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Plasma technology as a tool to decrease the sensitivity to water of fish protein films for food packaging
Accepted Manuscript Plasma technology as a tool to decrease the sensitivity to water of fish protein films for food packaging Viviane Patrícia Romani, Bradley Olsen, Magno Pinto Collares, Juan Rodrigo Meireles Oliveira, Carlos Prentice, Vilásia Guimarães Martins PII:
S0268-005X(18)32320-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.03.021
Reference:
FOOHYD 5000
To appear in:
Food Hydrocolloids
Received Date: 25 November 2018 Revised Date:
12 March 2019
Accepted Date: 12 March 2019
Please cite this article as: Romani, Viviane.Patrí., Olsen, B., Collares, M.P., Oliveira, J.R.M., Prentice, C., Martins, Vilá.Guimarã., Plasma technology as a tool to decrease the sensitivity to water of fish protein films for food packaging, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/ j.foodhyd.2019.03.021. 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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Plasma technology as a tool to decrease the sensitivity to water of fish protein films
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for food packaging
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Viviane Patrícia Romania, Bradley Olsenb, Magno Pinto Collaresc, Juan Rodrigo
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Meireles Oliveirac, Carlos Prenticea, Vilásia Guimarães Martinsa
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a
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Grande do Sul 96203-900, Brazil
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b
Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, United States
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c
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Grande, Rio Grande do Sul 96203-900, Brazil
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Institute of Mathematics, Statistics and Physics, Federal University of Rio Grande, Rio
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ABSTRACT
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School of Chemistry and Food, Federal University of Rio Grande, Rio Grande, Rio
Proteins films have been developed for use in food packaging materials in order to
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replace synthetic polymers because of environment pollution. Despite their advantages,
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the use of protein films in a wide range of food products is still limited due to their
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hydrophilic behavior. Thus, this study aimed to decrease the sensitivity of fish protein
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films to water through the application of glow discharge plasma. Plasma parameters
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(power, pressure and time of exposure) were studied according to a 23 factorial design,
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and the physicochemical properties of the films including moisture content, water vapor
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permeability, and solubility in water were evaluated. The microstructure and thermal
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properties of the films were also characterized. In general, glow discharge plasma
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caused different effects in films properties, such as cleaning and etching which were
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ACCEPTED MANUSCRIPT responsible for the changes observed in physicochemical properties. Power and time of
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exposure, as well as their interaction, were the most influent parameters. Decrease in
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water vapor permeability and solubility were observed in some treatments, which are
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important characteristics for a material to be used as food packaging. Then, the plasma
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setting might be changed through the adjustment of parameters of exposure according to
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the specificity of the application intended.
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Keywords: Biodegradable materials, Cold plasma, Glow discharge, Myofibrillar
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proteins, Sustainable films.
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1. Introduction
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Concerns about the environmental pollution and depletion of fossil
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resources due to the wide use of petroleum-based plastics have led to growing interest
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in polymers from sustainable and biodegradable resources (Félix, Lucio-Villegas,
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Romero, & Guerrero, 2016). Biopolymers obtained from agro-based sources, including
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proteins, polysaccharides and lipids, may be extracted from food industry
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wastes/byproducts as well as from low cost raw materials. Proteins attract more
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attention because of their structure, that has the capacity to form strong three-
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dimensional networks (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2016; Rhim,
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Park, & Ha, 2013). Animal proteins, such as myofibrillar proteins, are more promising
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due to their capacity to form slightly transparent films with excellent UV-light barrier
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properties in comparison to commercial wrap film of polyvinyl chloride, for example
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(Kaewprachu, Osako, Benjakul, & Rawdkuen, 2016; Kaewprachu, Rungraeng, Osako,
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& Rawdkuen, 2017).
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ACCEPTED MANUSCRIPT According to the literature, the properties of protein films are known as
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better than other macromolecules, mainly in terms of gases barrier and mechanical
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properties and biodegradation (Wittaya, 2012). However, despite these advantages, the
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hydrophilic nature of proteins still limits the commercial use of these materials for food
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packaging because their performance is compromised when in contact with humidity
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(Benbettaïeb, Gay, Karbowiak, & Debeaufort, 2016; Wihodo & Moraru, 2013). To be
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used as packaging material, it is important that the film protects the food from the
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external environment throughout the production process, during handling, transportation
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and storage, until it is delivered to the consumer (Han, 2005). High solubility in water
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and water vapor permeability of the protein films in comparison with synthetic plastics
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limit their use in a wide range of foods, such as dairy and meat products. For example,
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the passage of water through the packaging material is crucial because it is directly
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linked to the mechanisms of food deterioration, including lipid oxidation and microbial
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activity (Robertson, 2013). Therefore, it is of the utmost significance to find alternatives
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to decrease the sensitivity of these materials to humidity.
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Several strategies have been reported to improve film performance, such as
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the use of crosslinking agents, incorporation of reinforcing substances, blending with
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different materials and others. The use of environmentally friendly processes is
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important to achieve the required properties for the polymers without the use of
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additional components and waste generation. Cold plasma is an example of a dry
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process suitable for heat-sensitive materials that does not pollute the environment. It is a
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non-thermal technology, which consists of a gas mixture containing ions, radicals, free
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electrons and neutral species, with the capacity to start chemical reactions on the surface
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of materials and consequently changing their properties (Chu, 2002; Geyter & Morent,
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2012; Mahmoud, 2016; Pankaj et al., 2014a). The application of cold plasma in fish
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ACCEPTED MANUSCRIPT proteins films for food packaging has been studied previously by our group to
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understand the effects of treatment time on their physicochemical properties, including
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mechanical performance and optical properties (Romani et al., 2019). Here, the effects
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of operating parameters (pressure, power and time) of alternating current (AC) glow
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discharge plasma on protein films from fish are reported. They were studied aiming the
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improvement of films properties in terms of decreasing the sensitivity to water and
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water permeability.
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2. Material and Methods
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2.1 Raw material
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Whitemouth croaker (Micropogonias furnieri) muscle was used to obtain
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the protein isolates. The fish was obtained from processing industries in Rio Grande
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(RS), Brazil. The muscle was crushed and stored frozen at −18 °C until protein
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extraction.
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2.2 Obtaining the fish protein isolate
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Fish protein isolate (FPI) was obtained based on the method described by
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Nolsøe & Undeland (2009) through the pH-shifting process. This process consists in an
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alkaline solubilization followed by isoelectric precipitation of proteins. The fish muscle
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was homogenized with distilled water (ratio 5:1, v/w) to perform the alkaline
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solubilization at pH 12 for 20 min (4 °C). Then, a centrifugation was performed at
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7500×g for 15 min to separate the middle phase (soluble proteins) from lipids and
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isoelectric point; after 20 min the suspension was centrifuged at 7500×g for 15 min to
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get the pellet that was then dried and grounded. The separation process generated
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protein with 84.5% of purity determined according to the Association of Official
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Analytical Chemists (AOAC, 2000) method.
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2.3 Film preparation
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Fish protein films were prepared using 3% (w/v) of FPI through the casting
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technique as the method used by Romani, Prentice-Hernández, & Martins (2017). The
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pH of the mixture containing distilled water, proteins and 30% (w/w) of plasticizer
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(glycerol) was adjusted to 11.0. This mixture was heated to 80 ºC and held for 20 min in
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order to denature proteins. After cooling, it was cast in Petri dishes and left to dry at 40
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ºC in an oven with air circulation (Biopar, S150BA). After drying, the protein films
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were left in desiccators (50% relative humidity – RH controlled with a saturated sodium
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bromide (NaBr) solution) for 24 h before plasma treatment.
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2.4 Glow discharge plasma treatment
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The films were treated using an AC glow discharge plasma (Fig. 1). The
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film samples (3) were placed inside the plasma chamber (1) in a sample support (13)
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and the treatment was performed between two electrodes (2) with an inter-electrode
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distance of 13 cm. Initially, the plasma chamber was evacuated to 0.15 Pa and then dry
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air was filled up to the process procedure. After pressure stabilization, the glow
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discharge plasma was generated by application of AC power. One of the electrodes was
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electrodes. The effect of different sets of plasma treatment conditions was studied
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according to a 23 factorial design with three central points. The variables evaluated were
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power, pressure and time. The coded and real values of variables are presented in Table
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1.
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2.5 Film evaluation
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2.5.1
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Moisture content
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Moisture content was determined by the weight loss of the films after being
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subjected to a temperature of 105 ± 2 °C for 24 h in an oven (DeLeo, A15E) according
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to Antoniou, Liu, Majeed, & Zhong (2015). The moisture (%) was calculated
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considering the dry matter content of the film, which was obtained by the difference
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between the initial and final weight.
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2.5.2
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Film thickness (mm) was obtained using a digital micrometer (IP65, Insize)
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Thickness
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with precision of 0.001 mm. The measurements were made in ten different locations of
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the samples and the average was determined.
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2.5.3
Solubility in water
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method described by Gontard, Duchez, Cuq, & Guilbert (1994) with some
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modifications. Film discs of 2 cm in diameter were dried in an oven (DeLeo, A15E) at
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105 ± 2 °C to determine the initial dry weight. Then, the samples were immersed in 50
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mL of distilled water and continuously shaken (200 rpm) at 25 ºC for 24 h. After
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immersion, the samples were dried again to determine final dry weight. The weight of
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the soluble portion was determined considering the weight of the insoluble matter and
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the initial weight of the film and expressed as film solubility (%) according to Equation
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1.
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SW (%) =
(Wo - W) Wo
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Water vapor permeability
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2.5.4
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dry weight after the immersion.
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(1)
Where W0 is the dry weight of film before immersion in water and W is the
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The water vapor permeability (WVP) was determined according to the
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ASTM Standard Method E96-00 (ASTM, 2000). The test was performed using
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permeation cells containing silica as desiccant inside. The films were sealed on the cells
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and placed in desiccators containing distilled water. Weight measurements were
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evaluated at 24 h intervals during 7 days at 25 ºC. The WVP (g.mm/m².day.kPa) was
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obtained according to Equation 2 considering the weight gain, the thickness, the area of
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the films, the time and the vapor pressure difference:
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WVP (g.mm/m².day.kPa) =
W×L
(2)
A × t × ∆P
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Where W is the weight gain of the cell (g); L is the film thickness (mm); A is
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the exposed area of film (m²); t is the time of weight gain (day); and ∆P is the vapor
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pressure difference across the film (kPa).
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2.5.5
Microstructure
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The effect of AC glow discharge in morphology of films surface was
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observed through Scanning Electron Microscopy (SEM). Stubs were used to fix the film
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samples using double sided adhesive tape. The samples were coated with gold and
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images were obtained in a microscope (Jeol, JSM-6060) at a magnification of 1000×.
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Energy-dispersive X-ray spectroscopy (EDX) was conducted under 10 kV incident
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electron energy to identify changes in concentration of elements of films surface.
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The films were also evaluated through wide-angle X-ray diffraction
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(WAXS) using a Bruker, D8 Advance Diffractometer operating at a voltage of 40 kV
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and current of 30 mA, at incident angle of 2Ɵ, between 10 to 50º.
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2.5.6
Thermal characterization
Thermal modifications caused by AC glow discharge plasma in films were
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studied by differential scanning calorimetry (DSC) using a calorimeter (Shimadzu
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TGA-60). Film samples (3 mg ± 0.01) were sealed in aluminum pans and scanned at a
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heating rate of 10 ºC/min over a range of 30 to 200 ºC using a nitrogen flow rate of 50
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mL/min. The enthalpy values (∆H), glass transition (Tg) and melting (Tm) temperatures
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were obtained from the endothermic peaks present in the thermogram.
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3. Results and Discussion
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3.1 Effects of plasma treatment on moisture, solubility in water and water vapor
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permeability of the films
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The effects of the operating parameters power, pressure and time of AC
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glow discharge in fish protein films were studied specifically in the properties related to
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water, which limit the use of these materials in foods with high water activity. Table 2
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presents the results obtained for moisture content, solubility in water and water vapor
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permeability under the different conditions, and Fig. 2 shows the effects of the variables
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studied as well as their interactions on the properties evaluated.
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The moisture content of the films provides information about the way in
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which the studied variables affect affinity to water (Capitani et al., 2016). It is possible
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to observe from Fig. 2 that the time of plasma treatment is the most influent parameter
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in decreasing moisture content of films while the interaction effect between time and
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power led to the moisture increase.
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Solubility is defined as the affinity of a material for a substance,
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representing an important characteristic to be considered when studying packaging
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materials for foods (Han & Scanlon, 2005). The solubility in water (SW) of the films
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varied in the range between 29.9 and 45.6% (Table 2), which represent medium SW
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compared to other studies using biomacromolecules from agro-sources, such as chia
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protein and mucilage (58.7 to 80.8%) (Capitani et al., 2016), sunflower protein (86.3 to
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chicken skin gelatin incorporated with rice flour (82.9 to 93.7%) (Soo & Sarbon, 2018).
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The values obtained for SW in the present study are attributed to the hydrophilic nature
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of myofibrillar proteins from fish and the plasticizer used (glycerol). The interaction
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effect between the variables power and time influenced solubility of films causing its
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decrease, which has significant importance mainly for use in foods with high humidity.
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Potential applications of these films are governed by the solubility, for example meat
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and dairy products require water resistance due to the high-water activity of these foods.
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In contrast, high sensitivity to water is a functional advantage in the development of
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soluble sachets, such as edible films/coatings for pre-measured portions to be dissolved
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in water or hot food (Guilbert & Gontard, 2005; Yang, Paulson, & Nickerson, 2010).
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Besides that, high solubility in films represents an advantage in the view of
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biodegradability, however their capacity to protect dry foods from moisture of the
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environment as well as to prevent the water loss of some products is impaired (Stuchell
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& Krochta, 1994). The lowest solubility in water was observed for treatment T6,
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indicating the reduced affinity to water. This result obtained with the lowest power and
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time under study suggested the decrease of moisture content in the film probably
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without other pronounced effects such as etching and/or degradation of the matrix.
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The water vapor permeability (WVP) is another important characteristic to
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be considered in the use of a material to develop food packaging (Han & Scanlon,
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2005). Some foods, such as fruits and vegetables require reduced barrier to water vapor
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due to the respiration process, while snacks and biscuits, for example, need high barrier
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to maintain crunchiness. In the present study, the variables power and time, the
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interaction between power and time variables as well as the interaction between
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pressure and time of AC glow discharge plasma influenced the WVP of fish protein
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when higher power was used, in contrast to the other effects observed. The time of
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plasma treatment had the capacity to reduce the water vapor permeability, especially in
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the interaction with higher power and pressure. This is a positive trend mainly for the
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use of films in contact with food which low permeability is required. This behavior
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indicates that a considerable decrease of moisture in films surface might have occurred,
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as well as suggests the exposure of hydrophobic sites of amino acids in the protein
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sequence.
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The effects observed in the present study show that AC glow discharge is
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promising to decrease the sensitivity to water of fish protein films due to the lower
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solubility and water vapor permeability obtained in treatments T1 and T6, respectively.
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Different changes observed among the treatments with cold plasma might be explained
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by the different reactions that occur during the plasma treatment, such as cleaning,
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functionalization, crosslinking, etching and degradation. According to Poncin-Epaillard,
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Brosse, & Falher (1997), these reactions are governed by the reactive species generated
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by plasma and the energy absorbed by the surface of the material. Arolkar, Salgo,
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Kelkar-Mane, & Deshmukh (2015) reported that plasma leads to different effects during
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the treatment process; initially there is a cleaning of the surface removing low molecular
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weight fragments which are weakly linked and after this initial step the reactive species
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can cause etching or degradation processes as well as crosslinking of polymer chains. In
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protein films, these reactions are result of OH radicals alongside other reactive species
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formation in the cold plasma treatment. These species might cleave peptide and
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disulfide bonds, and oxidize amino acid chains (Mirmoghtadaie, Shojaee Aliabadi, &
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Hosseini, 2016).
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ACCEPTED MANUSCRIPT Lacroix & Cooksey (2005) reported that potential crosslinking in the film
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surface, by the increase in cohesion between polypeptide chains of the myofibrillar
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proteins, was effective in decreasing affinity to water, as might have contributed to
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some extent in the changes of some treatments in the present study. Other authors
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reported changes in hydrophilic properties of polymers after plasma treatment, such as
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Tenn et al. (2012), who observed a 28% decrease in water vapor permeability of
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ethylene vinyl alcohol (EVOH) after plasma application. According to Oh, Roh, & Min
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(2016), plasma treatment might induce modifications in films through the formation of
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oxygen and nitrogen groups on surface when carbon radicals are formed on polymer
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chains. This radical formation on the film surface lead to changes in its properties
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through the effects mentioned previously. As explained by Morent et al. (2010),
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crosslinking limits the mobility of polymer chains and the fraction of mobile groups
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affecting polymer properties. But despite that, the exposure of hydrophobic side chains
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due to bond scission of proteins might be responsible for the effects observed in the
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films of the present study as well as the moisture loss of the films surface due to the
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contribution of vacuum during plasma treatment.
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Cold plasmas are constituted of charged particles and excited/non-excited
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molecules (Misra, Tiwari, Raghavarao, & Cullen, 2011). Then, the role of power,
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meaning the applied voltage level, in affecting the properties of films is a result of the
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density of these particles produced in the plasma. According to Whitehead (2016),
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electrons are accelerated in an electric field through the gaseous medium for generation
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of plasma and a wide range of processes might occur when an electron collides with
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atoms or molecules. With higher power, more changes are expected in the material
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placed in the plasma chamber because more collisions and reactions occur. The time of
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plasma treatment is also extremely important because the longer the treatment, more
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reactions happen, as described previously the cleaning step followed by effects such as
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etching and degradation. In addition, the low pressure in the system of glow discharge
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plasma has an important function also due to the vacuum that might contribute for the
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moisture loss of films structure. In the factorial design performed in the present study, the treatments T1 and
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T6 showed lower water vapor permeability and solubility in water, respectively, that
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was the aim of the present work. Therefore, the parameters used in these treatments
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were defined as the optimal conditions. Then, in order to better understand the effects of
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AC glow discharge, the films treated according to these parameters were further
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characterized by their microstructure and thermal properties.
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3.4 Microstructure of AC glow discharge plasma treated films
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X-ray diffraction analysis (Fig. 3) showed predominantly amorphous
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structures which are characteristic of myofibrillar protein films. Changes were observed
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mainly in two diffraction peaks, centered at 2θ = 13º and 2θ = 19º, which according to
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the Bragg’s law correspond to distances of 6.85 Å and 4.73 Å, respectively. The smaller
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distance observed corresponds to the average backbone distance within the α-helix and
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larger distances are result of the α-helix packing or the distance among the near helices,
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as mentioned by Pankaj, Bueno-Ferrer, Misra, Bourke, & Cullen (2014). The
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application of AC glow discharge according to the treatments T1 and T6 led to small
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changes in the position of diffraction peaks and major changes in the peak intensities.
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For treatment T1, the peak centered at 19º presented a larger decrease in intensity,
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suggesting disruption of α-helix packing, which might have led to exposure of
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hydrophobic side chains consequently decreasing WVP. Regarding the film of treatment
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ACCEPTED MANUSCRIPT T6 (picture shown in Fig. S1), a similar decrease in both peaks was observed, that might
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indicate changes in molecular aggregates of helices as well as in the α-helix structure,
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reflecting in the changes of physicochemical properties studied. Other authors (Zhang et
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al., 2015) reported changes in secondary structures of proteins as a result of plasma
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treatment (for example in the amount of α-helix, β-sheet regions, as well as random
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coils), in agreement to the trends observed in XRD patterns of the present study.
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The effects of AC glow discharge plasma on the surface morphology of fish
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protein films were also evaluated using Scanning Electron Microscopy (images shown
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in Fig. S2). The surfaces of the films were predominantly smooth and homogeneous,
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and the plasma treatment caused a slightly increase in roughness of the surface with
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small protrusions due to the bombardment of reactive species in both treatments T1 and
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T6, however more pronounced in treatment T1. Other authors report that a rougher
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surface is result of the etching effect, that occurs due to physical and chemical
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processes, including chain scission, breakage of bonds, removal of fragments and
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degradation (Akishev et al., 2008; Mirabedini, Arabi, Salem, & Asiaban, 2007).
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Different studies of plasma treatment in films from biomacromolecules also reported
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increases in roughness of materials surface. Some examples include Pankaj et al. (2015)
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using gelatin films for plasma application, Chang & Chian (2013) evaluating surface
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modification effects on chitosan film biodegradability and protein adsorption and Song,
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Oh, Roh, Kim, & Min (2016) using plasma for improvement of polylactic acid films
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properties for food packaging.
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The study of the concentration of surface elements by EDX showed higher
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differences among treated and untreated samples compared to the differences between
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the plasma treatments (Table 3). These changes might indicate a disruption of surface
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molecular structure of films due to chain scission as well as etching effects, as also
ACCEPTED MANUSCRIPT reported by Hwang et al. (2003). As in the present study, Pankaj et al. (2015) observed
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increase in surface oxygenation after cold plasma treatment in gelatin films. However,
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these authors reported that this behavior was more dependent on the treatment time. A
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similar trend was found by Ramkumar et al. (2018) in low density polyethylene (LDPE)
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film surfaces with various gaseous plasma treatments.
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3.5 Effect of AC glow discharge plasma in thermal properties of fish protein films
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Thermal properties from DSC of control and plasma treated films showed
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endothermic peaks (Fig. 5). From DSC curves, it was observed that AC glow discharge
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caused more pronounced effects in the films properties of T1. The properties of glass
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transition temperature (Tg), melting temperature (Tm) and enthalpy (∆H) of fish protein
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films, as affected by glow discharge plasma, are presented in Table 4. Results observed
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by DSC analysis in general agree with the physicochemical performance obtained in the
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present study. AC glow discharge plasma was responsible for increasing Tg, Tm and ∆H,
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suggesting changes in chains mobility in the film matrix, as suggested previously due to
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the differences in water vapor passage through the film materials. As mentioned by
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Pankaj et al. (2014b), air plasma might generate etching effects on the materials surface,
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as in some treatments of the present factorial design; however, modifications in thermal
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properties also confirm that some moisture loss might occur in the film surface. It was
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responsible for modifications in Tg, Tm and ∆H because water acts as plasticizer
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increasing chains mobility, that was more pronounced mainly in T1.
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Some authors mentioned that cold plasma forms radicals on the surface of
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polymer films, which form crosslinked networks limiting the permeability of films due
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to the reduction in the network free volume. Other studies indicate that cold plasma
ACCEPTED MANUSCRIPT affects the solubility of polymers, but not the diffusivity of water molecules because the
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bulk properties of the material remain unchanged (Oh, Roh, & Min, 2016; Perez-Gago
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& Krochta, 2001; Stutz, Illers, & Mertes, 1990). From the results of the
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physicochemical properties in the present study, which were supported by
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microstructure and thermal characteristics, it can be inferred that changes in fish protein
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films depend on the plasma setting and the time of treatment because during the
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exposure of the material different reactions occur inside the plasma chamber.
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4. Conclusion
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The potential of AC glow discharge plasma to improve the performance of
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fish protein films was demonstrated in the present study. The plasma setting based on
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the factorial design performed (power, pressure and time of exposure) led to different
386
results for affinities with water (measured by water vapor permeability, solubility in
387
water and moisture content). These properties are important when using the material to
388
develop food packaging, then the effects observed are an advance for industrial
389
applications. As supported by microstructure and thermal analysis, the different
390
influences of AC glow discharge caused by the variation of the parameters studied
391
occur due to the different reactions that plasma causes, such as cleaning and etching.
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This study showed that power and time were the variables that had stronger
393
effects in the properties of fish protein films. Therefore, variation in the water vapor
394
permeability and solubility in water might be adjusted according to the application
395
required through the parameters of exposure in glow discharge plasma. Beyond that, the
396
modification of surface characteristics caused by plasma is advantageous for other
397
aspects in designing new materials for food packaging, such as the development of
ACCEPTED MANUSCRIPT 398
multilayer films or incorporation of active substances in the surface to extend shelf life
399
of food products. Overall, the use of AC glow discharge plasma to shape characteristics
400
of fish protein films is advantageous because it is an environmentally friendly
401
technology being used in a sustainable and non-polluting raw material.
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Acknowledgments
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The authors are grateful for the financial support by the Coordination for the
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Improvement of Higher Education Personnel (CAPES) and National Council of
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Research (CNPq) of Brazil. Also, this work was supported by the Serrapilheira Institute
408
(grant number Serra - 1709-20275). The authors also acknowledge CEME-SUL/FURG
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(Centro de Microscopia Eletrônica do Sul/Universidade Federal do Rio Grande/Brazil)
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for SEM and XRD analysis and MIT-Brazil Program.
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ACCEPTED MANUSCRIPT Table 1. Coded and real values of variables for plasma application in protein films. -1
0
+1
Power
5W
8W
11 W
Pressure
4 Pa
7 Pa
10 Pa
Time
1 min 3 min 5 min
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Variables
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Table 2. Factorial design of plasma treatment and results for moisture, solubility and
590
permeability.
Control
Solubility in
content (%)
water (%)
83.7 ± 1.3
38.4 ± 0.2
5.19 ± 0.29
permeability (g.mm/day.m².kPa)
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Treatment Power Pressure Time
Water vapor
Moisture
1
1
1
86.6 ± 2.5
36.9 ± 0.8
5.08 ± 0.03
T2
1
1
-1
83.3 ± 0.4
40.1 ± 0.1
6.23 ± 0.05
T3
1
-1
1
84.3 ± 0.6
37.8 ± 1.9
5.74 ± 0.04
T4
1
-1
-1
83.9 ± 1.4
45.6 ± 2.7
6.51 ± 0.12
T5
-1
1
1
81.0 ± 0.8
40.3 ± 0.6
6.07 ± 0.04
T6
-1
1
-1
84.8 ± 1.2
29.9 ± 3.5
5.81 ± 0.25
T7
-1
-1
1
T8
-1
-1
-1
T9
0
0
0
T10
0
0
0
T11
0
0
0
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43.7 ± 3.9
5.28 ± 0.14
84.2 ± 0.8
39.5 ± 1.1
5.36 ± 0.28
83.0 ± 0.9
42.2 ± 3.6
5.56 ± 0.24
83.3 ± 0.8
41.4 ± 0.5
5.45 ± 0.31
83.7 ± 1.4
44.6 ± 2.4
5.59 ± 0.08
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Table 3. Concentration of elements of control fish protein films and plasma treated
592
films. Weight (%) Protein film C
N
O
30.2 ± 0.92 13.8 ± 5.29 45.1 ± 2.37
Treatment 1
28.2 ± 0.73 11.4 ± 4.33 53.2 ± 1.96
Treatment 6
26.9 ± 0.76 11.8 ± 4.43 53.6 ± 1.98 Mean ± standard error.
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Control
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Table 4. DSC thermal characterization of control fish protein film and plasma treated
595
films. Protein film Tg (ºC) Tm (ºC) ∆H (J/g) 138.30
80.67
Treatment 1
119.96
162.15
116.17
Treatment 6
114.13
142.66
119.98
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Tg: Glass transition temperature; Tm: Melting temperature; ∆H: Enthalpy.
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Control
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ACCEPTED MANUSCRIPT Fig. 1. Schematic diagram of the plasma system. (1) Plasma vacuum chamber, (2) Electrodes, (3) Film sample and plasma sheath, (4) HV probe, (5) Power supply (0 – 15 kV), (6) Dry air cylinder, (7) Shunt resistor (current measurement), (8) Vacuum gauge,
(12) Vacuum meter, (13) Hollow glass sample support.
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(9) Oscilloscope, (10) Mechanical vacuum pump, (11) Turbo molecular vacuum pump,
Fig. 2. Pareto chart of the (a) moisture content, (b) solubility in water and (c) water
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vapor permeability. The black line indicates the critical level above which the variables
Fig. 3. Effect of AC glow discharge plasma in the X-ray diffraction pattern of fish protein films.
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Fig. 4. DSC curves of control fish protein film and plasma treated films.
ACCEPTED MANUSCRIPT Cold plasma was used to decrease the sensitivity to water of fish protein films
•
The films were plasma treated in different settings of power, pressure and time
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Power and time of exposure were the most influent parameters in films
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properties
Decrease in water vapor permeability and solubility in water were observed
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Glow discharge plasma effects can be adjusted through the parameters of
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S0268-005X(18)32320-8
DOI:
https://doi.org/10.1016/j.foodhyd.2019.03.021
Reference:
FOOHYD 5000
To appear in:
Food Hydrocolloids
Received Date: 25 November 2018 Revised Date:
12 March 2019
Accepted Date: 12 March 2019
Please cite this article as: Romani, Viviane.Patrí., Olsen, B., Collares, M.P., Oliveira, J.R.M., Prentice, C., Martins, Vilá.Guimarã., Plasma technology as a tool to decrease the sensitivity to water of fish protein films for food packaging, Food Hydrocolloids (2019), doi: https://doi.org/10.1016/ j.foodhyd.2019.03.021. 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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Plasma technology as a tool to decrease the sensitivity to water of fish protein films
2
for food packaging
3
Viviane Patrícia Romania, Bradley Olsenb, Magno Pinto Collaresc, Juan Rodrigo
5
Meireles Oliveirac, Carlos Prenticea, Vilásia Guimarães Martinsa
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6 7
a
8
Grande do Sul 96203-900, Brazil
9
b
Department of Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139, United States
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c
12
Grande, Rio Grande do Sul 96203-900, Brazil
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Institute of Mathematics, Statistics and Physics, Federal University of Rio Grande, Rio
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ABSTRACT
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School of Chemistry and Food, Federal University of Rio Grande, Rio Grande, Rio
Proteins films have been developed for use in food packaging materials in order to
17
replace synthetic polymers because of environment pollution. Despite their advantages,
18
the use of protein films in a wide range of food products is still limited due to their
19
hydrophilic behavior. Thus, this study aimed to decrease the sensitivity of fish protein
20
films to water through the application of glow discharge plasma. Plasma parameters
21
(power, pressure and time of exposure) were studied according to a 23 factorial design,
22
and the physicochemical properties of the films including moisture content, water vapor
23
permeability, and solubility in water were evaluated. The microstructure and thermal
24
properties of the films were also characterized. In general, glow discharge plasma
25
caused different effects in films properties, such as cleaning and etching which were
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ACCEPTED MANUSCRIPT responsible for the changes observed in physicochemical properties. Power and time of
27
exposure, as well as their interaction, were the most influent parameters. Decrease in
28
water vapor permeability and solubility were observed in some treatments, which are
29
important characteristics for a material to be used as food packaging. Then, the plasma
30
setting might be changed through the adjustment of parameters of exposure according to
31
the specificity of the application intended.
32
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Keywords: Biodegradable materials, Cold plasma, Glow discharge, Myofibrillar
34
proteins, Sustainable films.
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1. Introduction
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Concerns about the environmental pollution and depletion of fossil
39
resources due to the wide use of petroleum-based plastics have led to growing interest
40
in polymers from sustainable and biodegradable resources (Félix, Lucio-Villegas,
41
Romero, & Guerrero, 2016). Biopolymers obtained from agro-based sources, including
42
proteins, polysaccharides and lipids, may be extracted from food industry
43
wastes/byproducts as well as from low cost raw materials. Proteins attract more
44
attention because of their structure, that has the capacity to form strong three-
45
dimensional networks (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2016; Rhim,
46
Park, & Ha, 2013). Animal proteins, such as myofibrillar proteins, are more promising
47
due to their capacity to form slightly transparent films with excellent UV-light barrier
48
properties in comparison to commercial wrap film of polyvinyl chloride, for example
49
(Kaewprachu, Osako, Benjakul, & Rawdkuen, 2016; Kaewprachu, Rungraeng, Osako,
50
& Rawdkuen, 2017).
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52
better than other macromolecules, mainly in terms of gases barrier and mechanical
53
properties and biodegradation (Wittaya, 2012). However, despite these advantages, the
54
hydrophilic nature of proteins still limits the commercial use of these materials for food
55
packaging because their performance is compromised when in contact with humidity
56
(Benbettaïeb, Gay, Karbowiak, & Debeaufort, 2016; Wihodo & Moraru, 2013). To be
57
used as packaging material, it is important that the film protects the food from the
58
external environment throughout the production process, during handling, transportation
59
and storage, until it is delivered to the consumer (Han, 2005). High solubility in water
60
and water vapor permeability of the protein films in comparison with synthetic plastics
61
limit their use in a wide range of foods, such as dairy and meat products. For example,
62
the passage of water through the packaging material is crucial because it is directly
63
linked to the mechanisms of food deterioration, including lipid oxidation and microbial
64
activity (Robertson, 2013). Therefore, it is of the utmost significance to find alternatives
65
to decrease the sensitivity of these materials to humidity.
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Several strategies have been reported to improve film performance, such as
67
the use of crosslinking agents, incorporation of reinforcing substances, blending with
68
different materials and others. The use of environmentally friendly processes is
69
important to achieve the required properties for the polymers without the use of
70
additional components and waste generation. Cold plasma is an example of a dry
71
process suitable for heat-sensitive materials that does not pollute the environment. It is a
72
non-thermal technology, which consists of a gas mixture containing ions, radicals, free
73
electrons and neutral species, with the capacity to start chemical reactions on the surface
74
of materials and consequently changing their properties (Chu, 2002; Geyter & Morent,
75
2012; Mahmoud, 2016; Pankaj et al., 2014a). The application of cold plasma in fish
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ACCEPTED MANUSCRIPT proteins films for food packaging has been studied previously by our group to
77
understand the effects of treatment time on their physicochemical properties, including
78
mechanical performance and optical properties (Romani et al., 2019). Here, the effects
79
of operating parameters (pressure, power and time) of alternating current (AC) glow
80
discharge plasma on protein films from fish are reported. They were studied aiming the
81
improvement of films properties in terms of decreasing the sensitivity to water and
82
water permeability.
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2. Material and Methods
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2.1 Raw material
87
Whitemouth croaker (Micropogonias furnieri) muscle was used to obtain
89
the protein isolates. The fish was obtained from processing industries in Rio Grande
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(RS), Brazil. The muscle was crushed and stored frozen at −18 °C until protein
91
extraction.
94 95
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2.2 Obtaining the fish protein isolate
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Fish protein isolate (FPI) was obtained based on the method described by
96
Nolsøe & Undeland (2009) through the pH-shifting process. This process consists in an
97
alkaline solubilization followed by isoelectric precipitation of proteins. The fish muscle
98
was homogenized with distilled water (ratio 5:1, v/w) to perform the alkaline
99
solubilization at pH 12 for 20 min (4 °C). Then, a centrifugation was performed at
100
7500×g for 15 min to separate the middle phase (soluble proteins) from lipids and
ACCEPTED MANUSCRIPT insoluble matter. Soluble proteins were titrated to pH 5.5 until precipitation at the
102
isoelectric point; after 20 min the suspension was centrifuged at 7500×g for 15 min to
103
get the pellet that was then dried and grounded. The separation process generated
104
protein with 84.5% of purity determined according to the Association of Official
105
Analytical Chemists (AOAC, 2000) method.
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2.3 Film preparation
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Fish protein films were prepared using 3% (w/v) of FPI through the casting
110
technique as the method used by Romani, Prentice-Hernández, & Martins (2017). The
111
pH of the mixture containing distilled water, proteins and 30% (w/w) of plasticizer
112
(glycerol) was adjusted to 11.0. This mixture was heated to 80 ºC and held for 20 min in
113
order to denature proteins. After cooling, it was cast in Petri dishes and left to dry at 40
114
ºC in an oven with air circulation (Biopar, S150BA). After drying, the protein films
115
were left in desiccators (50% relative humidity – RH controlled with a saturated sodium
116
bromide (NaBr) solution) for 24 h before plasma treatment.
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2.4 Glow discharge plasma treatment
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The films were treated using an AC glow discharge plasma (Fig. 1). The
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film samples (3) were placed inside the plasma chamber (1) in a sample support (13)
122
and the treatment was performed between two electrodes (2) with an inter-electrode
123
distance of 13 cm. Initially, the plasma chamber was evacuated to 0.15 Pa and then dry
124
air was filled up to the process procedure. After pressure stabilization, the glow
125
discharge plasma was generated by application of AC power. One of the electrodes was
ACCEPTED MANUSCRIPT connected to the ground and high voltage input of 4.4 kV (60 Hz) was applied to the
127
electrodes. The effect of different sets of plasma treatment conditions was studied
128
according to a 23 factorial design with three central points. The variables evaluated were
129
power, pressure and time. The coded and real values of variables are presented in Table
130
1.
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131 132
2.5 Film evaluation
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2.5.1
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Moisture content
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Moisture content was determined by the weight loss of the films after being
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subjected to a temperature of 105 ± 2 °C for 24 h in an oven (DeLeo, A15E) according
138
to Antoniou, Liu, Majeed, & Zhong (2015). The moisture (%) was calculated
139
considering the dry matter content of the film, which was obtained by the difference
140
between the initial and final weight.
141
2.5.2
143
Film thickness (mm) was obtained using a digital micrometer (IP65, Insize)
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Thickness
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with precision of 0.001 mm. The measurements were made in ten different locations of
146
the samples and the average was determined.
147 148 149
2.5.3
Solubility in water
ACCEPTED MANUSCRIPT Solubility in water (SW) of the films was determined according to the
151
method described by Gontard, Duchez, Cuq, & Guilbert (1994) with some
152
modifications. Film discs of 2 cm in diameter were dried in an oven (DeLeo, A15E) at
153
105 ± 2 °C to determine the initial dry weight. Then, the samples were immersed in 50
154
mL of distilled water and continuously shaken (200 rpm) at 25 ºC for 24 h. After
155
immersion, the samples were dried again to determine final dry weight. The weight of
156
the soluble portion was determined considering the weight of the insoluble matter and
157
the initial weight of the film and expressed as film solubility (%) according to Equation
158
1.
159 160
SW (%) =
(Wo - W) Wo
× 100
161
Water vapor permeability
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2.5.4
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dry weight after the immersion.
164 165
(1)
Where W0 is the dry weight of film before immersion in water and W is the
162 163
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The water vapor permeability (WVP) was determined according to the
168
ASTM Standard Method E96-00 (ASTM, 2000). The test was performed using
169
permeation cells containing silica as desiccant inside. The films were sealed on the cells
170
and placed in desiccators containing distilled water. Weight measurements were
171
evaluated at 24 h intervals during 7 days at 25 ºC. The WVP (g.mm/m².day.kPa) was
172
obtained according to Equation 2 considering the weight gain, the thickness, the area of
173
the films, the time and the vapor pressure difference:
174
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WVP (g.mm/m².day.kPa) =
W×L
(2)
A × t × ∆P
176
Where W is the weight gain of the cell (g); L is the film thickness (mm); A is
178
the exposed area of film (m²); t is the time of weight gain (day); and ∆P is the vapor
179
pressure difference across the film (kPa).
180 181
2.5.5
Microstructure
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The effect of AC glow discharge in morphology of films surface was
184
observed through Scanning Electron Microscopy (SEM). Stubs were used to fix the film
185
samples using double sided adhesive tape. The samples were coated with gold and
186
images were obtained in a microscope (Jeol, JSM-6060) at a magnification of 1000×.
187
Energy-dispersive X-ray spectroscopy (EDX) was conducted under 10 kV incident
188
electron energy to identify changes in concentration of elements of films surface.
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The films were also evaluated through wide-angle X-ray diffraction
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(WAXS) using a Bruker, D8 Advance Diffractometer operating at a voltage of 40 kV
191
and current of 30 mA, at incident angle of 2Ɵ, between 10 to 50º.
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193 194 195
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2.5.6
Thermal characterization
Thermal modifications caused by AC glow discharge plasma in films were
196
studied by differential scanning calorimetry (DSC) using a calorimeter (Shimadzu
197
TGA-60). Film samples (3 mg ± 0.01) were sealed in aluminum pans and scanned at a
198
heating rate of 10 ºC/min over a range of 30 to 200 ºC using a nitrogen flow rate of 50
ACCEPTED MANUSCRIPT 199
mL/min. The enthalpy values (∆H), glass transition (Tg) and melting (Tm) temperatures
200
were obtained from the endothermic peaks present in the thermogram.
201 202
3. Results and Discussion
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3.1 Effects of plasma treatment on moisture, solubility in water and water vapor
205
permeability of the films
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The effects of the operating parameters power, pressure and time of AC
208
glow discharge in fish protein films were studied specifically in the properties related to
209
water, which limit the use of these materials in foods with high water activity. Table 2
210
presents the results obtained for moisture content, solubility in water and water vapor
211
permeability under the different conditions, and Fig. 2 shows the effects of the variables
212
studied as well as their interactions on the properties evaluated.
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The moisture content of the films provides information about the way in
214
which the studied variables affect affinity to water (Capitani et al., 2016). It is possible
215
to observe from Fig. 2 that the time of plasma treatment is the most influent parameter
216
in decreasing moisture content of films while the interaction effect between time and
217
power led to the moisture increase.
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Solubility is defined as the affinity of a material for a substance,
219
representing an important characteristic to be considered when studying packaging
220
materials for foods (Han & Scanlon, 2005). The solubility in water (SW) of the films
221
varied in the range between 29.9 and 45.6% (Table 2), which represent medium SW
222
compared to other studies using biomacromolecules from agro-sources, such as chia
223
protein and mucilage (58.7 to 80.8%) (Capitani et al., 2016), sunflower protein (86.3 to
ACCEPTED MANUSCRIPT 93.2%) (Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2013) and
225
chicken skin gelatin incorporated with rice flour (82.9 to 93.7%) (Soo & Sarbon, 2018).
226
The values obtained for SW in the present study are attributed to the hydrophilic nature
227
of myofibrillar proteins from fish and the plasticizer used (glycerol). The interaction
228
effect between the variables power and time influenced solubility of films causing its
229
decrease, which has significant importance mainly for use in foods with high humidity.
230
Potential applications of these films are governed by the solubility, for example meat
231
and dairy products require water resistance due to the high-water activity of these foods.
232
In contrast, high sensitivity to water is a functional advantage in the development of
233
soluble sachets, such as edible films/coatings for pre-measured portions to be dissolved
234
in water or hot food (Guilbert & Gontard, 2005; Yang, Paulson, & Nickerson, 2010).
235
Besides that, high solubility in films represents an advantage in the view of
236
biodegradability, however their capacity to protect dry foods from moisture of the
237
environment as well as to prevent the water loss of some products is impaired (Stuchell
238
& Krochta, 1994). The lowest solubility in water was observed for treatment T6,
239
indicating the reduced affinity to water. This result obtained with the lowest power and
240
time under study suggested the decrease of moisture content in the film probably
241
without other pronounced effects such as etching and/or degradation of the matrix.
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The water vapor permeability (WVP) is another important characteristic to
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243
be considered in the use of a material to develop food packaging (Han & Scanlon,
244
2005). Some foods, such as fruits and vegetables require reduced barrier to water vapor
245
due to the respiration process, while snacks and biscuits, for example, need high barrier
246
to maintain crunchiness. In the present study, the variables power and time, the
247
interaction between power and time variables as well as the interaction between
248
pressure and time of AC glow discharge plasma influenced the WVP of fish protein
ACCEPTED MANUSCRIPT films (Fig. 2). It is worth to note that the power had a positive effect, increasing WVP
250
when higher power was used, in contrast to the other effects observed. The time of
251
plasma treatment had the capacity to reduce the water vapor permeability, especially in
252
the interaction with higher power and pressure. This is a positive trend mainly for the
253
use of films in contact with food which low permeability is required. This behavior
254
indicates that a considerable decrease of moisture in films surface might have occurred,
255
as well as suggests the exposure of hydrophobic sites of amino acids in the protein
256
sequence.
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The effects observed in the present study show that AC glow discharge is
258
promising to decrease the sensitivity to water of fish protein films due to the lower
259
solubility and water vapor permeability obtained in treatments T1 and T6, respectively.
260
Different changes observed among the treatments with cold plasma might be explained
261
by the different reactions that occur during the plasma treatment, such as cleaning,
262
functionalization, crosslinking, etching and degradation. According to Poncin-Epaillard,
263
Brosse, & Falher (1997), these reactions are governed by the reactive species generated
264
by plasma and the energy absorbed by the surface of the material. Arolkar, Salgo,
265
Kelkar-Mane, & Deshmukh (2015) reported that plasma leads to different effects during
266
the treatment process; initially there is a cleaning of the surface removing low molecular
267
weight fragments which are weakly linked and after this initial step the reactive species
268
can cause etching or degradation processes as well as crosslinking of polymer chains. In
269
protein films, these reactions are result of OH radicals alongside other reactive species
270
formation in the cold plasma treatment. These species might cleave peptide and
271
disulfide bonds, and oxidize amino acid chains (Mirmoghtadaie, Shojaee Aliabadi, &
272
Hosseini, 2016).
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ACCEPTED MANUSCRIPT Lacroix & Cooksey (2005) reported that potential crosslinking in the film
274
surface, by the increase in cohesion between polypeptide chains of the myofibrillar
275
proteins, was effective in decreasing affinity to water, as might have contributed to
276
some extent in the changes of some treatments in the present study. Other authors
277
reported changes in hydrophilic properties of polymers after plasma treatment, such as
278
Tenn et al. (2012), who observed a 28% decrease in water vapor permeability of
279
ethylene vinyl alcohol (EVOH) after plasma application. According to Oh, Roh, & Min
280
(2016), plasma treatment might induce modifications in films through the formation of
281
oxygen and nitrogen groups on surface when carbon radicals are formed on polymer
282
chains. This radical formation on the film surface lead to changes in its properties
283
through the effects mentioned previously. As explained by Morent et al. (2010),
284
crosslinking limits the mobility of polymer chains and the fraction of mobile groups
285
affecting polymer properties. But despite that, the exposure of hydrophobic side chains
286
due to bond scission of proteins might be responsible for the effects observed in the
287
films of the present study as well as the moisture loss of the films surface due to the
288
contribution of vacuum during plasma treatment.
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Cold plasmas are constituted of charged particles and excited/non-excited
290
molecules (Misra, Tiwari, Raghavarao, & Cullen, 2011). Then, the role of power,
291
meaning the applied voltage level, in affecting the properties of films is a result of the
292
density of these particles produced in the plasma. According to Whitehead (2016),
293
electrons are accelerated in an electric field through the gaseous medium for generation
294
of plasma and a wide range of processes might occur when an electron collides with
295
atoms or molecules. With higher power, more changes are expected in the material
296
placed in the plasma chamber because more collisions and reactions occur. The time of
297
plasma treatment is also extremely important because the longer the treatment, more
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reactions happen, as described previously the cleaning step followed by effects such as
299
etching and degradation. In addition, the low pressure in the system of glow discharge
300
plasma has an important function also due to the vacuum that might contribute for the
301
moisture loss of films structure. In the factorial design performed in the present study, the treatments T1 and
303
T6 showed lower water vapor permeability and solubility in water, respectively, that
304
was the aim of the present work. Therefore, the parameters used in these treatments
305
were defined as the optimal conditions. Then, in order to better understand the effects of
306
AC glow discharge, the films treated according to these parameters were further
307
characterized by their microstructure and thermal properties.
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308 309
3.4 Microstructure of AC glow discharge plasma treated films
310
X-ray diffraction analysis (Fig. 3) showed predominantly amorphous
312
structures which are characteristic of myofibrillar protein films. Changes were observed
313
mainly in two diffraction peaks, centered at 2θ = 13º and 2θ = 19º, which according to
314
the Bragg’s law correspond to distances of 6.85 Å and 4.73 Å, respectively. The smaller
315
distance observed corresponds to the average backbone distance within the α-helix and
316
larger distances are result of the α-helix packing or the distance among the near helices,
317
as mentioned by Pankaj, Bueno-Ferrer, Misra, Bourke, & Cullen (2014). The
318
application of AC glow discharge according to the treatments T1 and T6 led to small
319
changes in the position of diffraction peaks and major changes in the peak intensities.
320
For treatment T1, the peak centered at 19º presented a larger decrease in intensity,
321
suggesting disruption of α-helix packing, which might have led to exposure of
322
hydrophobic side chains consequently decreasing WVP. Regarding the film of treatment
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ACCEPTED MANUSCRIPT T6 (picture shown in Fig. S1), a similar decrease in both peaks was observed, that might
324
indicate changes in molecular aggregates of helices as well as in the α-helix structure,
325
reflecting in the changes of physicochemical properties studied. Other authors (Zhang et
326
al., 2015) reported changes in secondary structures of proteins as a result of plasma
327
treatment (for example in the amount of α-helix, β-sheet regions, as well as random
328
coils), in agreement to the trends observed in XRD patterns of the present study.
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The effects of AC glow discharge plasma on the surface morphology of fish
330
protein films were also evaluated using Scanning Electron Microscopy (images shown
331
in Fig. S2). The surfaces of the films were predominantly smooth and homogeneous,
332
and the plasma treatment caused a slightly increase in roughness of the surface with
333
small protrusions due to the bombardment of reactive species in both treatments T1 and
334
T6, however more pronounced in treatment T1. Other authors report that a rougher
335
surface is result of the etching effect, that occurs due to physical and chemical
336
processes, including chain scission, breakage of bonds, removal of fragments and
337
degradation (Akishev et al., 2008; Mirabedini, Arabi, Salem, & Asiaban, 2007).
338
Different studies of plasma treatment in films from biomacromolecules also reported
339
increases in roughness of materials surface. Some examples include Pankaj et al. (2015)
340
using gelatin films for plasma application, Chang & Chian (2013) evaluating surface
341
modification effects on chitosan film biodegradability and protein adsorption and Song,
342
Oh, Roh, Kim, & Min (2016) using plasma for improvement of polylactic acid films
343
properties for food packaging.
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344
The study of the concentration of surface elements by EDX showed higher
345
differences among treated and untreated samples compared to the differences between
346
the plasma treatments (Table 3). These changes might indicate a disruption of surface
347
molecular structure of films due to chain scission as well as etching effects, as also
ACCEPTED MANUSCRIPT reported by Hwang et al. (2003). As in the present study, Pankaj et al. (2015) observed
349
increase in surface oxygenation after cold plasma treatment in gelatin films. However,
350
these authors reported that this behavior was more dependent on the treatment time. A
351
similar trend was found by Ramkumar et al. (2018) in low density polyethylene (LDPE)
352
film surfaces with various gaseous plasma treatments.
353 354
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3.5 Effect of AC glow discharge plasma in thermal properties of fish protein films
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355
Thermal properties from DSC of control and plasma treated films showed
357
endothermic peaks (Fig. 5). From DSC curves, it was observed that AC glow discharge
358
caused more pronounced effects in the films properties of T1. The properties of glass
359
transition temperature (Tg), melting temperature (Tm) and enthalpy (∆H) of fish protein
360
films, as affected by glow discharge plasma, are presented in Table 4. Results observed
361
by DSC analysis in general agree with the physicochemical performance obtained in the
362
present study. AC glow discharge plasma was responsible for increasing Tg, Tm and ∆H,
363
suggesting changes in chains mobility in the film matrix, as suggested previously due to
364
the differences in water vapor passage through the film materials. As mentioned by
365
Pankaj et al. (2014b), air plasma might generate etching effects on the materials surface,
366
as in some treatments of the present factorial design; however, modifications in thermal
367
properties also confirm that some moisture loss might occur in the film surface. It was
368
responsible for modifications in Tg, Tm and ∆H because water acts as plasticizer
369
increasing chains mobility, that was more pronounced mainly in T1.
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Some authors mentioned that cold plasma forms radicals on the surface of
371
polymer films, which form crosslinked networks limiting the permeability of films due
372
to the reduction in the network free volume. Other studies indicate that cold plasma
ACCEPTED MANUSCRIPT affects the solubility of polymers, but not the diffusivity of water molecules because the
374
bulk properties of the material remain unchanged (Oh, Roh, & Min, 2016; Perez-Gago
375
& Krochta, 2001; Stutz, Illers, & Mertes, 1990). From the results of the
376
physicochemical properties in the present study, which were supported by
377
microstructure and thermal characteristics, it can be inferred that changes in fish protein
378
films depend on the plasma setting and the time of treatment because during the
379
exposure of the material different reactions occur inside the plasma chamber.
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4. Conclusion
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The potential of AC glow discharge plasma to improve the performance of
384
fish protein films was demonstrated in the present study. The plasma setting based on
385
the factorial design performed (power, pressure and time of exposure) led to different
386
results for affinities with water (measured by water vapor permeability, solubility in
387
water and moisture content). These properties are important when using the material to
388
develop food packaging, then the effects observed are an advance for industrial
389
applications. As supported by microstructure and thermal analysis, the different
390
influences of AC glow discharge caused by the variation of the parameters studied
391
occur due to the different reactions that plasma causes, such as cleaning and etching.
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This study showed that power and time were the variables that had stronger
393
effects in the properties of fish protein films. Therefore, variation in the water vapor
394
permeability and solubility in water might be adjusted according to the application
395
required through the parameters of exposure in glow discharge plasma. Beyond that, the
396
modification of surface characteristics caused by plasma is advantageous for other
397
aspects in designing new materials for food packaging, such as the development of
ACCEPTED MANUSCRIPT 398
multilayer films or incorporation of active substances in the surface to extend shelf life
399
of food products. Overall, the use of AC glow discharge plasma to shape characteristics
400
of fish protein films is advantageous because it is an environmentally friendly
401
technology being used in a sustainable and non-polluting raw material.
403
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Acknowledgments
404
The authors are grateful for the financial support by the Coordination for the
406
Improvement of Higher Education Personnel (CAPES) and National Council of
407
Research (CNPq) of Brazil. Also, this work was supported by the Serrapilheira Institute
408
(grant number Serra - 1709-20275). The authors also acknowledge CEME-SUL/FURG
409
(Centro de Microscopia Eletrônica do Sul/Universidade Federal do Rio Grande/Brazil)
410
for SEM and XRD analysis and MIT-Brazil Program.
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References
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416 417 418
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AOAC, (2000). Official Methods of Analysis (17th ed.). Washington, DC: Association of Official Analytical Chemists. ASTM, (2000). Standard test methods for water vapor transmission of materials - E96-
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Akishev, Y., Grushin, M., Dyatko, N., Kochetov, I., Napartovich, A., Trushkin, N.,
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Duc, T. M., Descours, S. (2008). Studies on cold plasma–polymer surface
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Antoniou, J., Liu, F., Majeed, H., & Zhong, F. (2015). Characterization of tara gum
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air-plasma treatment on corn starch/poly(ε-caprolactone) films. Polymer
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https://doi.org/10.1016/j.polymdegradstab.2015.07.016
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Benbettaïeb, N., Gay, J. P., Karbowiak, T., & Debeaufort, F. (2016). Tuning the Functional Properties of Polysaccharide–Protein Bio-Based Edible Films by
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Chemical, Enzymatic, and Physical Cross-Linking. Comprehensive Reviews in
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Benbettaïeb, N., Karbowiak, T., Brachais, C. H., & Debeaufort, F. (2016). Impact of
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electron beam irradiation on fish gelatin film properties. Food Chemistry, 195, 11–
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ACCEPTED MANUSCRIPT Table 1. Coded and real values of variables for plasma application in protein films. -1
0
+1
Power
5W
8W
11 W
Pressure
4 Pa
7 Pa
10 Pa
Time
1 min 3 min 5 min
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Table 2. Factorial design of plasma treatment and results for moisture, solubility and
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permeability.
Control
Solubility in
content (%)
water (%)
83.7 ± 1.3
38.4 ± 0.2
5.19 ± 0.29
permeability (g.mm/day.m².kPa)
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Treatment Power Pressure Time
Water vapor
Moisture
1
1
1
86.6 ± 2.5
36.9 ± 0.8
5.08 ± 0.03
T2
1
1
-1
83.3 ± 0.4
40.1 ± 0.1
6.23 ± 0.05
T3
1
-1
1
84.3 ± 0.6
37.8 ± 1.9
5.74 ± 0.04
T4
1
-1
-1
83.9 ± 1.4
45.6 ± 2.7
6.51 ± 0.12
T5
-1
1
1
81.0 ± 0.8
40.3 ± 0.6
6.07 ± 0.04
T6
-1
1
-1
84.8 ± 1.2
29.9 ± 3.5
5.81 ± 0.25
T7
-1
-1
1
T8
-1
-1
-1
T9
0
0
0
T10
0
0
0
T11
0
0
0
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43.7 ± 3.9
5.28 ± 0.14
84.2 ± 0.8
39.5 ± 1.1
5.36 ± 0.28
83.0 ± 0.9
42.2 ± 3.6
5.56 ± 0.24
83.3 ± 0.8
41.4 ± 0.5
5.45 ± 0.31
83.7 ± 1.4
44.6 ± 2.4
5.59 ± 0.08
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Table 3. Concentration of elements of control fish protein films and plasma treated
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films. Weight (%) Protein film C
N
O
30.2 ± 0.92 13.8 ± 5.29 45.1 ± 2.37
Treatment 1
28.2 ± 0.73 11.4 ± 4.33 53.2 ± 1.96
Treatment 6
26.9 ± 0.76 11.8 ± 4.43 53.6 ± 1.98 Mean ± standard error.
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Table 4. DSC thermal characterization of control fish protein film and plasma treated
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films. Protein film Tg (ºC) Tm (ºC) ∆H (J/g) 138.30
80.67
Treatment 1
119.96
162.15
116.17
Treatment 6
114.13
142.66
119.98
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Tg: Glass transition temperature; Tm: Melting temperature; ∆H: Enthalpy.
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ACCEPTED MANUSCRIPT Fig. 1. Schematic diagram of the plasma system. (1) Plasma vacuum chamber, (2) Electrodes, (3) Film sample and plasma sheath, (4) HV probe, (5) Power supply (0 – 15 kV), (6) Dry air cylinder, (7) Shunt resistor (current measurement), (8) Vacuum gauge,
(12) Vacuum meter, (13) Hollow glass sample support.
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(9) Oscilloscope, (10) Mechanical vacuum pump, (11) Turbo molecular vacuum pump,
Fig. 2. Pareto chart of the (a) moisture content, (b) solubility in water and (c) water
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Fig. 3. Effect of AC glow discharge plasma in the X-ray diffraction pattern of fish protein films.
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Fig. 4. DSC curves of control fish protein film and plasma treated films.
ACCEPTED MANUSCRIPT Cold plasma was used to decrease the sensitivity to water of fish protein films
•
The films were plasma treated in different settings of power, pressure and time
•
Power and time of exposure were the most influent parameters in films
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Decrease in water vapor permeability and solubility in water were observed
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