Preparation and characterisation of zein films obtained by electrospraying

Accepted Manuscript Preparation and CHARACTERISATION of zein films obtained by electrospraying V.A. Gaona-Sánchez, G. Calderón-Domínguez, E. Morales-Sánchez, J.J. ChanonaPérez, G. Velázquez-de la Cruz, J.V. Méndez-Méndez, E. Terrés-Rojas, R.R. Farrera-Rebollo PII:

S0268-005X(15)00111-3

DOI:

10.1016/j.foodhyd.2015.03.003

Reference:

FOOHYD 2914

To appear in:

Food Hydrocolloids

Received Date: 11 September 2014 Revised Date:

18 January 2015

Accepted Date: 3 March 2015

Please cite this article as: Gaona-Sánchez, V., Calderón-Domínguez, G., Morales-Sánchez, E., Chanona-Pérez, J., Velázquez-de la Cruz, G., Méndez-Méndez, J., Terrés-Rojas, E., FarreraRebollo, R., Preparation and CHARACTERISATION of zein films obtained by electrospraying, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.03.003. 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.

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ACCEPTED MANUSCRIPT 1

PREPARATION AND CHARACTERISATION OF ZEIN FILMS OBTAINED BY

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ELECTROSPRAYING.

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VA. Gaona-Sánchez1, G. Calderón-Domínguez1*, E. Morales-Sánchez2, JJ. Chanona-Pérez1,

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G. Velázquez-de la Cruz2, JV. Méndez-Méndez3, E. Terrés-Rojas4, RR. Farrera-Rebollo1

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Ayala s/n, Casco de Santo Tomás, C.P. 11340, México D.F. MÉXICO. 2CICATA- Unidad

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Querétaro. Cerro Blanco No. 141. Col. Colinas del Cimatario, C.P. 76090, Santiago de

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Querétaro, Querétaro MÉXICO.3CNMN-IPN. Luis Enrique Erro s/n, U. Prof. Adolfo López

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ENCB-IPN. Departamento de Ingeniería Bioquímica. Prolongación de Carpio y Plan de

Mateos, 07738, México D.F. MÉXICO. 4IMP. Eje Central Lázaro Cárdenas 152, San Bartolo

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Atepehuacan, 07730. México D.F. México.

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* [email protected]

Abstract

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Electrospraying is a technique now applied for the production of food nanoparticles,

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nanofibres and nanocapsules, which could constitute a good alternative for the development

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of films. The goal of this work was to determine the electrospraying operation conditions and

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the zein concentration that would allow a film to be produced and also to evaluate the

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structural characteristics and barrier and thermal properties of films developed at different

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thicknesses. Results showed that films can be obtained by electrospraying, resulting possible

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to produce homogeneous films, without electrical arc formation during processing when

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using a 11.4 % zein solution, an electrical voltage of 7.8-8.7 kV and a distance of 1.5 ± 0.5

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cm between the nozzle and the deposit plaque. Regarding the effect of the production

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methods (electrospraying and casting) and the film thickness on film appearance, both

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variables had significant effect on colour parameters, observing a more yellowish colour

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(b*=72.3 ± 6.8) and lower transparency (%T=90.6 ± 0.21) at larger thicknesses (100 µm), and

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a smoother and more homogeneous surface (ESEM) when films were obtained by

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electrospraying. Water vapour permeability was only influenced by film thickness (1.51 x 10-

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electrospraying and casting films were different (222°C and 231°C respectively) and higher

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than that of zein powder (165°C), indicating a possible change in the film structure due to the

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process. Under the process conditions tested in this work, electrospraying can be considered

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as an alternative technique to produce films.

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Keywords: electrospraying; casting; zein films

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to 3.11 x 10-08 g/s-m-Pa) in both methodologies. The glass transition temperatures (Tg) for

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1. Introduction

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Zein is a biopolymer with the potential to generate biodegradable plastics. It is a primary

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maize protein and subproduct of the bioethanol industry (Biswas, Selling, Woods, & Evans,

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2009; Tihminlioglu, Atik, & Özen, 2010), and it is a non-toxic polymer that is biocompatible

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and biodegradable (Wang & Padua, 2003) and used to produce films with adequate

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characteristics in terms of permeability and thermal resistance (Ghanbarzadeh & Oromiehi.,

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2008; Tihminlioglu et al., 2010) that provide protection against microbial attack (Escamilla-

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García, et al., 2013). Zein films and zein particles can be used for the encapsulation of

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essential oils, aromas and flavours, for the controlled released of additives or as an active

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packaging material for food (Wang & Padua, 2005; Shukla & Cheryan, 2001).

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Although zein and other biopolymers have been successfully applied at a laboratory scale, the

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development of biodegradable and/or edible films and coatings through traditional techniques

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(casting, extrusion and coextrusion) tend to generate films with problems that include a lack

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of uniformity in the thickness and homogeneity (Acevedo, Pedreschi, Enrione, Osorio, &

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Aguilera, 2010). Therefore, new production alternatives have been researched that could be

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based on nanomanufacturing technologies (Fabra, Busolo, Lopez-Rubio, & Lagaron, 2013),

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ACCEPTED MANUSCRIPT which are rarely used for food. Examples of these technologies can be found in the literature,

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in particular for encapsulation (Klinkesorn, Sophanodora, Chinachoti, Decker, &

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McClements, 2005; Priya, Vijayalakshmi, & Raichur, 2011), enzyme immobilisation (Lei, Bi,

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& Yang, 2007), emulsion stabilisation (Guezey & McClements, 2006) and food packaging

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structure improvement (Tihminlioglu et al., 2010; Arcan & Yemenicioğlu, 2013).

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There are two types of methodologies reported for the development of micromaterials and

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nanomaterials: ''top-down'' and ''bottom-up'' (Reverchon & Adami, 2006; Sanguansri &

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Augustin, 2006); these technologies can also be applied to the production of microstructured

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or nanostructured biodegradable films (Jaworek & Sobczyk, 2008). The “top-down” approach

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is characterised by the production of nanomaterials through size reduction, such as through

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the application of a shear force. Processes such as homogenisation at high pressures and

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microfluidisation, ultrasonic emulsification and emulsification membranes are used to

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generate nanomaterials based on this approach. The “bottom-up” approach is related to the

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synthesis of nanometric length materials from molecular scale units (Reverchon & Adami,

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2006). This approach involves the ‘self-assembly’ of molecules, which requires a balance of

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attractive and repulsive forces between a pair of molecules to form the structure. These forces

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can be influenced by factors such as changes of temperature, concentration, pH, ionic strength

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of the system, mechanical force (pressure) or electric or magnetic field strength (Serizawa,

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Iida, Matsuno, & Kurita, 2006; McClements, 2006).

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Among these techniques, electrohydrodynamic processes, specifically electrospinning and

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electrospraying have been reported to be used in the elaboration of fibres and particles from

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the micro to the nanoscale (Anu Bhushani & Anandharamakrishnan, 2014). Both techniques

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are similar, differing mainly in the polymer solution concentration used, and as a result the

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product types obtained. Fibres are developed through electrospinning while particles by

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ACCEPTED MANUSCRIPT electrospraying, being these structures the result of the way the jet is ejected from the tip of

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the Taylor cone (Anu Bhushani & Anandharamakrishnan, 2014).

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From these techniques, electrospraying (electrohydrodynamic spraying), can be considered as

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a “top-down” methodology because the starting material is at bulk state, and when electrical

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forces are used, nanosized materials are obtained (Okutan, Terzi, & Altay, 2014). In this

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regard, Jayasinghe (2006) expressed that the route to form self-assembled nanostructures

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through electrospraying is predominantly a top-down fabricating technique for directing

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bottom-up assemblies, as construction blocks are produced from fine drops by a layer-by-

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layer deposition process to form a continuous coating on a substrate (Jaworek, 2007, Jaworek

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& Sobezyk, 2008). This process is considered efficient because at least 80-90% of the base

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material can be deposited (Jaworek, 2007; Jaworek & Sobczyk, 2008).

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In the electrospraying technique, the liquid is forced through a capillary tube (nozzle) by an

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electrical field that disperses the liquid into fine drops onto the substrate; this process can be

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performed in an ambient or gaseous atmosphere at low temperature without the need for

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reactors or high vacuum environments. This system can produce micrometric- or nanometric-

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sized drops, and their distribution can be monodisperse depending on the flow rate of the

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liquid and voltage in the capillary nozzle (Torres-Giner, Gimenez, & Lagarón, 2008; Jaworek,

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2007). However, the fact that these drops become electrically charged facilitates the control of

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their motion (including their deviation and focus) by means of an electric field.

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The main factors that influence the morphology of structures formed by an electric field are

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the solution properties (viscosity, conductivity and surface tension) and process parameters

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(voltage, distance to the collector, flow rate and environmental humidity) (Aceituno-Medina,

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Mendoza, Lagaron, & López-Rubio, 2013; Chakraborty, Liao, Adler, & Leong, 2009).

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In recent years, the use of electrospraying has focused on the electrospinning of nanofibres

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(Jiang, Reddy, & Yang, 2010; Miyoshi, Toyohara, & Minematsu, 2005; Torres-Giner et al.,

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ACCEPTED MANUSCRIPT 2008; Yao, Li, & Song, 2007) and encapsulation using zein or other materials (Jaworek,

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2007). In this respect, Fabra, López-Rubio, & Lagaron (2014) compared the effect of

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incorporating nanostructurated interlayers of zein by electrospinning on

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polyhydroxyalcanoates, while Kayaci & Uyar (2012) produced nanofibres containing

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ciclodextrins, also applying this technique. On the other hand, the production of zein

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nanoparticles has also been studied. In this regard, Gómez-Estaca, Balaguer, Gavara &

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Hernández-Munoz (2012) prepared zein nanoparticles with a compact spherical structure by

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electrohydrodynamic atomisation studying the effect of polymer concentration, flow rate and

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applied voltage. Many other works have been carried out to develop nanoparticles, mainly

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fibres and encapsulates, using different protein sources such as whey protein concentrate

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(López-Rubio, & Lagaron, 2012) soy protein isolate (Wang, Marcone, Barbut, & Lim, 2013),

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amaranth protein isolate (Aceituno-Medina et al., 2013), egg albumen (Wongsasulak,

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Patapeejumruswong, Weiss, Supaphol, & Yoovidhya, 2010), collagen, gelatine (Okutan,

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Terzi, & Altay,2014) and casein (Xie, & Hsieh, 2003). However, no information related to the

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production of zein films applying the electrospraying technique was found; therefore, the

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goal of the present work was to evaluate the feasibility of generating zein films through the

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electrospraying technique and to study the structural characteristics and barrier and thermal

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properties of the developed films.

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2. Materials and Methods

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2.1. Materials

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Maize zein (Z3625, Sigma-Aldrich, Mex) was used for the elaboration of films. It is

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composed by the α ( 22 and 24 kDa), β ( 24, 22, and 14 kDa), γ, and δ fractions as reported

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by Sigma-Aldrich catalogue and obtained by electrophoretic analysis(data not shown).

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Glycerol was employed as plasticiser (G5516, Sigma-Aldrich, Mex) and ethanol at 96% (v/v)

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as the dilution medium.

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2.2. Films obtained through electrospraying

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2.2.1. Preliminary tests

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Preliminary tests were performed to select the zein concentration and the equipment operation

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conditions under which samples could be produced with film characteristics similar to those

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obtained by the casting method. Based on the information published by Torres et al. (2008),

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Del Nobile et al., (2008) and Doğan, Özen, & Tıhmınlıoğlu (2008), different zein

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concentrations (29, 16, 11.4 and 8.8 g of zein/100 g of solution) were selected that

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corresponded to values reported for films obtained by traditional methods and nanofibres

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obtained by the electrospinning method. In all cases, the zein was mixed with ethanol ( 96%,

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density of 0.807 g/mL) and stirred (Corning PC 320 Hot Plate Stirrer, Tewksbury, MA, USA)

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for 10 minutes at 300 rpm. Subsequently, glycerol was added at 22.2% (w/w) based on zein

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powder and agitated for 10 more minutes. Finally, this solution was heated at 73 ± 2°C for 4

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minutes. The level of glycerol was selected based on reported casting studies (Trezza &

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Krochta, 2000; Del Nobile, Conte, A., Incoronato, & Panza,2008) where films made of

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protein were obtained using that or similar glycerol levels. Furthermore, Liang et. al., 2015,

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recently reported that to assure glycerol be acting as a plasticiser in zein films it must be

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added at levels equal or higher than 20%.

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The solution was fed into the electrospraying device, which consists of a syringe pump, X-

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axis linear actuator, stainless steel collector and variable high voltage power supply.

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The solution was then placed in the syringe pump, which consists of a 10 mL syringe and a

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linear actuator that pushes the plunger of the syringe to provide the required flow. The syringe

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needle is made of surgical grade stainless steel with an outer diameter of 32 thousandths of an

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ACCEPTED MANUSCRIPT inch (0.8 mm or code 21G) and an inner diameter of approximately half the outer diameter

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(0.4 mm) blunted by abrasion. The syringe was fixed horizontally, and the needle was

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electrically connected to the positive high voltage power supply (0 to 30 kV DC, 30A24-P4

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Brand Ultravolt, Ronkonkoma, NY, USA). The ground electrode was connected to the

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collector (stainless steel sheet of 9 × 9 cm2 with a 2D finish and 1/64" calibre). The injection

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flow rate, a constant value (IF = 14.5 mL/h), corresponds to a minimum value that assures the

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zein solution ejected, under the different process conditions, was deposited on the collector,

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forming the product (film or fibres); the electric current was read and recorded through an

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ammeter (HandHeld Multimeter, Model MUL-600, Brand Steren, China) incorporated into

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the power supply.

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The solutions were tested to determine the conditions at which the film would form, noting

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the responses as well as the distance of the tip of the needle to the collector (D) and the

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electric voltage (EV). The selection criteria were as follows: 1) aspersion feasibility, 2) non-

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formation of fibres, 3) non-generation of an electric arc and 4) homogeneity of the formed

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film. The selected process conditions (D, EV) and concentration of zein (CZ) were used in the

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subsequent trial.

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2.2.2. Characterisation of the zein solution

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The zein solution at the selected concentration was evaluated with respect to viscosity,

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conductivity, density and pH, to be used as control quality parameters for the process.

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Viscosity (µ). This parameter was determined using a rheometer (Anton Paar Physica MCR

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101, Graz, Austria) with a system of concentric cylinders and a paddle stirrer that was

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designed to measure viscosity in biopolymer solutions (ST24-2D/2V/2V-30). The test was

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conducted at a constant deformation speed of 100 s-1 and a temperature of 20°C, which are

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ACCEPTED MANUSCRIPT similar conditions to those reported by Kayaci & Uyar (2012). The viscosity was reported in

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Pascal-second (Pa s), and the measurements were performed in triplicate.

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Conductivity (σ). The two-point resistivity technique was followed (Wieder, 1978; Calixto &

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Sánchez, 2007) using a multimeter (LCR HiTester, HIOKI Model 3532-50; Nagano, Japan),

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which also recorded the resistance (R) values at three different frequencies (50 Hz, 100 Hz

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and 1000 Hz). Equation 1 was used for the calculation of σ.

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=  ∗ …………….……..Equation 1

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where σ (kΩ-1 m-1) is the conductivity, R (kΩ) is the measured resistance, L (m) is the distance

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between the two electrodes and A (m2) is the transversal area of the cell.

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The pH and density were evaluated following the 981.12 and 962.37 methods of the AOAC

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International (1995 & 1997), respectively.

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2.2.3. Films obtained by electrospraying

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Based on the preliminary tests, both the operational conditions and the zein solution

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concentrations at which a film was formed were used. Furthermore, the film thickness (FT)

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was varied to determine the effect that this parameter had on the physical (colour,

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transparency), structural (roughness and homogeneity), barrier (water vapour permeability)

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and thermal (glass transition temperature and other phase transition temperatures)

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characteristics, for which three thicknesses were selected (64.5 ± 4.5, 84.5 ± 4.5, 104.5 ± 4.5

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µm). The selection of these values was mainly based on the device capability, which did not

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allow to form films with thicknesses lower than 60 µm (bilayer), and the upper limit was the

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formation of a film composed of four thin layers, based on the number of times the spray was

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deposited on the same location of the collection plate. As a comparison samples, films of the

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same thickness and zein concentration were developed by the casting method. All of the

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samples were stored at room temperature inside a desiccator that contained a saturated

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solution of sodium bromide (NaBr) to maintain a relative humidity of 59.7% for subsequent

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analysis.

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2.2.4. Films obtained by casting

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The films were elaborated following the methodology described by Lai & Padua (1997) with

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certain modifications. The zein solution at the concentration chosen for electrospraying was

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poured into Teflon circular plates (7.1 cm in diameter) in different amounts (1.95 ± 0.31, 3.03

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± 0.43 and 3.76 ± 0.18 g) depending on the desired thickness. All of the solutions were

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subjected to kiln-drying (TERLAB, MAH25D, Mex.) at 35°C for 4.5 hours. The samples

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were stored and analysed using the methods described for films obtained by electrospraying.

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2.3. Characterisation of the films

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2.3.1. Thickness

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The thickness of the films was measured, as a film quality parameter, using a digital

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micrometer (Fowler 54-860-001 Electronic IP54, China) and following the methodology

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reported by Arzate-Vázquez, et al. (2011),. The films were placed between the micrometer

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spindle and anvil, and the measurement was performed at the first sign of contact between the

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film and spindle. Three independent samples (true replicates) of each film were evaluated at

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ten different positions. The mean value and standard deviation are reported. Only those

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samples which had the selected thicknesses (64.5 ± 4.5, 84.5 ± 4.5, 104.5 ± 4.5 µm ) were

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used for analysis.

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2.3.2. Colour

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ACCEPTED MANUSCRIPT From the films that were obtained and stored, colour was evaluated by means of a colorimeter

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(CR-400 Chroma Metre, Konica Minolta, Ramsey, NJ, USA). Prior to performing the

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measurements on the films, the colorimeter was calibrated with a reference plate (Y = 93.7, x

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= 0.3159, y = 0.3324), and the colour values were expressed in CIELab colour space

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coordinates, where L represents the luminosity, ± a* (green (-a) and red (+a)) and ± b* (blue

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(-b) and yellow (+b)) the chromatic components (Calderón-Dominguez, Vera-Dominguez,

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Farrera-Rebollo, Arana-Errasquin, & Mora-Escobedo, 2005). To perform the measurements,

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the films were placed on the calibration plate of the colorimeter (Escamilla-García et al.,

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2013). A total of five measurements per film were taken, and the evaluation was performed in

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triplicate. The transparency (Equation 2) of the films (%T) was determined assuming that a

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fully transparent film would generate the same luminosity values (L*) as those obtained from

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the blank calibration plate (L * 0 = 100) and that any difference would be the result of more

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opaque material (L * <100) (Escamilla et. al., 2013). Ten measurements were taken from each

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sample, and the samples were evaluated in triplicate. The values obtained from the

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colorimeter were analysed by the SigmaPlot 12.5 program (Systat Software, San Jose, CA,

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USA) and applying a two-way analysis of variance (ANOVA) and the Holm-Sidak method at

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a level of p < 0.05.

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L* = %T…………………………………… (Equation 2).

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2.3.3. Water vapour permeability

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The water vapour permeability (WVP) was determined using a modification of the standard

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gravimetric method known as the “cup method” or “test cell,” which is based on the

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American Society for Testing and Materials (ASTM) E 96-88 and ASTM E 96-92 methods

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with some variations (Mc Hugh, Avena‐Bustillos, & Krochta, 1993; Mali, Grossmann,

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García, Martino, & Zaritzky, 2006). For this test, the samples were trimmed to obtain a

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ACCEPTED MANUSCRIPT circular sample 51.1 mm in diameter, and the samples were placed in permeation cells. The

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permeation was measured while maintaining a temperature of 30°C and a relative humidity

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(RH) gradient of 100-0% through the films by placing distilled water on the permeation cell

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(100% RH) and anhydrous silica gel outside of the cells (0% RH). The change in weight of

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the cell was recorded every 30 minutes in an analytical balance with a precision of 0.0001 g

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(DENVER instrument, Goettingen, Germany) as a function of time.

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The data of the weight loss kinetics were used to calculate the permeability (Equation 3). All

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of the tests were performed in triplicate.

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 = ×%

 % 



×  =   

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……………………Equation 3

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where WVTR is the result obtained when dividing the value of the weight loss kinetic curve

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slope (g/s) by the area (m2) of the sample subjected to the test; S is the saturation vapour

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pressure in Pascals (Pa) at the test temperature; %HR1 and %HR2 are the relative humidity

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values in the test inside and outside the permeation cell, respectively, expressed as fractions;

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and L is the film thickness (m).

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2.3.4. Differential scanning calorimetry

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Thermal analysis (DSC) was performed in a differential scanning calorimeter (Mettler DSC1

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STAR System, Mettler Toledo, Switzerland) equipped with an oven with a FRS5 sensor. The

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samples of approximately 20 ± 1 mg were weighed (Denver Instruments balance, model APX.

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200, Bohemia, NY, USA) inside a 40 µL aluminium sample holder with a cover, which was

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hermetically sealed and then perforated (a hole at the cover centre). For the experiment, the

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heating rate was 5°C min-1 at a temperature interval of -10 to 400°C with nitrogen (N2) as the

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purging gas at a flow rate of 50 mL min-1 and with a cooling gas line at a flow rate of 2 mL

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ACCEPTED MANUSCRIPT min-1, which used an empty aluminium sample holder as the reference. The instrument was

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calibrated with an indium/zinc standard (99.98% purity, melting point of 156.61°C and

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enthalpy of fusion of 28.71 J g -1). Each sample was analysed in duplicate, and the glass

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transition temperature (Tg), as well as the temperatures at which other transitions were

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observed, are reported. Tg was determined as the point of intersection of the tangent drawn at

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the point of greatest slope on the transition curve with the extrapolated baseline prior to the

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transition (ASTM-D7426-08).

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2.3.5. Environmental scanning electron microscopy (ESEM)

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The zein films obtained by electrospraying and casting were studied by environmental

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scanning electron microscopy (ESEM). The samples were directly placed on a cylindrical

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aluminium microscope stubs using double-sided tape. The micrographs were collected with a

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XL30 ESEM environmental scanning electron microscope (Philips, Amsterdam, Holland)

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using an acceleration voltage of 25 kV, a secondary electron detector (GSE) and a chemical

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analysis detector (EDX, Winter Springs, FL, USA) to evaluate the sample homogeneity and

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ensure the non-contamination of the product during its manipulation.

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2.3.6. Atomic force microscopy (AFM)

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For this evaluation, an atomic force microscope diMultimode V was used (Veeco, Santa

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Barbara, CA, USA) with a diNANOSCOPE V controller using RTESP probes (Bruker,

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Camarillo, CA, USA) with a resonance frequency of 286–362 kHz and a force constant of

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20–80 N m−1 in tapping mode. A 0.5 cm × 0.5 cm section was split from each sample for

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attachment to a stainless steel disc using double-sided adhesive tape to measure five areas of

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different sizes (1 × 1, 2 × 2, 2.5 × 2.5 and 10 × 10 µm2) by scanning at a speed of 1.5 Hz. The

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roughness was calculated from the images using the square root of the height deviation (Rq,

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Equation 4). Also, the arithmetic mean of the height deviation absolute values (Ra, Equation

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5) were calculated using the Nano Scope Analysis 1.20 program (Veeco) and through the

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application of a flattening process (one degree).

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! = "

&

……………..…………………………………………….. Equation 4

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∑$%

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(

' = ∑& %,(|*+| ………………………………………………………….. Equation 5 &

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2.4. Statistical analysis

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The data were statistically evaluated by a two-way ANOVA and means compared by

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applying the Holm-Sidak test, using the SigmaPlot 12.5 program and a significant difference

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of p < 0.05. The reported values are the averages and standard deviations of at least three

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repetitions of each sample.

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3. Results and Discussion

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3.1. Preliminary tests

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Table 1 shows the results obtained during the preliminary tests that determined the zein

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concentration and operational conditions under which the film could be obtained.

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According to the results in Table 1, depending on the zein concentration (CZ), the product

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generated during the electrospraying process can present different types of structures. When

316

the CZ was used at 29%, the generation of fibres occurred (Figure 1A), which has been

317

reported by other researchers (Torres et al., 2008; Aceituno-Medina et al., 2013; Yao et al.,

318

2007), who indicate that the variation of structure is a function of the concentration. From the

319

same data, it can be observed that at lower concentrations (8.8% ≤ CZ ≤ 16.2%), the solution

320

could be atomised, resulting in the formation of films. However, even when a homogenous

321

structure is generated with a CZ of 16.2%, the formation of electric arcs was constant, and

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ACCEPTED MANUSCRIPT this concentration was discarded to avoid damage to the equipment. For the films produced

323

with a CZ of 8.8%, the structure was fragile and not homogenous (Figure 1B), which

324

prevented adequate handling for the subsequent analyses. When maintaining CZ at 11.4%, an

325

aspersion that promoted a continuous structure was obtained (Figure 1C). This structure was

326

similar to that of the films obtained by the casting method. There was also an absence of

327

electric arcs during the process. Therefore, the degree of homogeneity of the film, as well as

328

its structure, depends on the concentration of the solution used. Similar results have been

329

reported by Torres et al. (2008), Aceituno-Medina et al. (2013) and Yao et al. (2007),

330

although these results were reported for fibres.

331

At low electric voltage values (EV < 7 kV), the particles of the solution did not deposit on the

332

plate because the critical point for the solution could not be reached, which is similar to the

333

findings reported by Torres et al. (2008) and Miyoshi et al. (2005). At greater values (EV > 11

334

kV), the solvent evaporates on its way to the collector, thereby generating electric arcs. The

335

best results were observed in the interval of 7.8-8.7 kV.

336

At distances of 1.5 ± 0.5 cm between the ejector and plate (D), the formation of homogenous

337

films occurs without the generation of electric arcs and with a constant spraying. No studies

338

about the formation of films regarding this variable and electrospraying process were found in

339

the literature. However, Bock , Dargaville, & Woodruff ( 2012) reported for electrospraying

340

particle obtention, that small distances to the collector can impair full solvent evaporation,

341

resulting in the collapsing of particles, coalescence and broad size distributions, phenomenon

342

that could be related to the film formation.

343

Considering the results of the preliminary tests, the following values were chosen as process

344

parameters: CZ = 11.4%, EV = 8.3 ± 0.4 kV, D = 1.5 cm, and IF = 14.5 mL/h. Similarly, the

345

zein solution at the selected concentration was evaluated with regard to pH (7.3), viscosity

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(0.036±0.001Pa s), density (0.861 ± 0.02 g/cm3) and conductivity (29.812 ± 2.24 kΩ−1 m−1) to

347

establish the quality control parameters of the solution to be fed to the equipment.

348

3.2. Characterisation of films obtained by electrospraying

350

3.2.1. Colour

351

Colour is an important quality attribute in foods, as this parameter affects the acceptance or

352

rejection of the product by consumers; hence, when films are developed to be used on foods,

353

manufacturers look for transparency and colour attributes that do not change the final

354

appearance of the material to be covered. The results of the transparency (% T) and

355

chromaticity (a*; b*) measurements are shown in Figure 2. From the results, transparency

356

(%T , Figure 2A), did show significant difference (p < 0.05) between the electrospraying and

357

casting methods, except in the case of the thickest films (104.5 ± 4.5 µm), presenting smaller

358

transparency values as compared to the others. Regarding the effect of film thickness,

359

transparency was significantly changed from a maximum of 94.4 ± 0.25% for the 64.5 ± 4.5

360

µm film to a minimum of 89.3 ± 1.6%.

361

From the chromaticity statistical evaluation it was observed that the a* values (Figure 2B)

362

were not modified due to the elaboration method or film thickness (P = 0.587), whereas for

363

parameter b*, both the elaboration method and the thickness of the films showed a significant

364

effect (p < 0.05). These changes in the yellow tone could be related to the amount of solution

365

poured for the preparation of the films of different thicknesses, resulting in an increase of

366

zein β-carotenes and zeaxanthin, which produce a yellowish coloration (Kurilich & Juvick,

367

1999; Sessa, Eller, Palmquist, & Lawton, 2003; Assis, Scramin, Correa, de Britto, & Forato,

368

2012). The obtained "b" data also suggest that the electrospraying method tends to generate

369

less yellowish films as compared to those obtained by casting, which is a similar tendency to

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ACCEPTED MANUSCRIPT 370

that reported by Torres-Giner et al. (2008) and Fabra, Lopez-Rubio, & Lagaron (2014) for

371

zein nanostructures.

372

3.2.2. Water vapour permeability

374

The water vapour permeability (WVP) values varied from 1.51 x 10-08 to 2.57 x 10-08 (g)/(s-

375

m-Pa) for electrospraying and from 1.38 x 10-08 to 3.11 x 10-08 (g)/(s-m-Pa) for casting

376

(Figure 3); these values are within the interval reported by Wang & Padua (2005),

377

Ghanbarzadeh & Oromiehi (2008), Wang & Padua (2004), Wang, Geil & Padua (2005), who

378

mentioned that the zein films water vapour permeability are three orders above the

379

polyethylene synthetic films of low and high density and within the same order as gluten,

380

chitosan and methylcellulose films. This information is similar to the results obtained in this

381

study.

382

Using statistical analysis, it was observed that the permeability of the films is not influenced

383

by the elaboration method (p > 0.05) but it is by the thickness in a directly proportional

384

relationship. This effect can be explained by the exponential relationship between thickness

385

and RH at the film surface (Pereira, Souza, Cerqueira, Teixeira, & Vicente, 2010; Bertuzzi,

386

Castro-Vidaurre, Armada, & Gottifredi, 2007), whereas other authors attribute the effect to

387

changes in the film structure because of the swelling that water provokes in the polymer

388

(Park, Weller, Vergano, & Testin, 1993). Although it has been reported that the WVP is

389

independent of the thickness at the beginning of the diffusion process (McHugh et al., 1993),

390

it is recognised that the films developed based on biopolymers exhibit dependence

391

relationships between these two parameters.

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392 393

3.2.3. Differential scanning calorimetry

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ACCEPTED MANUSCRIPT The thermograms of the films produced by the electrospraying and casting methods, as well

395

as that of zein powder, are presented in Figure 4. These results show that the elaboration

396

method had an effect on the thermal characteristics of the materials. A Tg value of 222°C

397

(Figure 4, arrow “a”) was obtained by electrospraying, which is higher than that of zein

398

powder (165°C, arrow "c") and lower than the value obtained by casting (231°C, arrow "b").

399

In this regard, Sessa, Woods, Mohamed & Palmquist (2011) suggested that an increase in the

400

Tg value is related to more ordered zein molecules, while lower values provide more flexible

401

and less friable films, which could be related to a better workability when using in the

402

formulation the same glycerol concentration. Regarding the effect of the method, Taleb,

403

Didierjean, Jelsch, Mangeot, Capelle, & Aubry (1999) reported that the interaction of the

404

charged molecules with an external electric field might alter their distribution inside the

405

protein solution, that could change the characteristics of the film.

406

Furthermore, from our results other peaks in the temperature interval from 50 to 120°C can be

407

observed, that could be related to a loss of volatile compounds such as ethanol residues and

408

also to the relaxation, unfolding and denaturalisation of the protein through the rupture of

409

hydrogen bonds. Finally, the thermograms presented peaks in the temperature interval of 280

410

to 355°C that were most likely related to zein degradation (Neo, Swift, Ray, Gizdavic-

411

Nikolaidis, Jin, & Perera, 2013; Müller et al., 2011; Tillekeratne & Easteal, 2000).

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3.2.4. Environmental scanning electron microscopy

414

The micrographs of the film samples obtained by electrospraying (5a-5c) and casting (5d-5f)

415

at different thicknesses (64.5 ± 4.5 µm, 84.5 ± 4.5 µm and 104.5 ± 4.5 µm) can be observed in

416

Figure 6. In both cases, the homogeneity of the solution and certain imperfections in different

417

proportions can be observed depending on the elaboration method used. For the

418

electrospraying method (5a-5c), the number of imperfections (small holes and ridges,

17

ACCEPTED MANUSCRIPT indicated by arrows) was lower in comparison to the films produced by casting. This effect

420

can be related to a shorter drying time and more homogenous deposits, which give rise to a

421

smoother surface (Lai & Padua, 1997; Sessa et al., 2011). Furthermore, the homogeneity of

422

the film obtained by electrospraying was obvious at lower thicknesses, which highlighted the

423

presence of particles and imperfections when this parameter was increased.

424

In these micrographs, the presence of bright structures could also be observed that might

425

represent macromolecular aggregates (Figure 5, circles) of different origin to the raw

426

materials of the solution. Through an EDX analysis, it was confirmed that the bright

427

structures presented the same composition as other regions of lower brightness in the samples,

428

which confirms the homogeneity of the sample and non-contamination of the product during

429

its manipulation. Through these micrographs, no phase separation, formation of capsules or

430

flat or circular fibres were observed, which suggests that the CZ and electrospraying

431

parameters are appropriate for the elaboration of the films.

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3.2.5. Atomic force microscopy

434

Atomic force microscopy (AFM) was used to describe the surface morphology of the films

435

and calculate their roughness. For the films prepared via electrospraying (Figure 6A-D), the

436

images showed an absence of pores, thereby confirming a continuous surface without cracks

437

and a good structural integrity as observed by ESEM. The images also showed the presence of

438

aggregates and undulations of lower size in comparison to casting, which were most likely

439

caused by the ejection mechanism, composition, and type of polymer (zein solution) used

440

(Han, Xu, Lu, & Wang, 2013; Panchapakesan., Sozer, Dogan, Huang, & Kokini, 2012).

441

It has been reported that the zein aggregation/agglomeration ratio in the film surface is a

442

function of the type of zein used. According to Panchapakesan et al. (2012), among the four

443

zein fractions (α-, β-, γ- and δ,), the β-zein is the most hydrophilic, whereas the α fraction is

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ACCEPTED MANUSCRIPT the most hydrophobic and produces a more heterogeneous surface texture film in comparison

445

to one developed with a greater proportion of β-zein. Based on this statement, and considering

446

that our polymer was formed by the different protein fractions, there is a high possibility of

447

developing a more homogeneous film by electrospraying (Figure 7).

448

In this regard, the elaboration method significantly affected (p <0.05) the roughness data (Ra

449

and Rq, Figure 8), and in all cases, the samples obtained by electrospraying generated more

450

homogenous surfaces, which were reflected in the lower roughness values. This type of

451

conformation could generate different mechanical properties compared to those presented by

452

films produced by casting

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4. Conclusions

455

The electrospraying technique can be used for the development of more homogeneous films,

456

without affecting the water vapour barrier capacity, but with a different structure as compared

457

to the casting method, with the advantage of shorter processing times, as the film is produced

458

at the moment of contact of the solution on the collection plate giving rise to a new approach

459

in the film development area.

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Acknowledgements

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V.A. Gaona-Sánchez wishes to express its gratitude to CONACYT and PIFI-IPN for the

463

scholarship provided by the institutions. This research was funded through projects 20120417,

464

20120809, 20131518, 20140625 from the National Polytechnic Institute (IPN - Mexico) and

465

CONACYT 1668, 133162, 191389. The authors also wish to thank the Mexican Petroleum

466

Institute (IMP), and the Centre for Nanoscience and Micro and Nanotechnology (CNMN-

467

IPN) for the technical assistance.

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ACCEPTED MANUSCRIPT 469 470

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TE D

626

isolate-based fiber fortified with anthocyanin-rich red raspberry (< i> Rubus

633

strigosus) extracts. Food Research International, 52(2), 467-472.

634 635 636 637

AC C

632

59. Wang, Y., & Padua, G. W. (2003). Tensile properties of extruded zein sheets and extrusion blown films. Macromolecular Materials and Engineering, 288(11), 886-893.

60. Wang, Y., & Padua, G. W. (2004). Water sorption properties of extruded zein films. Journal of agricultural and food chemistry, 52(10), 3100-3105.

638

61. Wang, Y., Geil, P., & Padua, G. W. (2005). Effects of processing on the structure of

639

zein/oleic acid films investigated by X‐ray diffraction. Macromolecular bioscience,

640

5(12), 1200-1208.

26

ACCEPTED MANUSCRIPT 641 642

62. Wieder, H. H. (1978). Laboratory notes on electrical and galvanomagnetic measurements. Elsevier.

643

63. Wongsasulak, S., Patapeejumruswong, M., Weiss, J., Supaphol, P., & Yoovidhya, T.

644

(2010). Electrospinning of food-grade nanofibers from cellulose acetate and egg

645

albumen blends. Journal of food engineering, 98(3), 370-376. 64. Xie, J., & Hsieh, Y. L. (2003). Ultra-high surface fibrous membranes from

RI PT

646 647

electrospinning of natural proteins: casein and lipase enzyme. Journal of Materials

648

Science, 38(10), 2125-2133

SC

TE D

M AN U

mats. Journal of applied polymer science, 103(1), 380-385.

EP

650

65. Yao, C., Li, X., & Song, T. (2007). Electrospinning and crosslinking of zein nanofiber

AC C

649

27

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Table 1 Results of the preliminary test for the selection of operating conditions for the production of zein films by electrospraying.

Result

(kV)

29

7- 13

16.2

8-10

11.4

7.8-8.7

8.8

7-9

RI PT

solution

TE

Produced fibres (B = 5-15 cm) and electrical arc (D = 5-7.5 cm) for all values of D tested, and impeded stable ejection. Film uniformly but electrical arc occurred continuously (D <5); formed fibres (D> 5.0)

SC

g zein/100 g

Uniform films without generating electrical arcs (1cm
M AN U

CZ

Inhomogeneous films (1cm
CZ: concentration of the solution of zein; TE: working voltage; D: distance between

AC C

EP

TE D

emission source and receiver plate.

AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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AC C

EP

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Electrospraying can be used to produce zein films.

•

Electrospraying generates similar films to those produce by casting.

•

Electrosprayied zein generates different products as varying process conditions.

AC C

EP

TE D

M AN U

SC

RI PT

•