Microstructures
Obtained
by
Electrohydrodynamic Processing
Niloufar Sharif1 , Mohammad-Taghi Golmakani1* , Mehrdad Niakousari1 , Behrouz Ghorani2 ,
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Department of Food Science and Technology, School of Agriculture, Shiraz University, km
12
Shiraz-Esfahan
Highway,
P.
O.
Box:
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1
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Amparo Lopez-Rubio3*
71441-65186,
Shiraz,
Iran.
Emails:
2
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[email protected]; [email protected]; [email protected] Department of Food Nanotechnology, Research Institute of Food Science and Technology
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(RIFST), km 12 Mashhad-Quchan Highway, P. O. Box: 91895/157/356, Mashhad, Iran. Email: [email protected]
Food Quality and Preservation Department, IATA-CSIC, 46980 Paterna, Valencia, Spain.
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3
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Email: [email protected]
*Corresponding authors:
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- Amparo Lopez Rubio: Phone: (+34) 963900022; fax: (+34) 963636301. Email: [email protected] - Mohammad-Taghi Golmakani: Phone: (+98)71-36138243. Fax: (+98)71-32286110. Email: [email protected]
ACCEPTED MANUSCRIPT Abstract This paper presents a comprehensive study on the electrohydrodynamic processing of gliadin to develop food-grade delivery systems with different morphologies. The effects of biopolymer concentration, applied voltage and solution flow-rate on particle morphology, molecular organisation,
crystallinity and
thermal properties were investigated.
Gliadin
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concentration influenced the apparent viscosity and conductivity of the solutions, giving raise
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to particle morphologies at 10 wt.% gliadin and beaded-free fibers above 25 wt.% gliadin. In
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general, increasing the voltage resulted in smaller average sizes of the obtained structures, while no significant differences in morphology were observed amongst the tested flow rates.
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Interestingly, the amide I position in the FTIR reflected changes in protein conformation which could be correlated with the final morphology attained. Moreover, the acetic acid used
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for solution preparation disrupted the original amino acid chain packing of the gliadin fraction, being the electrospun/electrosprayed samples amorphous. These gliadin-based
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microparticles and microfibers obtained could serve as food-grade delivery vehicles.
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Keywords: Electrospinning; Electrospraying; Gluten; Gliadin, XRD; Plant Proteins
ACCEPTED MANUSCRIPT 1. Introduction The development of delivery systems for bioactive components is a topic of increased interest in the food area, as it provides a number of advantages, such as increasing the solubility of certain bioactive compounds, protecting labile ingredients and/or favoring a gradual bioactive release (Chen, Remondetto, & Subirade, 2006; Joye, Davidov-Pardo, & McClements, 2014). different
techniques
used
for the
development
of delivery
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Amongst the
systems,
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electrohydrodynamic processes, namely electrospinning and electrospraying, are rapidly
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emerging as promising technologies to obtain fibers and particles at the micron, submicron and nanoscales (Mendes, Stephansen, & Chronakis, 2017). The key advantages of the so-
of the
electrospun
fiber
mats,
their
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obtained fibers and particles are their large surface-to-volume ratio and, in the specific case small pore
size
and
high
porosity.
These
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electrohydrodynamic processed structures can be exploited for a wide variety of applications including tissue engineering wound healing (M. Liu, Duan, Li, Yang, & Long, 2017), drug 2014), encapsulation,
immobilization of enzymes, filtration and
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delivery (Hu et al.,
fabrication of food packaging materials, among others (Bhushani & Anandharamakrishnan,
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2014; Ghorani & Tucker, 2015). As widely reported, in an electrohydrodynamic process, a polymer or biopolymer solution is drawn from a nozzle towards a grounded collector by the
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application of an external electric field (Karthikeyan, Guhathakarta, Rajaram, & Korrapati, 2012; Neo, Ray, Easteal, Nikolaidis, & Quek, 2012). During the flight between the nozzle and the collector, the jet is elongated and the solvent evaporates, producing dry continuous fibers in the case of electrospinning, while in the case of electrospraying, the jet breaks down into fine droplets, subsequently producing solid nano- or microparticles upon solvent evaporation (Bhushani & Anandharamakrishnan, 2014). Many parameters can influence fiber/particle formation and morphology during an electrohydrodynamic process including solution properties (mainly viscosity, electrical conductivity and surface tension), process
ACCEPTED MANUSCRIPT conditions (applied voltage, fluid flow rate, distance from nozzle to collector) and ambient conditions (like temperature and humidity) (Ghorani & Tucker, 2015; Mendes et al., 2017). Specifically, solution properties can be tailored to produce different morphologies using electrohydrodynamic processing, as they depend on the type of polymer/biopolymer used, its concentration, the solvent employed to dissolve/disperse the polymer and the polymer-
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solvent interactions (Pérez-Masiá, López-Rubio, & Lagarón, 2013).
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For food-related applications, the use of GRAS materials is a requirement and, thus, the use
biocompatible,
have
attracted
increasing
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of biopolymers such as proteins, which are generally renewable, biodegradable and attention
as
the
raw
materials
for
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electrohydrodynamic processing (Ago, Okajima, Jakes, Park, & Rojas, 2012). Plant storage proteins, including co-products from the industrial processing of cereal crops, would be
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interesting raw materials for fabricating cost-effective electrospun/electrosprayed structures (Y. Wang & Chen, 2012b), as they are considered more “environmentally economical” when
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compared with animal-derived proteins (X. Wang, Yu, Li, Bligh, & Williams, 2015). Wheat
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gluten, a by-product of wheat starch industry has two main fractions; monomeric gliadins and polymeric glutenins (Feng, Wu, Wang, & Liu, 2017). Gliadins are important storage prolamins in wheat kernel, which are complex glycoproteins rich in proline and glutamine
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(Veloso, Dias, Rodrigues, & Peres, 2016). These wheat-derived proteins are of particular interest for the development of food-grade delivery systems because they are highly hydrophobic, unlike many other food proteins, thus enabling to encapsulate hydrophobic ingredients without the need for oil phases (Duclairoir, Orecchioni, Depraetere, Osterstock, & Nakache,
2003).
Moreover,
gliadin
nanoparticles
have
bioadhesive
ability
through
electrostatic interactions and hydrogen bonding to the intestinal membrane (Tarhini, GreigeGerges, & Elaissari, 2017), fact of outmost importance for bioactive delivery applications.
ACCEPTED MANUSCRIPT Although gliadin nanoparticles have been developed for drug delivery applications mainly using emulsification and desolvation techniques (Gulfam et al., 2012; Soares et al., 2011; Xu et al., 2017), there are only few works in which gliadin-based structures have been prepared using electrohydrodynamic techniques (Gulfam et al., 2012; Xu et al., 2017) and none of them have explored how the processing parameters affect structure formation through this
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technique. Therefore, the objective of this study was to evaluate how solution properties
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(surface tension, viscosity and electrical conductivity) and process parameters (applied
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voltage and fluid flow rate) affected the morphology of the gliadin structures obtained through electrohydrodynamic processing. The molecular organisation (Fourier Transform analysis),
crystallinity (X-Ray diffraction) and
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Infrared
scanning calorimetry) of the generated micro-
and submicro-
(differential
structures were also
2. Materials and Methods
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2.1. Materials
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characterized.
thermal properties
Wheat gluten was obtained from a local shop (Shiraz, Iran). Acetic acid was purchased from Scharlau company (Barcelona, Spain). All other chemicals used were of analytical grade,
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unless otherwise specified.
2.2. Gliadin extraction
The gliadin fraction was extracted using the method reported by Hong et al. (2016) with slight modifications. Briefly, samples of dried gluten powder (20 g) were gently stirred in an ethanol/water mixture (70/30 v/v; gluten/solvent ratio of 1/12) for 4h at 20ºC. The suspension was centrifuged to collect the gliadin fraction at 10000 g for 10 min. Finally, the ethanol was evaporated at ambient conditions. The extraction yield of gliadin from wheat gluten powder
ACCEPTED MANUSCRIPT was 37.5%. The protein content as determined using the Kjeldahl method was 89.8% on a dry matter basis.
2.3. Solution preparation for electrohydrodynamic processing For the electrohydrodynamic process, gliadin solutions were prepared in pure acetic acid (Y.
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Wang & Chen, 2012b) dissolving the protein at various concentrations (5, 10, 15, 20, 25, 30
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and 35%, w/v). Greater concentrations gave rise to too viscous solutions that could not be
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processed. The various solutions were stirred at room temperature until the protein was
2.4. Characterization of gliadin solutions
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completely dissolved.
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The apparent viscosity, surface tension and electrical conductivity of the gliadin solutions at the different concentrations were evaluated. The surface tension of the solutions was
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measured using the Wilhemy plate method in an EasyDyne K20 tensiometer (Krüss GmbH, Hamburg, Germany) after calibration of the equipment with deionized water. The electrical
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conductivity of the solutions was measured using a conductivity meter XS Con6 (Labbox, Barcelona, Spain). The apparent viscosity of the gliadin solutions was determined by a
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rotation viscometer VISCO BASIC PLUS L from Fungilab S.A. (Sant Feliu de Llobregat, Spain) at 30 rpm using the TL5 spindle. All measurements were made at 25ºC. Experiments were performed in triplicate.
2.5. Electrohydrodynamic processing of the gliadin solutions The various gliadin solutions were processed using an electrohydrodynamic apparatus equipped with a variable high voltage 0-35 kV power supplier (Spinner-3X-Advance, ANSTCO, Iran). Gliadin solutions were loaded into 10 mL disposable plastic syringes and
ACCEPTED MANUSCRIPT they were electrospun/electrosprayed under a steady flow-rate using a stainless-steel needle (inner diameter of 0.9 mm) which was connected to the electrode of the high voltage power supply. Two different voltages (15 and 18 kV) and fluid flow rates (0.5 and 1 ml/h) were applied to see how these process parameters affected the morphology of the obtained gliadin structures. Tip to collector distance was kept constant at 100 mm, as preliminary tests
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demonstrated a proper solution drying at this specific distance. The obtained structures were
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collected on aluminum foil attached to the surface of the collector and kept overnight under
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the hood to evaporate any solvent residues. All experiments were performed at ambient
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conditions.
2.6. Morphological characterization of the gliadin structures
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The morphologies of electrosprayed/electrospun gliadin structures were observed with a TESCAN-Vega 3 scanning electron microscope (SEM) (TESCAN, Czech Republic). SEM
under
vacuum (Q
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was conducted at an accelerating voltage of 20 kV after sputter coating the samples with gold 150R-ES;
Quorum Technologies,
UK). Image
analysis
software
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(Digimizer, MedCalc Software, Belgium) was used to determine fiber/particle diameters from the SEM micrographs in their original magnification. Size distributions were obtained from a
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minimum of 100 measurements.
2.7. Fourier Transform Infrared (FT-IR) analysis of the gliadin structures Fourier transform infrared spectroscopy (FTIR) analyses of electrosprayed/electrospun structures were conducted using an FTIR spectrometer (model FTIR-8400S; Shimadzu Corp., Japan)
to
investigate
the
molecular
organization
of
the
samples.
The
electrosprayed/electrospun structures (ca. 2 mg) were ground and dispersed in spectroscopic grade potassium bromide (KBr; ca. 130 mg). A pellet was then formed by compressing the
ACCEPTED MANUSCRIPT samples at 150 MPa and the samples were analyzed in transmission mode. Samples were scanned from 400 to 4000 cm-1 at 2 cm-1 resolution.
2.8. X-ray Diffraction (XRD) X-ray Diffraction (XRD) analyses were performed using a X-ray Diffractometer (model D8-
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ADVANCE, Bruker, Germany) with Cu Kα radiation in the 2θ range of 5-80° at 40 mV and
2.9. Differential Scanning Calorimetry (DSC)
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40 mA.
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Differential scanning calorimetry (DSC) was conducted on a Jade DSC (PerkinElmer, USA). The thermal analyses were conducted within a temperature range from 35°C to 250°C at a
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2.10 Statistical analysis
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heating rate of 10°C/min under nitrogen gas flow at a flow rate of 50 ml/min.
The obtained data was expressed as the mean ± standard deviation of triplicate
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determinations. Statistical significance among treatments were evaluated with analysis of variance (one-way ANOVA with Tukey’s post hoc test), using SPSS 24 (SPSS Inc., Chicago,
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IL, USA) statistical software. Tukey’s multiple range tests were applied to determine the significance of differences between mean values (p<0.05).
3. Results and Discussion The development of delivery structures derived from natural sources is of great interest in the area of functional foods for bioactive protection and controlled release (Chen et al., 2006; Gómez-Mascaraque, Lagarón, & López-Rubio, 2015; Li, Lim, & Kakuda, 2009; Noruzi, 2016). In this work, the aim was to understand how different solution and processing
ACCEPTED MANUSCRIPT parameters affected the electrohydrodynamic processing of gliadin extracted from wheat gluten using a food-grade solvent (acetic acid). This particular protein fraction has special interest for food applications, given its hydrophobic character, which makes it suitable for hydrophobic bioactive encapsulation (Davidov-Pardo & McClements, 2015). The different
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conditions tested are compiled in Table 1.
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3.1. Solution properties and morphology of electrohydrodynamic processed gliadin
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structures
To better understand how solution properties affected the morphology of gliadin structures
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obtained by electrohydrodynamic processing, seven different concentrations of gliadin in acetic acid were investigated. Table 1 compiles the solution properties and it can be clearly
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observed that even at the lowest protein concentration, the surface tension was low enough to allow the electrohydrodynamic processing, thus, not being a restrictive parameter in this case.
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The surface tension of water is around 70 mN/m and needs to be decreased to facilitate the elongation of the jet to allow for a stable electrohydrodynamic process. In general,
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incorporation of proteins at low concentrations have been observed to decrease surface tension below 45 mN/m, which is sufficiently low for processing the solutions in a stable
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mode through electrospinning/electrospraying (Gómez-Mascaraque et al., 2015). In contrast, both apparent viscosity and electrical conductivity were seen to greatly vary depending on gliadin concentration. As expected, when the concentration of the gliadin in pure acetic acid was increased, apparent viscosity also increased (Table 1). Regarding the electrical conductivity, increasing the gliadin concentration up to 25 wt%, results in increased conductivity. However, a decrease in conductivity was observed when further increasing gliadin concentration. This decrease in the conductivity of protein solutions at high protein
ACCEPTED MANUSCRIPT concentrations has been previously observed for zein solutions (Kayaci & Uyar, 2012) and could be ascribed to the very high viscosity of these solutions which limits electron mobility. Figure 1 shows the SEM images of the various gliadin structures obtained applying a voltage of 15 kV. Gliadin concentration of 5 wt.% was insufficient to generate any type of structure through electrohydrodynamic processing. This can be explained by the lack of biopolymer
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entanglements in this specific solution, also reflected in its low apparent viscosity (Table 1).
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In contrast, gliadin particles were obtained when doubling the concentration to 10 wt.%. At
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such low biopolymer concentration, due to Rayleigh instability, the governing process was electrospraying (Ghorani & Tucker, 2015). Increasing the protein concentration, the process
changed
from
electrospraying
to
electrospinning.
This
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electrohydrodynamic
phenomenon occurred at a concentration of 15 wt.%, where nanofibers with beaded structures
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were formed. This is in accordance with the results of Y. Wang and Chen (2012a) who reported beaded electrospun structures at this specific gliadin concentration. Apparently, at
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this specific gliadin concentration, because of the increased molecular entanglements, the fluid jet breaking up into particles was prevented (Gómez-Mascaraque, Sanchez, & López-
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Rubio, 2016). In addition, it has been reported that higher polymer concentrations lead to increased interactions between polymer and solvent, which favors the elongation of the fluid
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facilitating chain entanglements and, thus, giving raise to electrospun fibers (Li et al., 2009). However, as reported by Woerdeman et al. (2005), fiber formation by electrospinning is a result of not only chain entanglements, but also to the presence of reversible junctions in the protein (specifically the breaking and re-forming of disulfide bonds that occur via thiol/disulfide interchange reaction). Therefore, in contrast with gluten spinnability, where the presence of the highest molecular weight glutenin chains were responsible for the lower threshold concentration for fiber formation (only 5% w/v in 1,1,1,3,3,3-hexafluoro-2propanol- HFIP) (Woerdeman et al., 2005), in the case of the lower molecular weight gliadin
ACCEPTED MANUSCRIPT fraction used in this study, 30 wt.% gliadin was needed to generate beaded-free fiber structures. In general, proteins must be more or less unfolded in order to be electrospinnable and, in this case, acetic acid was a proper solvent to fulfill this requirement (Nieuwland et al., 2013). The voltage applied during electrohydrodynamic processing is another parameter that has
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been seen to affect the morphology, shape and size of final structures obtained. The same
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gliadin solutions were processed increasing the voltage to 18 kV (cf. Figure 2). This higher
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voltage had distinct influence on the morphology and diameter of electrospun/electrosprayed gliadin structures. In general, and in agreement with previous knowledge about the effect of
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voltage on particle size, the average diameter of particles and fibers was decreased by increasing the voltage. The diameter of particles at electrical potential of 15 kV and 18 kV
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were 817 nm and 467 nm, respectively (flow rate 1 ml/h). In other words, at higher voltage, particles of smaller diameters were formed. According to Ghorani, Alehosseini, and
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Tucker (2017), the application of high voltages results in a higher whipping of the solution
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and a faster evaporation of the solvent during the flight towards the collector and, thus, to formation of the smaller average sizes of particles and/or fibers. However, the behavior of gliadin solutions with the greatest protein concentration (i.e. 35 wt.%) was different. Thicker
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tubular and branched microfibers (with average diameters 5440 nm) were formed in this case, which has been observed before in previous works and has been ascribed to increased ejection of solutions from the nozzle (only observed for high concentrations and high voltages), thus resulting in thicker electrospun fibers (Bhardwaj & Kundu, 2010; Heikkilä & Harlin, 2008). It is also worthwhile pointing out that, at a gliadin concentration of 35 wt.%, the electrical conductivity of the solution was lower, which as explained before may be due to limited electron mobility as a consequence of the high solution viscosity (cf. Table 1).
ACCEPTED MANUSCRIPT According to Tan, Inai, Kotaki, and Ramakrishna (2005) a less electrically conductive spinning solution may stretch less, resulting in fiber products with larger diameters. Finally, fluid flow rate was the other processing parameter we varied, because it determines the amount of fluid transfer rate and jet velocity, thus affecting the spinnability of the samples. Generally, results on increasing the flow rate at different concentration revealed that
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it mainly affected bead formation. In other words, lower flow rates resulted in the less beaded
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electrospun structures. According Zong et al. (2002), due to lack of sufficient time for
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evaporation of solvent prior reaching to collector, higher fluid rates results in beaded fibers. In addition, at high voltage and high gliadin concentration, increasing the fluid flow rate
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resulted in branched microfibers. It has been reported that at a higher critical value, fluid flows can result in the formation of junction zones between the deposited polymer structures
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(branched electrospun structures). This is due to the insufficient time available to allow for complete solvent evaporation, causing the fibers to fuse together (Kriegel, Arrechi, Kit,
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McClements, & Weiss, 2008). In this study, decreasing the flow rate from 1 to 0.5 mL/h had no major impacts on the morphology of the structures obtained and, thus, the best conditions
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for particle and fiber formation were set at gliadin concentrations of 10 and 25 wt.% respectively, with an applied voltage of 18 kV and a fluid flow rate of 1 mL/h. The diameter
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of particles and fibers were 468 nm and 256 nm, respectively. These formulations and processing conditions were selected for further characterization.
3.2. FTIR spectroscopy To investigate the changes in molecular organisation of the gliadin structures after electrohydrodynamic processing, electrospun and electrosprayed samples were analyzed using FTIR spectroscopy. The Amide I band of proteins and polypeptides in the region ranging from 1600 to 1700 cm-1 has long been used in the study of the secondary structures
ACCEPTED MANUSCRIPT of proteins (Gu & Wang, 2013). This arises from the amide bonds that link the amino acids. The absorption associated with amide I band is related to stretching vibration of C=O bond of the amide in which the vibrational frequency reflects the hydrogen bond strength between C=O bond and N-H bond. In other words, this vibrational frequency reflects secondary structures such as α-helix of proteins and polypeptides (Secundo & Guerrieri, 2005).
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As observed in other proteins, electrohydrodynamic processing seems to enhance molecular
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order of the amino acids chains, reflected in more defined and narrower spectral bands
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(Gómez-Mascaraque et al., 2015). In fact, band width is a measure of conformational freedom and, thus, the narrowing of spectral bands confirms the formation of more rigid
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(structured) particles/fibers. Apart from these obvious changes, when comparing the spectrum of unprocessed gliadin with those from the electrosprayed/electrospun structures, shifts in the
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amide I band were observed (cf. Figure 3). Interestingly, while a shift towards lower wave numbers was observed for the electrosprayed particles obtained from the 10 wt.% gliadin
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solution (from 1660 cm-1 to 1654 cm-1 for the unprocessed and electrosprayed gliadin, respectively), this amide I band was displaced towards higher wave numbers (1666 cm-1 ) in
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the electrospun structures from the 25 wt.% gliadin solution. It should be stressed that this shifts reflect small conformational changes in the protein, as similarly reported for other
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proteins in acid conditions (Aceituno-Medina, Lopez-Rubio, Mendoza, & Lagaron, 2013). However, the interesting observation is that for several proteins studied (Hirota, Goto, & Mizuno, 1997; Stephens et al., 2005), when fibers are formed through electrospinning, a shift towards higher wavenumbers is observed, which is generally correlated with an increase in the α-helical and random coil configuration (Yang et al., 2009). In contrast, less extended conformations, which are correlated with lower wavenumber positions of the amide I band normally correlated with -sheet configurations, are generally observed when particles are
ACCEPTED MANUSCRIPT obtained through electrospraying (Aceituno-Medina et al., 2013; Gaona-Sánchez et al., 2015).
3.3. X-ray Diffraction The XRD diffraction patterns of pure gliadin and electrospun structures are presented in
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Figure 4. The diffraction pattern of the extracted gliadin showed two broad peaks with
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characteristic distances of 2θ~11° and ~18 (corresponding to 1.61 nm and 0.98 nm,
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respectively). Similar results were also reported in some other studies (Gulfam et al., 2012; Xu et al., 2017). After electrohydrodynamic processing, the diffraction patterns of the
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structures obtained showed a broad diffraction band around 2θ~14°, which is characteristic of amorphous materials. This result indicates that the original amino acid chain packing of the
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extracted protein was lost during the processing giving raise to amorphous materials. This result contrasts with the one from Gulfam et al. (2012), where they did not observe any
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change in the XRD patterns, probably due to the conditions used to process the materials which did not alter the physical structure of the proteins (they used ethanol instead of acetic
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acid to process the gliadin material).
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3.4. Differential Scanning Calorimetry The DSC analysis was conducted to determine the thermal properties of the obtained gliadin structures after electrohydrodynamic process. The DSC thermograms of pure gliadin as well as the electrospun structures are presented in Figure 5. The DSC thermogram of pure gliadin exhibited a single endothermic at 87 °C which as often termed as dehydration temperature (TD), which reflects the loss of bound water from the materials (H. Wang et al., 2016). In agreement with observations for other prolamins, no endothermic peak was observed for pure gliadin (Huang et al., 2013; Yilmaz et al., 2016). The values of TD for particles and fibers
ACCEPTED MANUSCRIPT were 81 and 80 °C, respectively, i.e. the high surface area of the generated materials facilitated water evaporation. Moreover, from the normalized heat flow curves, the electrospun/electrosprayed samples contained less sorbed water (as the area under the curve was significantly lower). This may indicate that less functional groups from gliadin were available for water sorption. In addition, a glass transition temperature (Tg) value of 179 °C
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was observed in the thermogram of pure gliadin, slightly higher than that previously reported
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(Duclairoir et al., 2003). A shift towards higher temperatures in the Tg was observed for the
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electrosprayed (Tg of 204ºC) and electrospun structures (Tg of 202ºC) (cf. arrows and insets in Figure 5). This result is in agreement with previous results obtained for zein, in
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which a significant Tg increase from 165ºC to 222ºC was observed after electrospraying this protein (Gaona-Sánchez et al., 2015). This Tg increase could be correlated with the increased
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molecular order observed through FTIR (see section 3.2), in which the more rigid structures
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would require higher temperatures for this second order transition.
In this study,
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4. Conclusions
the effects of solution properties and process parameters on the
electrohydrodynamic
processing
of
gliadin
solutions
have
been
studied.
Gliadin
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concentration, which mainly affected apparent viscosity and conductivity was the main factor that determined the morphology of the structures obtained through electrohydrodynamic processing. Increasing the applied voltage generally led to reduced size of the structures, while fluid flow rate did not significantly affect the process in the conditions studied. 10 wt.% gliadin
solutions
resulted
in particle
formation
through
electrospraying,
while
increasing the solution concentration above 25wt.% led to the formation of almost beadedfree fibers. Interestingly, amide I band position in the FTIR spectra is indicative of conformational changes related to the formation of either particles or fibers. The use of acetic
ACCEPTED MANUSCRIPT acid for processing the protein, led to the disruption of the original amino acid chain packing, as observed from the XRD diffractograms. However, the increased molecular order observed from the FTIR spectra affected the thermal properties of the obtained structures, increasing their glass transition temperature in comparison with the unprocessed extracted gliadin. The overall results presented in this work demonstrate the suitability of acetic acid for the
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electrohydrodynamic processing of gliadin fractions, showing the versatility of the technique
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which allows the development of different morphologies (fibers or particles), which can be
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further used as bioactive delivery vehicles.
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Acknowledgements
The authors gratefully acknowledge the financial support of the Research Affairs Office at
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Shiraz University (Grant #93GCU1M1981). The authors acknowledge financial support from the Spanish Ministry of Economy, Industry and Competitiveness MINECO (AGL2015-
None.
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References
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Declaration of interest
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63855-C2-1-R project) and the financial support of EU FEDER funds.
Aceituno-Medina, M., Lopez-Rubio, A., Mendoza, S., & Lagaron, J. M. (2013). Development of novel ultrathin structures based in amaranth (Amaranthus hypochondriacus) protein isolate through electrospinning. Food Hydrocolloids, 31(2), 289-298. doi:https://doi.org/10.1016/j.foodhyd.2012.11.009 Ago, M., Okajima, K., Jakes, J. E., Park, S., & Rojas, O. J. (2012). Lignin -based electrospun nanofibers reinforced with cellulose nanocrystals. Biomacromolecules, 13 (3), 918-926. doi:10.1021/bm201828g Bhardwaj, N., & Kundu, S. C. (2010). Electrospinning: a fascinating fiber fabrication technique. Biotechnology advances, 28 (3), 325-347. Bhushani, J. A., & Anandharamakrishnan, C. (2014). Electrospinning and electrospraying techniques: Potential food based applications. Trends in food science & technology, 38 (1), 21-33.
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Chen, L., Remondetto, G. E., & Subirade, M. (2006). Food protein-based materials as nutraceutical delivery systems. Trends in food science & technology, 17 (5), 272-283. Davidov-Pardo, G., & McClements, D. J. (2015). Nutraceutical delivery systems: Resveratrol encapsulation in grape seed oil nanoemulsions formed by spontaneous emulsification. Food chemistry, 167 , 205-212. doi:https://doi.org/10.1016/j.foodchem.2014.06.082 Duclairoir, C., Orecchioni, A.-M., Depraetere, P., Osterstock, F., & Nakache, E. (2003). Evaluation of gliadins nanoparticles as drug delivery systems: a study of three different drugs. International journal of pharmaceutics, 253 (1-2), 133-144. Feng, J., Wu, S., Wang, H., & Liu, S. (2017). Gliadin nanoparticles stabilized by a combination of thermally denatured ovalbumin with gemini dodecyl O glucoside: The modulating effect of cosurfactant. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 516 , 94-105. Gaona-Sánchez, V., Calderon-Dominguez, G., Morales-Sanchez, E., Chanona-Perez, J. J., Velazquez-De La Cruz, G., Mendez-Mendez, J. V., . . . Farrera-Rebollo, R. R. (2015). Preparation and characterisation of zein films obtained by electrospraying. Food Hydrocolloids, 49 , 1-10. Ghorani, B., Alehosseini, A., & Tucker, N. (2017). 8 - Nanocapsule formation by electrospinning A2 - Jafari, Seid Mahdi Nanoencapsulation Technologies for the Food and Nutraceutical Industries (pp. 264-319): Academic Press. Ghorani, B., & Tucker, N. (2015). Fundamentals of electrospinning as a novel delivery vehicle for bioactive compounds in food nanotechnology. Food Hydrocolloids, 51 , 227-240. Gómez-Mascaraque, L. G., Lagarón, J. M., & López-Rubio, A. (2015). Electrosprayed gelatin submicroparticles as edible carriers for the encapsulation of polyphenols of interest in functional foods. Food Hydrocolloids, 49 , 42-52. Gómez-Mascaraque, L. G., Sanchez, G., & López-Rubio, A. (2016). Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds. Carbohydrate polymers, 150 , 121-130. Gu, L., & Wang, M. (2013). Effects of protein interactions on properties and microstructure of zein–gliadin composite films. Journal of Food Engineering, 119(2), 288-298. Gulfam, M., Kim, J.-e., Lee, J. M., Ku, B., Chung, B. H., & Chung, B. G. (2012). Anticancer drug-loaded gliadin nanoparticles induce apoptosis in breast cancer cells. Langmuir, 28(21), 8216-8223. Heikkilä, P., & Harlin, A. (2008). Parameter study of electrospinning of polyamide-6. European Polymer Journal, 44 (10), 3067-3079. Hirota, N., Goto, Y., & Mizuno, K. (1997). Cooperative α‐ helix formation of β‐ lactoglobulin and melittin induced by hexafluoroisopropanol. Protein science, 6(2), 416-421. Hong, N. V., Trujillo, E., Puttemans, F., Jansens, K. J., Goderis, B., Van Puyvelde, P., . . . Van Vuure, A . W. (2016). Developing rigid gliadin based biocomposites with high mechanical performance. Composites Part A: Applied Science and Manufacturing, 85 , 76-83.
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Hu, X., Liu, S., Zhou, G., Huang, Y., Xie, Z., & Jing, X. (2014). Electrospinning of polymeric nanofibers for drug delivery applications. Journal of controlled release, 185 , 12-21. Huang, W., Zou, T., Li, S., Jing, J., Xia, X., & Liu, X. (2013). Drug-loaded zein nanofibers prepared using a modified coaxial electrospinning process. Aaps Pharmscitech, 14(2), 675-681. Joye, I. J., Davidov-Pardo, G., & McClements, D. J. (2014). Nanotechnology for increased micronutrient bioavailability. Trends in food science & technology, 40(2), 168-182. Karthikeyan, K., Guhathakarta, S., Rajaram, R., & Korrapati, P. S. (2012). Electrospun zein/eudragit nanofibers based dual drug delivery system for the simultaneous delivery of aceclofenac and pantoprazole. International journal of pharmaceutics, 438 (1), 117-122. Kayaci, F., & Uyar, T. (2012). Electrospun zein nanofibers incorporating cyclodextrins. Carbohydrate polymers, 90 (1), 558-568. Kriegel, C., Arrechi, A., Kit, K., McClements, D., & Weiss, J. (2008). Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Critical reviews in food science and nutrition, 48 (8), 775-797. Li, Y., Lim, L. T., & Kakuda, Y. (2009). Electrospun zein fibers as carriers to stabilize (−)‐ epigallocatechin gallate. Journal of food science, 74 (3), 233-240. Liu, H., Ding, X., Zhou, G., Li, P., Wei, X., & Fan, Y. (2013). Electrospinning of nanofibers for tissue engineering applications. Journal of Nanomaterials, 2013, 1-11. Liu, M., Duan, X.-P., Li, Y.-M., Yang, D.-P., & Long, Y.-Z. (2017). Electrospun nanofibers for wound healing. Materials Science and Engineering: C, 76 , 1413-1423. Mendes, A. C., Stephansen, K., & Chronakis, I. S. (2017). Electrospinning of food proteins and polysaccharides. Food Hydrocolloids, 68 , 53-68. Neo, Y. P., Ray, S., Easteal, A. J., Nikolaidis, M. G., & Quek, S. Y. (2012). Influence of solution and processing parameters towards the fabrication of electrospun zein fibers with sub-micron diameter. Journal of Food Engineering, 109 (4), 645-651. Nieuwland, M., Geerdink, P., Brier, P., Van Den Eijnden, P., Henket, J. T., Langelaan, M. L., . . . Martin, A. H. (2013). Food-grade electrospinning of proteins. Innovative Food Science & Emerging Technologies, 20 , 269-275. Noruzi, M. (2016). Electrospun nanofibres in agriculture and the food industry: A review. Journal of the Science of Food and Agriculture, 96 (14), 4663-4678. Pérez-Masiá, R., López-Rubio, A., & Lagarón, J. M. (2013). Development of zeinbased heat-management structures for smart food packaging. Food Hydrocolloids, 30 (1), 182-191. Secundo, F., & Guerrieri, N. (2005). ATR-FT/IR study on the interactions between gliadins and dextrin and their effects on protein secondary structure. Journal of agricultural and food chemistry, 53 (5), 1757-1764. Soares, R. M., Patzer, V. L., Dersch, R., Wendorff, J., da Silveira, N. P., & Pran ke, P. (2011). A novel globular protein electrospun fiber mat with the addition of polysilsesquioxane. International journal of biological macromolecules, 49 (4), 480-486.
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Stephens, J. S., Fahnestock, S. R., Farmer, R. S., Kiick, K. L., Chase, D. B., & Rab olt, J. F. (2005). Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy. Biomacromolecules, 6(3), 1405-1413. Tan, S., Inai, R., Kotaki, M., & Ramakrishna, S. (2005). Systematic parameter study for ultra-fine fiber fabrication via electrospinning process. Polymer, 46 (16), 6128-6134. Tarhini, M., Greige-Gerges, H., & Elaissari, A. (2017). Protein-based nanoparticles: From preparation to encapsulation of active molecules. International journal of pharmaceutics, 522 (1-2), 172-197. Veloso, A. C. A., Dias, L. G., Rodrigues, L. R., & Peres, A. M. (2016). Chapter 18 Gliadins in Foods and the Electronic Tongue A2 - Méndez, María Luz Rodríguez Electronic Noses and Tongues in Food Science (pp. 179-188). San Diego: Academic Press. Wang, H., Hao, L., Niu, B., Jiang, S., Cheng, J., & Jiang, S. (2016). Kinetics and Antioxidant Capacity of Proanthocyanidins Encapsulated in Zein Electrospun Fibers by Cyclic Voltammetry. Journal of agricultural and food chemistry, 64(15), 3083-3090. Wang, X., Yu, D.-G., Li, X.-Y., Bligh, S. A., & Williams, G. R. (2015). Electrospun medicated shellac nanofibers for colon-targeted drug delivery. International journal of pharmaceutics, 490 (1-2), 384-390. Wang, Y., & Chen, L. (2012a). Electrospinning of prolamin proteins in acetic acid: the effects of protein conformation and aggregation in solution. Macromolecular Materials and Engineering, 297 (9), 902-913. Wang, Y., & Chen, L. (2012b). Fabrication and characterization of novel assembled prolamin protein nanofabrics with improved stability, mechanical property and release profiles. Journal of Materials Chemistry, 22 (40), 21592-21601. Woerdeman, D. L., Ye, P., Shenoy, S., Parnas, R. S., Wnek, G. E., & Trofimova, O. (2005). Electrospun fibers from wheat protein: investigation of the interplay between molecular structure and the fluid dynamics of the electrospinning process. Biomacromolecules, 6 (2), 707-712. Xu, Y., Li, J.-J., Yu, D.-G., Williams, G. R., Yang, J.-H., & Wang, X. (2017). Influence of the drug distribution in electrospun gliadin fibers on drug-release behavior. European Journal of Pharmaceutical Sciences, 106 , 422-430. Yang, X., Wu, D., Du, Z., Li, R., Chen, X., & Li, X. (2009). Spectroscopy study on the interaction of quercetin with collagen. Journal of agricultural and food chemistry, 57(9), 3431-3435. Yilmaz, A., Bozkurt, F., Cicek, P. K., Dertli, E., Durak, M. Z., & Yilmaz, M. T. (2016). A novel antifungal surface-coating application to limit postharvest decay on coated apples: Molecular, thermal and morphological properties of electrospun zein–nanofiber mats loaded with curcumin. Innovative Food Science & Emerging Technologies, 37 , 74-83. Zong, X., Kim, K., Fang, D., Ran, S., Hsiao, B. S., & Chu, B. (2002). Structure and process relationship of electrospun bioabsorbable nanofiber membranes. Polymer, 43(16), 4403-4412.
ACCEPTED MANUSCRIPT Table
1.
Solution
concentrations
used,
properties
of the
different
solutions
and
electrospinning parameters investigated Gliadin Concentration (wt. %)
Viscosity (mPa.s 30 rpm, 25 ° C)
Surface tension (mN.m-1 )
Electrical Conductivity (μs.cm-1 )
5
61.73±1.06g
28.03±0.11d
19.68±0.16f
Applied Voltage (kV)
Feed rate (ml.h1 )
Distance (mm)
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15 and 0.5 100 18 and 1 10 100.57±0.30f 28.13±0.06cd 49.25±0.08e 15 and 0.5 100 18 and 1 15 179.04±0.87e 28.01±0.35cd 102.55±2.19d 15 and 0.5 100 18 and 1 20 321.24±1.95d 28.40±0.17cd 129.70±0.85b 15 and 0.5 100 18 and 1 25 522.54±0.56c 28.80±0.37bc 147.75±1.48a 15 and 0.5 100 18 and 1 30 628.27±1.32b 29.17±0.24ab 129.25±0.78b 15 and 0.5 100 18 and 1 35 788.01±2.16a 29.73±0.46a 119.10±0.14c 15 and 0.5 100 18 and 1 Data are displayed in means ± standard deviation of three replications (P < 0.05); means in each column bearing different superscripts are significantly different (P < 0.05).
ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. SEM images of electrosprayed /electrospun structures obtained at the conditions of 15 kV, 1 mL/h from gliadin solutions of (a) 10 wt%, (b) 15 wt%, (c) 20 wt%,(d) 25 wt%, (e) 30 wt% and (f) 35 wt%. Figure 2. SEM images of electrosprayed /electrospun structures obtained at the conditions of
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18 kV, 1 mL/h from gliadin solutions of (a) 10 wt%, (b) 15 wt%, (c) 20 wt%, (d) 25 wt%, (e)
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30 wt% and (f) 35 wt%.
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Figure 3. FTIR spectra of unprocessed gliadin (solid line), electrosprayed gliadin (dashed line) and electrospun gliadin (dotted line). Inset shows the magnification of the Amide I band
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with arrows pointing to the respective band shifts.
Figure 4. XRD diffraction patterns of: (A) unprocessed gliadin; (B) electrosprayed gliadin;
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and (C) electrospun gliadin.
Figure 5. DSC thermograms of pure gliadin (solid line), electrosprayed gliadin (dashed line)
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and electrospun gliadin (dotted line). Thermograms have been offset for clarity. Arrows point out to the Tg of the materials and insets show a magnification of the curves where the change
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in specific heat is observed.
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Gliadin microparticles/microfibers were obtained by electrohydrodynamic processing Acetic acid was used as a food-grade solvent to develop the protein microstructures The effects of processing parameters on morphology and properties were studied
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obtained
Niloufar Sharif, Mohammad-Taghi Golmakani, Niakousari, Behrouz Ghorani, Amparo Lopez-Rubio
by
Mehrdad
PII: DOI: Reference:
S0963-9969(18)30814-7 doi:10.1016/j.foodres.2018.10.027 FRIN 7997
To appear in:
Food Research International
Received date: Revised date: Accepted date:
6 May 2018 27 September 2018 7 October 2018
Please cite this article as: Niloufar Sharif, Mohammad-Taghi Golmakani, Mehrdad Niakousari, Behrouz Ghorani, Amparo Lopez-Rubio , Food-grade gliadin microstructures obtained by electrohydrodynamic processing. Frin (2018), doi:10.1016/ j.foodres.2018.10.027
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Food-Grade
Gliadin
Microstructures
Obtained
by
Electrohydrodynamic Processing
Niloufar Sharif1 , Mohammad-Taghi Golmakani1* , Mehrdad Niakousari1 , Behrouz Ghorani2 ,
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Department of Food Science and Technology, School of Agriculture, Shiraz University, km
12
Shiraz-Esfahan
Highway,
P.
O.
Box:
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1
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Amparo Lopez-Rubio3*
71441-65186,
Shiraz,
Iran.
Emails:
2
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[email protected]; [email protected]; [email protected] Department of Food Nanotechnology, Research Institute of Food Science and Technology
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(RIFST), km 12 Mashhad-Quchan Highway, P. O. Box: 91895/157/356, Mashhad, Iran. Email: [email protected]
Food Quality and Preservation Department, IATA-CSIC, 46980 Paterna, Valencia, Spain.
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3
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Email: [email protected]
*Corresponding authors:
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- Amparo Lopez Rubio: Phone: (+34) 963900022; fax: (+34) 963636301. Email: [email protected] - Mohammad-Taghi Golmakani: Phone: (+98)71-36138243. Fax: (+98)71-32286110. Email: [email protected]
ACCEPTED MANUSCRIPT Abstract This paper presents a comprehensive study on the electrohydrodynamic processing of gliadin to develop food-grade delivery systems with different morphologies. The effects of biopolymer concentration, applied voltage and solution flow-rate on particle morphology, molecular organisation,
crystallinity and
thermal properties were investigated.
Gliadin
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concentration influenced the apparent viscosity and conductivity of the solutions, giving raise
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to particle morphologies at 10 wt.% gliadin and beaded-free fibers above 25 wt.% gliadin. In
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general, increasing the voltage resulted in smaller average sizes of the obtained structures, while no significant differences in morphology were observed amongst the tested flow rates.
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Interestingly, the amide I position in the FTIR reflected changes in protein conformation which could be correlated with the final morphology attained. Moreover, the acetic acid used
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for solution preparation disrupted the original amino acid chain packing of the gliadin fraction, being the electrospun/electrosprayed samples amorphous. These gliadin-based
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microparticles and microfibers obtained could serve as food-grade delivery vehicles.
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Keywords: Electrospinning; Electrospraying; Gluten; Gliadin, XRD; Plant Proteins
ACCEPTED MANUSCRIPT 1. Introduction The development of delivery systems for bioactive components is a topic of increased interest in the food area, as it provides a number of advantages, such as increasing the solubility of certain bioactive compounds, protecting labile ingredients and/or favoring a gradual bioactive release (Chen, Remondetto, & Subirade, 2006; Joye, Davidov-Pardo, & McClements, 2014). different
techniques
used
for the
development
of delivery
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Amongst the
systems,
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electrohydrodynamic processes, namely electrospinning and electrospraying, are rapidly
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emerging as promising technologies to obtain fibers and particles at the micron, submicron and nanoscales (Mendes, Stephansen, & Chronakis, 2017). The key advantages of the so-
of the
electrospun
fiber
mats,
their
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obtained fibers and particles are their large surface-to-volume ratio and, in the specific case small pore
size
and
high
porosity.
These
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electrohydrodynamic processed structures can be exploited for a wide variety of applications including tissue engineering wound healing (M. Liu, Duan, Li, Yang, & Long, 2017), drug 2014), encapsulation,
immobilization of enzymes, filtration and
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delivery (Hu et al.,
fabrication of food packaging materials, among others (Bhushani & Anandharamakrishnan,
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2014; Ghorani & Tucker, 2015). As widely reported, in an electrohydrodynamic process, a polymer or biopolymer solution is drawn from a nozzle towards a grounded collector by the
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application of an external electric field (Karthikeyan, Guhathakarta, Rajaram, & Korrapati, 2012; Neo, Ray, Easteal, Nikolaidis, & Quek, 2012). During the flight between the nozzle and the collector, the jet is elongated and the solvent evaporates, producing dry continuous fibers in the case of electrospinning, while in the case of electrospraying, the jet breaks down into fine droplets, subsequently producing solid nano- or microparticles upon solvent evaporation (Bhushani & Anandharamakrishnan, 2014). Many parameters can influence fiber/particle formation and morphology during an electrohydrodynamic process including solution properties (mainly viscosity, electrical conductivity and surface tension), process
ACCEPTED MANUSCRIPT conditions (applied voltage, fluid flow rate, distance from nozzle to collector) and ambient conditions (like temperature and humidity) (Ghorani & Tucker, 2015; Mendes et al., 2017). Specifically, solution properties can be tailored to produce different morphologies using electrohydrodynamic processing, as they depend on the type of polymer/biopolymer used, its concentration, the solvent employed to dissolve/disperse the polymer and the polymer-
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solvent interactions (Pérez-Masiá, López-Rubio, & Lagarón, 2013).
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For food-related applications, the use of GRAS materials is a requirement and, thus, the use
biocompatible,
have
attracted
increasing
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of biopolymers such as proteins, which are generally renewable, biodegradable and attention
as
the
raw
materials
for
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electrohydrodynamic processing (Ago, Okajima, Jakes, Park, & Rojas, 2012). Plant storage proteins, including co-products from the industrial processing of cereal crops, would be
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interesting raw materials for fabricating cost-effective electrospun/electrosprayed structures (Y. Wang & Chen, 2012b), as they are considered more “environmentally economical” when
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compared with animal-derived proteins (X. Wang, Yu, Li, Bligh, & Williams, 2015). Wheat
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gluten, a by-product of wheat starch industry has two main fractions; monomeric gliadins and polymeric glutenins (Feng, Wu, Wang, & Liu, 2017). Gliadins are important storage prolamins in wheat kernel, which are complex glycoproteins rich in proline and glutamine
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(Veloso, Dias, Rodrigues, & Peres, 2016). These wheat-derived proteins are of particular interest for the development of food-grade delivery systems because they are highly hydrophobic, unlike many other food proteins, thus enabling to encapsulate hydrophobic ingredients without the need for oil phases (Duclairoir, Orecchioni, Depraetere, Osterstock, & Nakache,
2003).
Moreover,
gliadin
nanoparticles
have
bioadhesive
ability
through
electrostatic interactions and hydrogen bonding to the intestinal membrane (Tarhini, GreigeGerges, & Elaissari, 2017), fact of outmost importance for bioactive delivery applications.
ACCEPTED MANUSCRIPT Although gliadin nanoparticles have been developed for drug delivery applications mainly using emulsification and desolvation techniques (Gulfam et al., 2012; Soares et al., 2011; Xu et al., 2017), there are only few works in which gliadin-based structures have been prepared using electrohydrodynamic techniques (Gulfam et al., 2012; Xu et al., 2017) and none of them have explored how the processing parameters affect structure formation through this
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technique. Therefore, the objective of this study was to evaluate how solution properties
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(surface tension, viscosity and electrical conductivity) and process parameters (applied
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voltage and fluid flow rate) affected the morphology of the gliadin structures obtained through electrohydrodynamic processing. The molecular organisation (Fourier Transform analysis),
crystallinity (X-Ray diffraction) and
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Infrared
scanning calorimetry) of the generated micro-
and submicro-
(differential
structures were also
2. Materials and Methods
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2.1. Materials
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characterized.
thermal properties
Wheat gluten was obtained from a local shop (Shiraz, Iran). Acetic acid was purchased from Scharlau company (Barcelona, Spain). All other chemicals used were of analytical grade,
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unless otherwise specified.
2.2. Gliadin extraction
The gliadin fraction was extracted using the method reported by Hong et al. (2016) with slight modifications. Briefly, samples of dried gluten powder (20 g) were gently stirred in an ethanol/water mixture (70/30 v/v; gluten/solvent ratio of 1/12) for 4h at 20ºC. The suspension was centrifuged to collect the gliadin fraction at 10000 g for 10 min. Finally, the ethanol was evaporated at ambient conditions. The extraction yield of gliadin from wheat gluten powder
ACCEPTED MANUSCRIPT was 37.5%. The protein content as determined using the Kjeldahl method was 89.8% on a dry matter basis.
2.3. Solution preparation for electrohydrodynamic processing For the electrohydrodynamic process, gliadin solutions were prepared in pure acetic acid (Y.
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Wang & Chen, 2012b) dissolving the protein at various concentrations (5, 10, 15, 20, 25, 30
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and 35%, w/v). Greater concentrations gave rise to too viscous solutions that could not be
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processed. The various solutions were stirred at room temperature until the protein was
2.4. Characterization of gliadin solutions
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completely dissolved.
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The apparent viscosity, surface tension and electrical conductivity of the gliadin solutions at the different concentrations were evaluated. The surface tension of the solutions was
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measured using the Wilhemy plate method in an EasyDyne K20 tensiometer (Krüss GmbH, Hamburg, Germany) after calibration of the equipment with deionized water. The electrical
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conductivity of the solutions was measured using a conductivity meter XS Con6 (Labbox, Barcelona, Spain). The apparent viscosity of the gliadin solutions was determined by a
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rotation viscometer VISCO BASIC PLUS L from Fungilab S.A. (Sant Feliu de Llobregat, Spain) at 30 rpm using the TL5 spindle. All measurements were made at 25ºC. Experiments were performed in triplicate.
2.5. Electrohydrodynamic processing of the gliadin solutions The various gliadin solutions were processed using an electrohydrodynamic apparatus equipped with a variable high voltage 0-35 kV power supplier (Spinner-3X-Advance, ANSTCO, Iran). Gliadin solutions were loaded into 10 mL disposable plastic syringes and
ACCEPTED MANUSCRIPT they were electrospun/electrosprayed under a steady flow-rate using a stainless-steel needle (inner diameter of 0.9 mm) which was connected to the electrode of the high voltage power supply. Two different voltages (15 and 18 kV) and fluid flow rates (0.5 and 1 ml/h) were applied to see how these process parameters affected the morphology of the obtained gliadin structures. Tip to collector distance was kept constant at 100 mm, as preliminary tests
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demonstrated a proper solution drying at this specific distance. The obtained structures were
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collected on aluminum foil attached to the surface of the collector and kept overnight under
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the hood to evaporate any solvent residues. All experiments were performed at ambient
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conditions.
2.6. Morphological characterization of the gliadin structures
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The morphologies of electrosprayed/electrospun gliadin structures were observed with a TESCAN-Vega 3 scanning electron microscope (SEM) (TESCAN, Czech Republic). SEM
under
vacuum (Q
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was conducted at an accelerating voltage of 20 kV after sputter coating the samples with gold 150R-ES;
Quorum Technologies,
UK). Image
analysis
software
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(Digimizer, MedCalc Software, Belgium) was used to determine fiber/particle diameters from the SEM micrographs in their original magnification. Size distributions were obtained from a
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minimum of 100 measurements.
2.7. Fourier Transform Infrared (FT-IR) analysis of the gliadin structures Fourier transform infrared spectroscopy (FTIR) analyses of electrosprayed/electrospun structures were conducted using an FTIR spectrometer (model FTIR-8400S; Shimadzu Corp., Japan)
to
investigate
the
molecular
organization
of
the
samples.
The
electrosprayed/electrospun structures (ca. 2 mg) were ground and dispersed in spectroscopic grade potassium bromide (KBr; ca. 130 mg). A pellet was then formed by compressing the
ACCEPTED MANUSCRIPT samples at 150 MPa and the samples were analyzed in transmission mode. Samples were scanned from 400 to 4000 cm-1 at 2 cm-1 resolution.
2.8. X-ray Diffraction (XRD) X-ray Diffraction (XRD) analyses were performed using a X-ray Diffractometer (model D8-
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ADVANCE, Bruker, Germany) with Cu Kα radiation in the 2θ range of 5-80° at 40 mV and
2.9. Differential Scanning Calorimetry (DSC)
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40 mA.
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Differential scanning calorimetry (DSC) was conducted on a Jade DSC (PerkinElmer, USA). The thermal analyses were conducted within a temperature range from 35°C to 250°C at a
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2.10 Statistical analysis
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heating rate of 10°C/min under nitrogen gas flow at a flow rate of 50 ml/min.
The obtained data was expressed as the mean ± standard deviation of triplicate
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determinations. Statistical significance among treatments were evaluated with analysis of variance (one-way ANOVA with Tukey’s post hoc test), using SPSS 24 (SPSS Inc., Chicago,
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IL, USA) statistical software. Tukey’s multiple range tests were applied to determine the significance of differences between mean values (p<0.05).
3. Results and Discussion The development of delivery structures derived from natural sources is of great interest in the area of functional foods for bioactive protection and controlled release (Chen et al., 2006; Gómez-Mascaraque, Lagarón, & López-Rubio, 2015; Li, Lim, & Kakuda, 2009; Noruzi, 2016). In this work, the aim was to understand how different solution and processing
ACCEPTED MANUSCRIPT parameters affected the electrohydrodynamic processing of gliadin extracted from wheat gluten using a food-grade solvent (acetic acid). This particular protein fraction has special interest for food applications, given its hydrophobic character, which makes it suitable for hydrophobic bioactive encapsulation (Davidov-Pardo & McClements, 2015). The different
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conditions tested are compiled in Table 1.
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3.1. Solution properties and morphology of electrohydrodynamic processed gliadin
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structures
To better understand how solution properties affected the morphology of gliadin structures
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obtained by electrohydrodynamic processing, seven different concentrations of gliadin in acetic acid were investigated. Table 1 compiles the solution properties and it can be clearly
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observed that even at the lowest protein concentration, the surface tension was low enough to allow the electrohydrodynamic processing, thus, not being a restrictive parameter in this case.
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The surface tension of water is around 70 mN/m and needs to be decreased to facilitate the elongation of the jet to allow for a stable electrohydrodynamic process. In general,
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incorporation of proteins at low concentrations have been observed to decrease surface tension below 45 mN/m, which is sufficiently low for processing the solutions in a stable
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mode through electrospinning/electrospraying (Gómez-Mascaraque et al., 2015). In contrast, both apparent viscosity and electrical conductivity were seen to greatly vary depending on gliadin concentration. As expected, when the concentration of the gliadin in pure acetic acid was increased, apparent viscosity also increased (Table 1). Regarding the electrical conductivity, increasing the gliadin concentration up to 25 wt%, results in increased conductivity. However, a decrease in conductivity was observed when further increasing gliadin concentration. This decrease in the conductivity of protein solutions at high protein
ACCEPTED MANUSCRIPT concentrations has been previously observed for zein solutions (Kayaci & Uyar, 2012) and could be ascribed to the very high viscosity of these solutions which limits electron mobility. Figure 1 shows the SEM images of the various gliadin structures obtained applying a voltage of 15 kV. Gliadin concentration of 5 wt.% was insufficient to generate any type of structure through electrohydrodynamic processing. This can be explained by the lack of biopolymer
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entanglements in this specific solution, also reflected in its low apparent viscosity (Table 1).
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In contrast, gliadin particles were obtained when doubling the concentration to 10 wt.%. At
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such low biopolymer concentration, due to Rayleigh instability, the governing process was electrospraying (Ghorani & Tucker, 2015). Increasing the protein concentration, the process
changed
from
electrospraying
to
electrospinning.
This
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electrohydrodynamic
phenomenon occurred at a concentration of 15 wt.%, where nanofibers with beaded structures
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were formed. This is in accordance with the results of Y. Wang and Chen (2012a) who reported beaded electrospun structures at this specific gliadin concentration. Apparently, at
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this specific gliadin concentration, because of the increased molecular entanglements, the fluid jet breaking up into particles was prevented (Gómez-Mascaraque, Sanchez, & López-
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Rubio, 2016). In addition, it has been reported that higher polymer concentrations lead to increased interactions between polymer and solvent, which favors the elongation of the fluid
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facilitating chain entanglements and, thus, giving raise to electrospun fibers (Li et al., 2009). However, as reported by Woerdeman et al. (2005), fiber formation by electrospinning is a result of not only chain entanglements, but also to the presence of reversible junctions in the protein (specifically the breaking and re-forming of disulfide bonds that occur via thiol/disulfide interchange reaction). Therefore, in contrast with gluten spinnability, where the presence of the highest molecular weight glutenin chains were responsible for the lower threshold concentration for fiber formation (only 5% w/v in 1,1,1,3,3,3-hexafluoro-2propanol- HFIP) (Woerdeman et al., 2005), in the case of the lower molecular weight gliadin
ACCEPTED MANUSCRIPT fraction used in this study, 30 wt.% gliadin was needed to generate beaded-free fiber structures. In general, proteins must be more or less unfolded in order to be electrospinnable and, in this case, acetic acid was a proper solvent to fulfill this requirement (Nieuwland et al., 2013). The voltage applied during electrohydrodynamic processing is another parameter that has
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been seen to affect the morphology, shape and size of final structures obtained. The same
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gliadin solutions were processed increasing the voltage to 18 kV (cf. Figure 2). This higher
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voltage had distinct influence on the morphology and diameter of electrospun/electrosprayed gliadin structures. In general, and in agreement with previous knowledge about the effect of
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voltage on particle size, the average diameter of particles and fibers was decreased by increasing the voltage. The diameter of particles at electrical potential of 15 kV and 18 kV
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were 817 nm and 467 nm, respectively (flow rate 1 ml/h). In other words, at higher voltage, particles of smaller diameters were formed. According to Ghorani, Alehosseini, and
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Tucker (2017), the application of high voltages results in a higher whipping of the solution
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and a faster evaporation of the solvent during the flight towards the collector and, thus, to formation of the smaller average sizes of particles and/or fibers. However, the behavior of gliadin solutions with the greatest protein concentration (i.e. 35 wt.%) was different. Thicker
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tubular and branched microfibers (with average diameters 5440 nm) were formed in this case, which has been observed before in previous works and has been ascribed to increased ejection of solutions from the nozzle (only observed for high concentrations and high voltages), thus resulting in thicker electrospun fibers (Bhardwaj & Kundu, 2010; Heikkilä & Harlin, 2008). It is also worthwhile pointing out that, at a gliadin concentration of 35 wt.%, the electrical conductivity of the solution was lower, which as explained before may be due to limited electron mobility as a consequence of the high solution viscosity (cf. Table 1).
ACCEPTED MANUSCRIPT According to Tan, Inai, Kotaki, and Ramakrishna (2005) a less electrically conductive spinning solution may stretch less, resulting in fiber products with larger diameters. Finally, fluid flow rate was the other processing parameter we varied, because it determines the amount of fluid transfer rate and jet velocity, thus affecting the spinnability of the samples. Generally, results on increasing the flow rate at different concentration revealed that
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it mainly affected bead formation. In other words, lower flow rates resulted in the less beaded
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electrospun structures. According Zong et al. (2002), due to lack of sufficient time for
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evaporation of solvent prior reaching to collector, higher fluid rates results in beaded fibers. In addition, at high voltage and high gliadin concentration, increasing the fluid flow rate
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resulted in branched microfibers. It has been reported that at a higher critical value, fluid flows can result in the formation of junction zones between the deposited polymer structures
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(branched electrospun structures). This is due to the insufficient time available to allow for complete solvent evaporation, causing the fibers to fuse together (Kriegel, Arrechi, Kit,
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McClements, & Weiss, 2008). In this study, decreasing the flow rate from 1 to 0.5 mL/h had no major impacts on the morphology of the structures obtained and, thus, the best conditions
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for particle and fiber formation were set at gliadin concentrations of 10 and 25 wt.% respectively, with an applied voltage of 18 kV and a fluid flow rate of 1 mL/h. The diameter
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of particles and fibers were 468 nm and 256 nm, respectively. These formulations and processing conditions were selected for further characterization.
3.2. FTIR spectroscopy To investigate the changes in molecular organisation of the gliadin structures after electrohydrodynamic processing, electrospun and electrosprayed samples were analyzed using FTIR spectroscopy. The Amide I band of proteins and polypeptides in the region ranging from 1600 to 1700 cm-1 has long been used in the study of the secondary structures
ACCEPTED MANUSCRIPT of proteins (Gu & Wang, 2013). This arises from the amide bonds that link the amino acids. The absorption associated with amide I band is related to stretching vibration of C=O bond of the amide in which the vibrational frequency reflects the hydrogen bond strength between C=O bond and N-H bond. In other words, this vibrational frequency reflects secondary structures such as α-helix of proteins and polypeptides (Secundo & Guerrieri, 2005).
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As observed in other proteins, electrohydrodynamic processing seems to enhance molecular
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order of the amino acids chains, reflected in more defined and narrower spectral bands
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(Gómez-Mascaraque et al., 2015). In fact, band width is a measure of conformational freedom and, thus, the narrowing of spectral bands confirms the formation of more rigid
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(structured) particles/fibers. Apart from these obvious changes, when comparing the spectrum of unprocessed gliadin with those from the electrosprayed/electrospun structures, shifts in the
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amide I band were observed (cf. Figure 3). Interestingly, while a shift towards lower wave numbers was observed for the electrosprayed particles obtained from the 10 wt.% gliadin
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solution (from 1660 cm-1 to 1654 cm-1 for the unprocessed and electrosprayed gliadin, respectively), this amide I band was displaced towards higher wave numbers (1666 cm-1 ) in
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the electrospun structures from the 25 wt.% gliadin solution. It should be stressed that this shifts reflect small conformational changes in the protein, as similarly reported for other
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proteins in acid conditions (Aceituno-Medina, Lopez-Rubio, Mendoza, & Lagaron, 2013). However, the interesting observation is that for several proteins studied (Hirota, Goto, & Mizuno, 1997; Stephens et al., 2005), when fibers are formed through electrospinning, a shift towards higher wavenumbers is observed, which is generally correlated with an increase in the α-helical and random coil configuration (Yang et al., 2009). In contrast, less extended conformations, which are correlated with lower wavenumber positions of the amide I band normally correlated with -sheet configurations, are generally observed when particles are
ACCEPTED MANUSCRIPT obtained through electrospraying (Aceituno-Medina et al., 2013; Gaona-Sánchez et al., 2015).
3.3. X-ray Diffraction The XRD diffraction patterns of pure gliadin and electrospun structures are presented in
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Figure 4. The diffraction pattern of the extracted gliadin showed two broad peaks with
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characteristic distances of 2θ~11° and ~18 (corresponding to 1.61 nm and 0.98 nm,
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respectively). Similar results were also reported in some other studies (Gulfam et al., 2012; Xu et al., 2017). After electrohydrodynamic processing, the diffraction patterns of the
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structures obtained showed a broad diffraction band around 2θ~14°, which is characteristic of amorphous materials. This result indicates that the original amino acid chain packing of the
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extracted protein was lost during the processing giving raise to amorphous materials. This result contrasts with the one from Gulfam et al. (2012), where they did not observe any
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change in the XRD patterns, probably due to the conditions used to process the materials which did not alter the physical structure of the proteins (they used ethanol instead of acetic
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acid to process the gliadin material).
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3.4. Differential Scanning Calorimetry The DSC analysis was conducted to determine the thermal properties of the obtained gliadin structures after electrohydrodynamic process. The DSC thermograms of pure gliadin as well as the electrospun structures are presented in Figure 5. The DSC thermogram of pure gliadin exhibited a single endothermic at 87 °C which as often termed as dehydration temperature (TD), which reflects the loss of bound water from the materials (H. Wang et al., 2016). In agreement with observations for other prolamins, no endothermic peak was observed for pure gliadin (Huang et al., 2013; Yilmaz et al., 2016). The values of TD for particles and fibers
ACCEPTED MANUSCRIPT were 81 and 80 °C, respectively, i.e. the high surface area of the generated materials facilitated water evaporation. Moreover, from the normalized heat flow curves, the electrospun/electrosprayed samples contained less sorbed water (as the area under the curve was significantly lower). This may indicate that less functional groups from gliadin were available for water sorption. In addition, a glass transition temperature (Tg) value of 179 °C
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was observed in the thermogram of pure gliadin, slightly higher than that previously reported
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(Duclairoir et al., 2003). A shift towards higher temperatures in the Tg was observed for the
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electrosprayed (Tg of 204ºC) and electrospun structures (Tg of 202ºC) (cf. arrows and insets in Figure 5). This result is in agreement with previous results obtained for zein, in
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which a significant Tg increase from 165ºC to 222ºC was observed after electrospraying this protein (Gaona-Sánchez et al., 2015). This Tg increase could be correlated with the increased
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molecular order observed through FTIR (see section 3.2), in which the more rigid structures
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would require higher temperatures for this second order transition.
In this study,
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4. Conclusions
the effects of solution properties and process parameters on the
electrohydrodynamic
processing
of
gliadin
solutions
have
been
studied.
Gliadin
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concentration, which mainly affected apparent viscosity and conductivity was the main factor that determined the morphology of the structures obtained through electrohydrodynamic processing. Increasing the applied voltage generally led to reduced size of the structures, while fluid flow rate did not significantly affect the process in the conditions studied. 10 wt.% gliadin
solutions
resulted
in particle
formation
through
electrospraying,
while
increasing the solution concentration above 25wt.% led to the formation of almost beadedfree fibers. Interestingly, amide I band position in the FTIR spectra is indicative of conformational changes related to the formation of either particles or fibers. The use of acetic
ACCEPTED MANUSCRIPT acid for processing the protein, led to the disruption of the original amino acid chain packing, as observed from the XRD diffractograms. However, the increased molecular order observed from the FTIR spectra affected the thermal properties of the obtained structures, increasing their glass transition temperature in comparison with the unprocessed extracted gliadin. The overall results presented in this work demonstrate the suitability of acetic acid for the
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electrohydrodynamic processing of gliadin fractions, showing the versatility of the technique
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which allows the development of different morphologies (fibers or particles), which can be
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further used as bioactive delivery vehicles.
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Acknowledgements
The authors gratefully acknowledge the financial support of the Research Affairs Office at
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Shiraz University (Grant #93GCU1M1981). The authors acknowledge financial support from the Spanish Ministry of Economy, Industry and Competitiveness MINECO (AGL2015-
None.
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References
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Declaration of interest
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63855-C2-1-R project) and the financial support of EU FEDER funds.
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ACCEPTED MANUSCRIPT Table
1.
Solution
concentrations
used,
properties
of the
different
solutions
and
electrospinning parameters investigated Gliadin Concentration (wt. %)
Viscosity (mPa.s 30 rpm, 25 ° C)
Surface tension (mN.m-1 )
Electrical Conductivity (μs.cm-1 )
5
61.73±1.06g
28.03±0.11d
19.68±0.16f
Applied Voltage (kV)
Feed rate (ml.h1 )
Distance (mm)
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15 and 0.5 100 18 and 1 10 100.57±0.30f 28.13±0.06cd 49.25±0.08e 15 and 0.5 100 18 and 1 15 179.04±0.87e 28.01±0.35cd 102.55±2.19d 15 and 0.5 100 18 and 1 20 321.24±1.95d 28.40±0.17cd 129.70±0.85b 15 and 0.5 100 18 and 1 25 522.54±0.56c 28.80±0.37bc 147.75±1.48a 15 and 0.5 100 18 and 1 30 628.27±1.32b 29.17±0.24ab 129.25±0.78b 15 and 0.5 100 18 and 1 35 788.01±2.16a 29.73±0.46a 119.10±0.14c 15 and 0.5 100 18 and 1 Data are displayed in means ± standard deviation of three replications (P < 0.05); means in each column bearing different superscripts are significantly different (P < 0.05).
ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. SEM images of electrosprayed /electrospun structures obtained at the conditions of 15 kV, 1 mL/h from gliadin solutions of (a) 10 wt%, (b) 15 wt%, (c) 20 wt%,(d) 25 wt%, (e) 30 wt% and (f) 35 wt%. Figure 2. SEM images of electrosprayed /electrospun structures obtained at the conditions of
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18 kV, 1 mL/h from gliadin solutions of (a) 10 wt%, (b) 15 wt%, (c) 20 wt%, (d) 25 wt%, (e)
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30 wt% and (f) 35 wt%.
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Figure 3. FTIR spectra of unprocessed gliadin (solid line), electrosprayed gliadin (dashed line) and electrospun gliadin (dotted line). Inset shows the magnification of the Amide I band
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with arrows pointing to the respective band shifts.
Figure 4. XRD diffraction patterns of: (A) unprocessed gliadin; (B) electrosprayed gliadin;
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and (C) electrospun gliadin.
Figure 5. DSC thermograms of pure gliadin (solid line), electrosprayed gliadin (dashed line)
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and electrospun gliadin (dotted line). Thermograms have been offset for clarity. Arrows point out to the Tg of the materials and insets show a magnification of the curves where the change
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in specific heat is observed.
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FIGURE 1
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FIGURE 5
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Gliadin microparticles/microfibers were obtained by electrohydrodynamic processing Acetic acid was used as a food-grade solvent to develop the protein microstructures The effects of processing parameters on morphology and properties were studied
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