Food Hydrocolloids 113 (2021) 106471

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Food Hydrocolloids
journal homepage: http://www.elsevier.com/locate/foodhyd

Physicochemical and microstructural properties of composite edible film

obtained by complex coacervation between chitosan and whey
protein isolate
Loleny Tavares a, b, Hiléia K.S. Souza a, 1, **, Maria Pilar Gonçalves a, Cristina M.R. Rocha a, c, *
a
REQUIMTE-LAQV, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal
b
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, No. 9500, CEP 91501970, Porto Alegre, RS, Brazil
c
CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of this work was to study the potential application of edible films obtained by complex coacervation
Whey protein isolate promoted by the electrostatic interactions between positively charged chitosan (CH) 3% (w/w) and negatively
Chitosan charged whey protein isolate (WPI) 3% (w/w). Preliminary assays of turbidimetry were made in order to find the
Edible films
optimal CH-to-WPI mass ratio for the complex coacervation. The maximum turbidity was obtained in the CH:WPI
Complex coacervation
mass ratio of 0.1:1 (w/w). The dispersions of CH/WPI (both at 3% (w/w)), WPI 5% (w/w) and CH 3% (w/w)
were analyzed by Cryo-scanning electron microscopy (Cryo-SEM) and the micrograph of CH/WPI coacervate
presented a more compact network structure than dispersions of individual biopolymers. The composite CH/WPI
films were prepared, characterized and their performance and physicochemical properties were compared with
those of CH or WPI films, in terms of water vapor permeability (WVP), mechanical properties, solubility, sorption
isotherms, optical properties, scanning electron microscopy (SEM) imaging, Fourier-transform infrared spec­
troscopy (FTIR) and differential scanning calorimetry (DSC). Results revealed that the incorporation of small
amounts of CH in the matrix of WPI led to form a composite film with higher tensile strength, lower deformation,
malleability, flexibility, solubility and WVP in comparison to the mono-component WPI and CH films. The CH
incorporation resulted in a decrease in equilibrium moisture content of the CH/WPI film and the Guggenheim-
Anderson-de Boer (GAB) model of sorption data indicated isotherms of type II. All the films presented a ho­
mogeneous structure, color transparency, which is desired in food applications and packaging technology.

1. Introduction (Alizadeh-Sani, Ehsani, Moghaddas Kia, & Khezerlou, 2019; Cazón,

Velazquez, Ramírez, & Vázquez, 2017). Though the final aim is not to
The low plastics rate of degradation and their consequent accumu­ fully replace traditional non-biodegradable plastics, as there are still
lation in the environment has given rise to serious ecological problems many hurdles to be overcome, the use of edible films with friendly
and many debates in recent times (Lippolis et al., 2017; Luyt & Malik, polymers may help to lowering the environmental pollution and
2019). The global annual production of plastics is expected to reach 330 simultaneously increase the shelf-life and quality of food product
million tonnes in 2020, and approximately 20% of this production is (Hosseini e Gómez-Guillén, 2018). The biopolymers used in the formu­
used in packaging manufacture in the food industry due to the plastics lation of edible films are by-products derived from renewable agricul­
durability, design flexibility, lightness and low price (Ferreira, Alves, & tural resources or industrial by-products (Salgado, Ortiz, Musso, Di
Coelhoso, 2016; Hosseini & Gómez-Guillén, 2018). Edible films or Giorgio, & Mauri, 2015). Edible films are classified into three categories:
coatings have been studied as potential alternatives for plastic pack­ a) hydrocolloids that include proteins (whey protein, casein, gelatin,
aging due to their biodegradability, renewable and non-toxic properties wheat gluten, soy protein, and so on) and polysaccharides (alginates,

* Corresponding author. CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, 4710-057, Portugal.
** Corresponding author. REQUIMTE-LAQV, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, Porto, 4200-465, Portugal.
E-mail addresses: hileia.souza@fc.up.pt (H.K.S. Souza), cmrochainv@gmail.com (C.M.R. Rocha).
1
REQUIMTE-LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007, Porto,
Portugal.

https://doi.org/10.1016/j.foodhyd.2020.106471
Received 17 June 2020; Received in revised form 21 October 2020; Accepted 9 November 2020
Available online 11 November 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

cellulose and derivatives, starch, chitosan, among others) (Kaewprachu study were: a) to optimize the conditions to promote the complex
& Rawdkuen, 2016); b) lipids (waxes, acylglycerols, and fatty acids) coacervation between positively charged chitosan (CH) and negatively
(Dehghani, Hosseini, & Regenstein, 2018); and c) composites (mixtures charged whey protein isolate (WPI); and b) to study the influence of the
containing two or more components in order to produce films with mechanism of complex coacervation on the physicochemical properties
improved physicochemical properties) (Salgado et al., 2015). of the CH/WPI film when compared to the properties of the
Biodegradable films are thin layers of edible biodegradable polymers mono-component CH and WPI films. For comparison purposes, film
that can be applied to the food stuff (e.g food packaging), to reduce the formation with thermally induced denaturation of whey proteins was
rate of oxygen absorption, migration of lipids, gasses and aromas, and to also considered, as it is the most common method used in literature for
prevent the development of microorganisms during storage (Cazón WPI-based films.
et al., 2017; Tharanathan, 2003). Edible films with functional agents
such as antimicrobials, antioxidants and enzymes have also been used to 2. Material and methods
improve the shelf life and quality of the food products (Tavassoli-Ka­
frani, Shekarchizadeh, & Masoudpour-Behabadi, 2016). 2.1. Materials and reagents
According to literature, edible films can be formed by three mecha­
nisms: simple coacervation, complex coacervation and gelation or A commercial whey protein isolate (WPI, LACTOPRODAN DI-9224)
thermal coagulation (Kester & Fennema, 1986). The complex coacer­ was purchased from Arla Foods Ingredients Ambh (Denmark). Chitosan
vation results from electrostatic interactions between oppositely (CH) with a degree of deacetylation (DD) of 92% was obtained from
charged polysaccharides and proteins, leading to the formation of one Primex (Siglufjordur, Iceland). Sodium acetate trihydrate (CH3COO­
phase enriched in both polymers and another enriched in solvent Na⋅3H2O) (99.5%) and glacial acetic acid (CH3COOH) were purchased
(Rocha, Souza, Magalhães, Andrade, & Gonçalves, 2014). The complex from Merck. Purified water, produced by a Milli-Q filtration system, was
formation is influenced by different physicochemical parameters, such used for the preparation of all solutions.
as pH, total biopolymers concentration, ionic strength of medium, bio­
polymers mixing ratio, temperature, and agitation time (Schmitt & 2.2. Solutions preparation
Turgeon, 2011).
Chitosan (CH) is a natural polycationic polysaccharide obtained from The acetate buffer solutions were prepared by dissolving appropriate
the alkaline deacetylation of chitin at high temperatures (above 80 ◦ C) amounts of CH3COOH and CH3COONa in water and keeping the ionic
(Muxika, Etxabide, Uranga, Guerrero, & de la Caba, 2017). CH is strength equal to 0.100 mol.L− 1. The CH (0.1 and 3% (w/w)) solutions
composed of β-(1–4)-linked D-glucosamine and N-acetyl-d-glucosamine were prepared by dissolving weighted amounts of the respective solid
randomly distributed (Tavares & Noreña, 2019). The unique properties sample in acetate buffer at pH 5.5, whereas the WPI (0.1 and 3% (w/w))
of CH, such as biodegradability, non-toxicity, biocompatibility and solutions were prepared at pH 6.0. The final pH was adjusted with
anti-fungal properties make it suitable for edible film formulations and CH3COOH or NaOH, when needed. The CH solutions were centrifuged at
other different applications (Mujtaba et al., 2019; Tavares & Noreña, 13000 rpm for 15 min in order to remove impurities. CH and WPI so­
2019). CH also stands out of others biopolymers for presenting positive lutions were stirred for 4 h and subsequently stored in the refrigerator
charge at biological pH, while most of the polysaccharides are nega­ for 24 h until complete solubilization.
tively charged under the same conditions, which allows complex coac­
ervation by electrostatic interactions with a wide range of proteins 2.3. Coacervation conditions (preliminary assays)
(Tavares, Barros, Vaghetti, & Noreña, 2019; Tavares & Noreña, 2019).
Whey protein isolate (WPI) is a globular protein mixture obtained 2.3.1. Turbidity measurements
from cheese and casein manufacture, with a high content of hydro­ Turbidity measurements were performed according to the method
phobic, sulfhydryl groups inside the native globular structure and high described by Bastos et al. (2010), with minor modifications. Aliquots
proportion of surface hydrophilic groups (Zhang et al., 2016). WPI is (2–200 μL) of CH 0.1% (w/w) solution at pH 5.5 were added stepwise to
used as a food ingredient due to its nutritional value, gelation capacity, 0.9 mL of WPI 0.1% (w/w) solution at pH 6.0. The turbidity (or optical
and viscosity (Livney, 2010; Tavares et al., 2019). The films obtained dispersion, O.D.) of CH/WPI solutions was measured using an AGILENT
from WPI are characterized by transparency, flexibility, lack of odor and 8453 UV–visible spectrophotometer, at a wavelength of 400 nm. The
taste, favoring its acceptance by consumers (Bourtoom, 2008; Ramos influence of CH:WPI ratio (% weight (w/w)) was determined in
et al., 2013). triplicate.
As mentioned before, composites films have been used to deliver
complementary functional properties to the films over the individual 2.3.2. Cryo-scanning electron microscopy (Cryo-SEM)
biopolymer properties. For instance, the CH/WPI system was studied by The observation and analysis of the microstructure of CH/WPI (both
Ferreira, Nunes, Delgadillo, and Silva (2009). The authors used condi­ at 3% (w/w)), WPI 5% (w/w) and CH 3% (w/w) solutions were per­
tions where both biopolymers were positively charged. Di Pierro et al. formed at CEMUP, Porto, Portugal as previously described by Rocha
(2006) studied films using dispersions of chitosan-whey protein at pH et al. (2014). The solutions of biopolymers were frozen in slushy nitro­
5.0. However, the pH value was near the isoelectric point, pI, of WPI gen (− 210 ◦ C) and then transferred to an ALTO 2500 Cryo preparation
(4.5–5.5) and the global charge of WPI was neutral, not allowing sig­ chamber at − 150 ◦ C. The resulting frozen samples were fractured and
nificant electrostatic interactions between CH and WPI. Jiang et al. the ice formed was removed by sublimation at − 90 ◦ C for 1.5 min. The
(2016) used chemically modified CH and crosslinking to improve the samples were coated with a sputtered Au–Pd thin film at − 150 ◦ C for 40
properties of the composite CH/WPI film. The mixture of CH and WPI s, from a sputter head using ultrapure Argon gas. Analyses were per­
has been studied in other applications, such as encapsulation and edible formed at − 150 ◦ C in a JEOL JSM 6301F scanning electron microscope
film formulations (Bastos, Gonçalves, Andrade, Araújo, & Rocha Leão, (Japan) equipped with a Gatan ALTO 2500 Cryo preparation chamber
2012; Di Pierro et al., 2006; Di Pierro, Sorrentino, Mariniello, Giosa­ using an accelerating voltage of 15 kV and working distances of 15 mm.
fatto, & Porta, 2011; Ferreira, Nunes, Delgadillo, & Lopes-da-Silva,
2009; Kurek, Galus, & Debeaufort, 2014; Souza, Bai, Gonçalves, & 2.3.3. Preparation of CH, WPI and CH/WPI films
Bastos, 2009; bib_Tavares_et_al_2019Tavares et al., 2019; Tavares & CH films were prepared using the CH 3% (w/w) solution. WPI films
Noreña, 2019, 2021). However, to our knowledge, the use for film were prepared using the WPI solution prepared at 5% (w/w) instead of
production of CH/WPI mixtures obtained by the mechanism of complex 3% (w/w), since the films obtained from WPI 3% (w/w) appeared
coacervation was not reported. Therefore, the main objectives of this shrunk, curled and were difficult to remove from the Petri dish, thus

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

hampering their characterization. WPI 5% (w/w) film forming solutions 2.4.4. Solubility in water
were used to produce films at pH 6 (WPI-6) and pH 7 (WPI-7). For WPI- The water solubility of the films was tested according to a similar
6, the film-forming solution was prepared without any heat treatment. procedure elaborated by Rhim, Gennadios, Weller, Carole, and Hanna
Nevertheless, for WPI-7, the films were obtained by a gelation mecha­ (1998). The samples were cut into 2.5 × 2.5 cm strips. These specimens
nism described by Kurek et al. (2014). Briefly, the film-forming solution were dried in an oven at 105 ◦ C for 24 h, weighed (mi), and inserted, for
was prepared in distilled water at pH 7.0, being the solution submitted 24 h, into a beaker containing 50 mL of distilled water, at room tem­
to an heat treatment at 70 ◦ C during 20 min, which induces unfolding of perature. Then, the wet films were placed back in the oven to dry at
the globular whey proteins and exposure of internal sulfhydryl and 105 ◦ C for 24 h. Subsequently, they were weighed again to obtain the
hydrophobic groups, promoting cross-linking process via intermolecular final weight (mf). All the procedures were performed in triplicate.
disulfide bonds (Pérez-Gago, Nadaud, & Krochta, 1999).
For the CH/WPI film, a CH 3% (w/w) solution was added to WPI 3% 2.4.5. Sorption isotherms
(w/w) solution according the optimum CH:WPI ratio determined by The water sorption isotherms were determined gravimetrically as
turbidity measurements. The CH/WPI stock solution thus obtained was previously described by Sousa et al. (2014). Pieces of films (2.5 × 2.5
agitated at 200 rpm for 20 min. Thereafter, two phases were formed: a cm), previously dried in a vacuum oven at 60 ◦ C for 48 h, were weighed
solvent-rich phase (supernatant) and a dense biopolymer-rich phase and put into hermetic test chambers containing different saturated
(CH/WPI coacervate phase). The supernatant was discarded and the aqueous salt solutions at water activities varying between 0.113 and
precipitated CH/WPI coacervate was recovered. The coacervate dry 0.903, and stored at room temperature The samples were weighed
weight was determined using a vacuum oven at 105 ◦ C for 24 h. Then, periodically until they reached constant weight (i.e. were considered at
the appropriate amount of plasticizer (glycerol) was added (30 wt% of equilibrium). Duplicate measurements were performed for each film
plasticizer per dry weight of CH/WPI coacervate) to the CH/WPI coac­ formulation. The Guggenheim-Anderson-de Boer (GAB) model (Ander­
ervate. The mixture was then homogenized in a homogenizer (Ultra- son, 1946; Boer, 1968) (Eq. (2)), was used to fit the experimental
Turrax®, IKA) at 5000 rpm for 5 min, followed by sonication for 15 min, sorption data.
in order to remove small bubbles formed upon stirring. Finally, a fixed
C⋅k⋅X0 ⋅aw
amount (30 g) of the mixture was spread on polyoxyethylene methylene Xe = (2)
[(1 − k⋅aw )⋅(1 − k⋅aw + C⋅k⋅aw )]
(POM) Petri dishes and allowed to dry in a climatic test chamber at 25 ◦ C
and 40% of relative humidity for 24 h. where Xe is the equilibrium moisture content on a dry basis, X0 repre­
The WPI-6, WPI-7 and CH films were obtained following the same sents the monolayer moisture content (on a dry basis), C, Guggenheim
procedure described for the CH/WPI films, namely using the same constant, is a constant dependent on the water sorption heat in the first
plasticizer content (30 wt% of glycerol), the same amount (30 g) of film- and subsequent layers, k is a parameter reflecting the sorption heat of
forming solution to spread on Petri dishes and the same drying method. the multilayer as well as the heat of condensation of water vapor, and aw
represents water activity.
2.4. Films characterization The experimental sorption data was fitted with Eq. (2), by per­
forming a non-linear estimation using Statistica version 8 software
2.4.1. Thickness measurement (StatSoft, Tulsa, OK, USA), thus obtaining the corresponding GAB
The thickness (x) for each type of film was evaluated using a thick­ parameters.
ness measuring device (Absolute Digimatic Indicator, model ID-F150,
Mitutoyo Co., Japan) with a resolution of 1 μm, and the measure­ 2.4.6. Optical properties
ments were performed at five random positions. Optical measurements were performed in a Minolta colorimeter
(Minolta CR 400, Tokyo, Japan). The CIELab scale was used to deter­
2.4.2. Mechanical properties mine values of color difference (ΔE), luminance (L*), which is expressed
The tensile strength and elongation at break were determined using a as a percentage (from 0 for black to 100 for white) and ranges of color
TAXT2 Texture Analyzer and the analyses were performed according to (a* and b*, where a* ranges from green (− ) to red (+) and b* from blue
the standard method ASTM D 882–89. Films strips 8.0 cm long and 1.5 (− ) to yellow (+)) of each film. The color difference (ΔE) was calculated
cm wide were fixed on the grips and subjected to extensional deforma­ according to Eq. (3):
tion with the application of a displacement rate of 50 mm/min until √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
their rupture. Six replicates were performed in each case. ΔE = (L* − L)2 + (a* − a)2 + (b* − b)2 (3)

2.4.3. Water vapor permeability where L, a, and b represent the standard values of the white calibration
Measurement of the water vapor transmission rate (WVTR) and plate.
calculation of the water vapor permeability (WVP) were performed The opacity (%) of the films was evaluated using a Minolta color­
gravimetrically based on ASTM E96-95 method previously described in imeter (Minolta CR 400, Tokyo calibrated with a standard white back­
ASTM (1995). The samples were cut in a circular form with 80 mm ground (Yw) and a standard black background (Yb). The opacity values
diameter and placed in each sealed cell containing anhydrous calcium were calculated according to Eq. (4):
chloride (RH = 2%). The cells were weighed periodically, and the water
vapor permeability (WVP) in g m− 1s− 1 Pa− 1, of each film was deter­ Opacity =
Yb
× 100 (4)
mined according to Eq. (1): Yw

Δm⋅x Both color difference (ΔE) and opacity parameters were measured
WVP = (1) seven times at different spots of each film.
A⋅Δt⋅ΔP

where Δm is the weight gain (g), x is the film thickness, A is the area of 2.4.7. Scanning electron microscopy (SEM)
exposed film (0.003 m2) exposed for a time Δt (s) to a water vapor partial The analysis of the microstructure of the films was made in an
pressure differential ΔP (Pa). The water vapor permeability of each film electronic scanning microscope FEI Quanta 400 FEG located in CEMUP
was determined in triplicate. (Materials Center at the University of Porto). The samples were fixed on
aluminum supports with double-sided carbon tape and coated with a
fine Au–Pd layer. The upper surface and the cross section of the films

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

were analyzed at an acceleration voltage of 5 kV, working distances based films (WPI-6 and WPI-7) was also evaluated.
(WD) between 10.9 and 11.7 mm and a magnification level of x10000.

2.4.8. Fourier-transform infrared spectroscopy 3.1. Characterization of CH/WPI coacervate-forming solutions

FTIR spectra of the films were acquired using an ALPHA II- Bruker
spectrometer (Ettlingen, Germany), with Diamond ATR (Attenuated 3.1.1. Turbidity measurements
Total Reflectance), at room temperature. FTIR spectra of films were Though CH/WPI interactions strongly depend on pH, previous works
recorded using the abrasive pad of the ATR accessory, pouring a small already established the working pH range for electrostatic interaction to
piece of sample onto the pad. All spectra were registered between 4000 occur in this system: higher than the isoelectric point of the proteins
and 400 cm− 1 and collected at a resolution of 4 cm− 1; 24 scans were involved (for WPI, as already referred above, between 4.5 and 5.5), and
averaged before Fourier transformation. lower than pKa of 6.5 for CH (Bastos et al., 2010). WPI has amphoteric
nature due to the presence of both amino (NH2) and carboxyl (COOH)
2.4.9. Differential scanning calorimetry functional groups, while CH has positive charge at acidic conditions due
The thermal properties of CH, WPI-6, WPI-7 and CH/WPI were to its positive amino groups (NH+ 3 ). Therefore, the working pH was set at
evaluated through differential scanning calorimetry (DSC) using a Per­ 6.0 (pH > pI of WPI and pH < pKa of CH), which promotes electrostatic
kinElmer DSC 6000 equipment. Film samples (approx. 5 mg) were interactions between negative carboxyl groups (COO− ) of WPI and
cooled to 0 ◦ C. Analysis was carried out from 0 ◦ C to 250 ◦ C at a heating positive amino groups (NH+ 3 ) for CH (Tavares et al., 2019; Tavares &
rate of 10 ◦ C∙min− 1, under a nitrogen atmosphere (purge gas flow of 20 Noreña, 2019).
mL min− 1). The turbidity was determined at various CH-to-WPI ratios by addi­
tion of CH 0.1% (w/w) aliquots to the WPI solution 0.1% (w/w). It was
found that, when the CH-to-WPI ratio was increased, the turbidity
2.5. Statistical analyses increased until maximum interaction between CH and WPI reached a
plateau at an O.D. around 1.059, at CH-to-WPI mass ratio of 0.10 (w/w)
The data were analyzed by Analysis of Variance (ANOVA) and dif­ (Fig. 1). The turbidity increased due to clots and coacervate formation
ferences between means were analyzed by Tukey’s test at a significance containing CH/WPI complexes (Bastos et al., 2010), until the plateau,
level of P ≤ 0.05, using SAS (version 9.3) software. when CH molecules are saturated with WPI and no more insoluble
complexes are formed. After that, the O.D. remained practically un­
3. Results and discussion changed due to a maximum interaction between the positive charges of
CH and negative charges of WPI (stoichiometrically equal). The
The results section was divided in two main parts: in the first one, the maximum interaction remained constant even with the addition of
maximum interaction between CH and WPI, which is associated with the larger amounts of CH, meaning that the formed complexes remained
formation of aggregates suspended in the liquid matrix, was studied stable. According to Espinosa-Andrews, Sandoval-Castilla,
aiming to select the best complex coacervation condition to be used in Vázquez-Torres, Vernon-Carter, and Lobato-Calleros (2010), at the
the CH/WPI film production; in the second part, CH/WPI films were point of maximum interaction, the ionized groups of the oppositely
prepared, characterized and their performance and physicochemical charged biopolymers are mutually neutralized, leading to the formation
properties were compared with those of WPI or CH films alone. In of coacervate as insoluble complex.
addition, the effect of thermally induced denaturation of whey proteins The turbidity of the solutions is attributed to the appearance of
on the mechanical properties and water vapor permeability of WPI scattering particles or droplets in the medium, related to the coacervate

Fig. 1. Influence of CH:WPI mas ratio (% weight w/w) on the turbidity of WPI 0.1% (w/w) solutions with aliquots (2–200 μL) of CH 0.1% (w/w) solutions.

4
L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

formation. Ducel, Richard, Saulnier, Popineau, and Boury (2004) re­ or WPI films alone.
ported values of maximum turbidity of the same order of magnitude
(0.02 mol/mol) between mixtures of WPI 0.5% (w/w) and CH 0.4%
(w/w). However, Tavares and Noreña (2019) determined the coacer­ 3.2. Films properties
vation yield of the CH/WPI system by gravimetric analyses and found
maximum coacervate yield at CH-to-WPI ratio of 0.2:1 (w/w), instead of 3.2.1. Thickness and mechanical properties
0.1:1 (w/w). Nevertheless, the degree of deacetylation of CH (DD = 83, The thickness is a crucial factor for studying the physical properties
94 and 96%) and the technique used by these authors (gravimetry) were of films, since it directly affects others physicochemical properties such
different from those used in the present study (DD = 92% and turbi­ WVP or mechanical properties (Khodaei, Oltrogge, & Hamidi-Esfahani,
dimetry, respectively). Different deacetylation degrees and molecular 2020). The average film thickness (x) measured for the CH, WPI, WPI-D
weights may lead to different molecular interactions with WPI mole­ and CH/WPI films is presented in Table 1. The highest value was ob­
cules (Tavares & Noreña, 2019). According to the described above, and tained for the CH/WPI film, which can be correlated with its higher
to guarantee that the chosen parameters are already in the plateau re­ compact structure. The thickness of the WPI-6 films was smaller than
gion, CH/WPI mass ratio of 0.1: 1 (w/w) (slightly above the beginning of WPI-7 films, indicating that the covalent cross-linking promoted by the
the plateau, Fig. 1) was selected for the production of the composite heat denaturation of the whey proteins allow to increase the thickness.
films of CH/WPI by complex coacervation. Edible films with good mechanical properties are essential for food
packaging and preservation functions during product transport and
3.1.2. Cryo-SEM storage. The mechanical properties of films are influenced by the nature
The Cryo-SEM technique allows the visualization and morphological and chemical structure of film-forming components and structural
characterization of coacervates structure in their native environment cohesion (Eghbal et al., 2016; Ramos et al., 2013). The measured values
before the process of dry-down to films, where substantial structural of the tensile strength (TS) and elongation at break (E) are given in
changes occur. The results from Cryo-SEM are shown in Fig. 2. The Table 1. Tensile strength is related to the mechanical resistance of films
different solution composition clearly led to different network structure, associated to the cohesion forces between chains, and elongation at
mesh, arrangement and bonds in solution, corresponding to different break measures the ability of the films to extend before breaking (Galus
morphologies. The CH 3% (Fig. 2A) showed a continuous and homo­ & Lenart, 2013). The tensile strength of CH/WPI composite films was
geneous network with well-defined but much larger matrix structure. In significantly (p < 0.05) greater than the tensile strength obtained from
the case of WPI 5% (Fig. 2B) the structure is disperse and no network is the mono-component films (WPI-6, WPI-7 and CH). This increase is
observed. There was no formation of a homogenous structure. The CH/ mainly due to the reinforcement of the WPI matrix with small amounts
WPI coacervate (Fig. 2C) presented a balanced and compact structure, of incorporated CH, which had significant (p < 0.05) effect on the me­
with continuous network and tight net mesh (close to one another), chanical parameters of CH/WPI films. The electrostatic interaction
indicating synergistic effects of complex coacervation between CH and
WPI. The compact network structure mainly result due to the electro­ Table 1
static interactions between CH and WPI, providing the formation of Thickness (x), Tensile strength (TS), elongation at break (EAB) and water vapor
coacervates with three-dimensional layer-layer stacking (Souza et al., permeability (WVP) for the films composed by CH 3% (w/w), WPI-6 and WPI-7
2009). However, according to Jho, Yoo, Lin, Han, and Hwang (2017), in both prepared at 5% (w/w), and CH/WPI mass ratio of 0.1:1 (w/w)a.
some systems of complex coacervation the resulting dense coacervate Films x (mm) Mechanical property WVP (g m− 1 − 1
s Pa− 1) ×
may not present a network with homogeneous phase, but phase sepa­ 10− 9
ration into a biopolymers complex-rich phase and a bulk-like water EAB (%) TS (MPa)
microphase, at nanoscale length for phase separation.
CH 0.27 ± 25.42 ± 1.24 ± 0.88 ± 0.07c
The results of the coacervate network structure demonstrated that 0.02b 4.74b 0.27c
the electrostatic interactions between CH and WPI allow reinforcing the WPI-6 0.20 ± 42.36 ± 0.19 ± 1.05 ± 0.07b
molecular structure of CH/WPI complex. Similar behavior is reported by 0.01d 7.89a 0.05d
Rocha et al. (2014), for whey protein/agar insoluble complexes, with WPI-7 0.25 ± 9.34 ± 1.71c 2.04 ± 1.78 ± 0.33a
0.04c 0.31b
whey proteins distributed randomly and homogeneously through the
CH/ 0.35 ± 4.60 ± 0.45d 3.87 ± 1.02 ± 0.06b
agar gel matrix. WPI 0.03a 0.46a
The CH/WPI coacervate produced by complex coacervation showed a
Means with the same letter, in the same column, did not differ significantly
homogeneous and compact structure and the physicochemical proper­
(p ≤ 0.05), according to the Tukey test.
ties of produced films were determined and compared with those of CH

Fig. 2. Low-temperature scanning electron microscopy (Cryo-SEM) of solutions: A) CH 3% (w/w); B) WPI-6 prepared at 5% (w/w) and C) CH/WPI coacervate with
mass ratio of 0.1:1 (w/w). Amplifications of x5000. The accelerating voltage was 15 kV and working distance 15 mm in all cases.

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

between the positively charged CH (amino groups) and negatively the values ranging from 19 to 32% reported by Homez-Jara et al. (2018)
charged WPI (carboxyl groups) improved rigidity and created a stronger for CH films with different concentrations (0.5, 1.0 and 1.5%) and
network. This also resulted in a stiffer structure with decreased defor­ drying temperatures (2, 25 and 40 ◦ C). de Morais Lima et al. (2017)
mation at break, probably due to the lower malleability and flexibility of reported solubility of 22% for CH 1.5% (w/v) film dried at 38 ◦ C while
these films, promoted by the better homogenization and formation of 7.6% was reported by Baron, Pérez, Salcedo, Córdoba, and Sobral
thinner layers due to the electrostatic interaction between CH and WPI. (2017) for CH 2% (w/v) film dried at 30 ◦ C. These differences can be
The TS value achieved for the system of CH/WPI (obtained by the ascribed to differences in the molecular weight and degree of deacety­
complex coacervation method) was also higher than reported values for lation of CH as well as to differences in the CH concentration and
the same system where film formation was achieved by other mecha­ plasticizer content. The hydrophilic characteristic of WPI-6 films
nisms, namely: Ferreira et al. (2009) that studied the influence of the favored their interaction with OH water groups resulting in swelling and
protein amount in CH/WPI films, and reported that the increasing total solubility in water, which was also reported by (Ferreira et al.,
protein proportion caused decreasing in the elongation at break and 2009). Gounga, Xu, and Wang (2007) reported solubility of 50.58% for
tensile strength due to the formation of less cohesive structures. Kurek WPI films prepared by gelation mechanism (pH 7 and denaturation at
et al. (2014) also reported CH/WPI values of tensile strength and elon­ 90 ◦ C for 30 min). This relatively low solubility was ascribed to the high
gation at break of 0.60 MPa ± 1.67 and 33.90 ± 6.37%, respectively. In interaction between hydrophobic regions and existence of intermolec­
this study reported by Kurek et al. (2014), both biopolymers were ular disulfide bonds promoted by heat treatment. This effect of protein
positively charged, and there was no electrostatic interactions between denaturation by heat treatment on the solubility of WPI films was re­
them and the complex coacervation did not take place. WPI-7 film ported by Silva, Mauro, Gonçalves, and Rocha (2016). These authors
showed higher TS and EAB values than WPI-6 film, indicating that the obtained solubility of 62.04 and 59.97% for films made with two
covalent cross-linking via intermolecular disulfide bonds promoted by different heat treatments on the film forming solutions: heating until
globular whey protein denaturation increased the mechanical properties 75 ◦ C and heating until 75 ◦ C and keeping at 75 ◦ C for 10 min,
of the WPI based films. These results are mainly influenced by the mo­ respectively.
lecular structure of the produced films, since the WPI-7 films were made The CH/WPI films showed low solubility in water due to the elimi­
of cross-linked protein strands, whereas WPI-6 films have a more nation of charges on their surface. This result indicate the formation of
random structure in which cohesion is mainly due to hydrogen bonding films with more hydrophobic nature, with low interactions between
(bib_Pérez_Gago_et_al_1999Pérez-Gago et al., 1999). surface adsorption sites and water molecules. The solubility of CH films
was significantly (p < 0.05) higher than the solubility of CH/WPI films.
3.2.2. Water vapor permeability Differences in surface charge (protonated amine groups in CH and null
The WVP is one of the most important properties of edibles films. The charge resulting from electrostatic interactions in CH/WPI films) may
water content present in products influence significantly the change in explain this result. The complex coacervation between CH and WPI
the rates of deteriorative reactions and WVP reflects the capacity of the resulted in CH/WPI films with low affinity to interact with water mol­
film (when applied as package in food protection) to control the transfer ecules, which is beneficial in cases where product integrity and water
of moisture between food and its surrounding (Homez-Jara et al., 2018; resistance are desired. This is a major advantage, as high solubility is
Kurek et al., 2014). CH/WPI films showed a better protection barrier for usually a significant drawback in the industrial application of
the water vapor when compared to the WPI-7 films produced by gelation hydrocolloid-based films.
mechanism (Table 1). It is expected that CH/WPI films present more
hydrophobic characteristics due to the canceling of charges promoted by 3.2.4. Sorption isotherms
electrostatic interactions, which may limit biopolymer segmental Sorption isotherms provide significant information on how the films
mobility and contribute to reduce the WVP, making the film less will behave when exposed to different humidity conditions. The mois­
permeable to water vapor. The WVP values for CH/WPI and WPI-6 films ture sensitivity of films was evaluated at 25 ◦ C, over a wide range of aw.
did not differ significantly (p > 0.05), which may indicate that WPI had Experimental sorption data are presented in Fig. 3.
higher influence than CH in the obtained WVP value for CH/WPI film.
Furthermore, CH films presented the lower WVP, which may indicate
that incorporating of CH will also decrease WVP of WPI films. In fact, it
has been reported that the WVP of CH/WPI films increased with the
amount of CH present due to polymer-polymer interaction (Ferreira
et al., 2009). Kurek et al. (2014) reported WVP with values ranging from
0.55 to 3.72 × 10− 10 g m− 1 s− 1 Pa− 1 for CH/WPI films dried in three
different relative humidity (RH) gradient conditions (33–0%, 75–30%,
and 100–30%), respectively. The RH = 40% and mechanism of coac­
ervation utilized in our study as well as differences in biopolymers
concentration could explain the results’ differences. The WPI-7 films
showed higher values of WVP than the WPI-6 films, indicating changes
in the molecular structure of the WPI based films. The WVP properties of
both CH and WPI based films have been studied by different authors:
Kokoszka, Debeaufort, Lenart, and Voilley (2010) studied the behavior
of WPI films with four concentrations of protein at four different RH
gradients, and concluded that the WVP values of the films increased with
the WPI concentration, except for high RH gradient. Homez-Jara et al.
(2018) reported results of WVP for CH films ranging between 0.27 ×
10− 11 g m− 1s− 1 Pa− 1 and 92.2 × 10− 11 g m− 1s− 1 Pa− 1, and concluded
that CH concentration did not have a consistent effect on the WVP
values. Fig. 3. Equilibrium moisture content (Xe) vs water activity (aw) of experimental
sorption data at 25 ◦ C for the films composed by CH 3% (w/w) (squares), WPI-6
3.2.3. Solubility prepared at 5% (w/w) (triangles) and CH/WPI coacervate with mass ratio of
The water solubility of CH films was 41.82%, which is higher than 0.1:1 (w/w) (circles), with the respective fits using the GAB model (Eq. (2)).

6
L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

The equilibrium moisture content, Xe, increased slowly with that water molecules not bonded to the monolayer will be able to move
increasing aw up to around 0.7. For aw > 0.7, a steep increase of Xe was through the film. This results in a WVP slightly higher than for CH films
observed for all films studied (Fig. 3). and equivalent to WPI-6 films.
The higher solubility, water affinity and water retention of CH 3%
(w/w) and WPI 5% (w/w) films resulted in higher Xe, at a given aw, 3.2.5. Color
when compared to CH/WPI film (Fig. 3). The color attributes hold a prime role in product appeal and con­
Experimental sorption data was fitted to the GAB model (Eq. (2)). sumer acceptability (Ramos et al., 2013). All the films were visually
Sigmoid curves, typical of hydrophilic materials, were obtained in all homogenous and semi-transparent (Fig. 4). The incorporation of CH into
cases (Fig. 3). Overall, a suitable adjustment of the experimental data is WPI matrix resulted in more yellowish CH/WPI films with positive b*
obtained, as suggested by the good correlation coefficients (R2 > 0.99) value (Table 2). The results of color parameters indicated that CH had
and range of k values (0 < k < 1) (Sousa et al., 2014) (Table 3). higher influence than WPI in the color parameters of the CH/WPI films.
The amount of water retained at the primary sorption sites of the The color change in CH/WPI films could be attributed to the original
films was quantified by the monolayer moisture content, X0 (on a dry color of CH that had a slightly yellow appearance (Ojagh, Rezaei,
basis) (Table 3). This parameter is related to the composition and Razavi, & Hosseini, 2010) and/or due to the stronger network and
chemical structure of each film. The estimated values were higher for coacervation mechanism. According to Ebrahimi, Koocheki, Milani, and
pure WPI (0.217) and CH (0.137) films. CH/WPI film presented the Mohebbi (2016), the amount and type of interactions between poly­
lowest value (0.089), suggesting less available bonding sites at the saccharide and protein may change the films’ color properties.
monolayer, probably due to the lower hydrophilicity and null surface The films total color difference (ΔE) was not significantly different
charge resulting from electrostatic interactions between WPI and CH. (p < 0.05). The less transparent staining of the composite films was due
The X0 values for CH films are in accordance with the results reported by to the yellowish color of CH. In fact, the opacity values are similar (p >
Souza, Campiña, Sousa, Silva, and Gonçalves (2013), where the values 0.05) for CH/WPI films and CH films. Furthermore, though differences
of X0 was influenced by the interstitial space existing in the matrices of are statistically significant for WPI films, all films were visually trans­
the films, which increased the rate of diffusion of the water into the parent. The color of the composite films is in accordance with other
monolayer. The parameter C, related to the energy difference between studies reported in literature with high values of L* and variation of a*
adsorption in the form of a monolayer and/or in several multilayers, can and b* parameters (Chillo et al., 2008; Córdoba & Sobral, 2017; Kurek
be used to quantify the strength of the established bonds between the et al., 2014).
water molecules and the hydrophilic sites at the monolayer of the
polymeric film: the greater the C value, the stronger the bonds (Xiao, 3.2.6. Film microstructure
Lim, & Tong, 2012). The scanning electron microscopy (SEM) technique was used to
C is higher for the CH/WPI films (1.697; Table 3). Adsorption of a study the film microstructure, and existence of possible gaps and cracks
monolayer is favored with respect to a larger multilayer formation in their structures. SEM images of films surface are represented in
(stronger bonds are formed), resulting from mixing the two biopolymers Fig. 5A (CH), Fig. 5B (WPI-6) and Fig. 5C (CH/WPI), and the films cross-
(bib_Silva_et_al_2016Silva et al., 2016). For CH and WPI films, C was section are represented in Fig. 5D (CH), Fig. 5E (WPI-6) and Fig. 5F (CH/
lower (1.183 and 1.095, respectively) than for CH/WPI film. The higher WPI). For the SEM images of films surface, the structure of the surface of
value of C for WPI film, relative to CH film, suggests that the interaction CH/WPI is dense, cohesive and homogeneous, probably due to the
at the primary sorption sites was stronger in the former case. For all the incorporation of CH in the polymer matrix of the WPI. Unlike the results
films studied, the k range values, 0 < k < 1 (Table 3), indicates that the reported by Ferreira et al. (2009), the samples didn’t display the phase
water in the upper layers was less structured than pure water (Xiao et al., segregation typical of WPI heat-gelled systems, one phase rich in CH and
2012). This also reflects the solubility and WVP of the films. Overall, other rich in WPI, confirming the thermodynamic compatibility be­
films behavior towards water is the result of the net contribution of tween this biopolymers promoted by complex coacervation. All the films
different factors such as the surface affinity of the film towards water, sowed structure with some heterogeneity and roughness. However,
the size of the films pores and the affinity of the inner films to water, not when compared CH/WPI film with the CH and WPI-6 films, the latter
always working towards the same direction. For instance, in the first showed opaque zones in their structures due essentially to the formation
case the effect on WVP is positive (the highest affinity towards water the of small, non-dissoluble air bubbles. This result is consistent with the
higher WVP would be), but in the last case it may be the opposite (as formation of CH and WPI films with poor mechanical properties and
moving through the film is more difficult due to higher affinity of hy­ more fragile and weak microstructure. Talón et al. (2017) showed
drophilic structures towards water). Similarly, though the values of X0 similar results for CH films, which presented a
are significantly lower for the CH/WPI film, suggesting lower interac­ heterogeneously-fractured surface related with crystalline regions. Silva
tion at the primary layer sites and lower interstitial space available; the et al. (2016) reported WPI films with a continuous smooth surface and
C value are considerably higher, suggesting favored adsorption of a homogeneous structure. For the cross-section morphology, Fig. 5D (CH),
monolayer when compared to a larger multilayer formation, meaning Fig. 5E (WPI-6) and Fig. 5F (CH/WPI) films showed similar morphology

Fig. 4. Photographs of films composed by CH 3% (w/w), WPI-6 prepared at 5% (w/w), and CH/WPI coacervate with mass ratio of 0.1:1 (w/w).

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

Table 2
Solubility and optical properties for the films composed by CH 3% (w/w), WPI-6 prepared at 5% (w/w), and CH/WPI mass ratio of 0.1:1 (w/w)a.
Films Solubility in water (%) Optical propriety

Color Opacity (%)

a* b* L* ΔE

CH 41.82 ± 1.20c − 1.32 ± 0.25b 9.46 ± 2.02a 96.91 ± 1.29a,b 7.98 ± 2.07a 14.66 ± 1,14a
WPI-6 100a 1.33 ± 0.02a − 4.78 ± 0.29b 99.8 ± 0.15a 7.19 ± 0.31a 12.85 ± 0.65b
CH/WPI 14.58 ± 0.94b − 0.85 ± 0.13b 9.34 ± 0.65a 92.94 ± 0.77b 8.89 ± 0.76a 14.58 ± 0.94a
a
Means with the same letter, in the same column, did not differ significantly (p ≤ 0.05), according to the Tukey test.

between the peak intensity in the specific region of wavenumber. The

Table 3
FTIR spectrum of CH film indicated broad band in the range from ~3737
GAB parameters for the films composed by CH 3% (w/w), WPI-6 prepared at 5%
to 3000 cm− 1 and centred at around ~3280 cm− 1 as result of stretching
(w/w) and CH/WPI mass ratio of 0.1:1 (w/w)**.
vibrations of O–H and N–H groups. The two weak peaks at ~3937 cm− 1
Films C k X0 R2 and ~3882 cm− 1 indicate the C–H stretching, ~1566 cm− 1 corre­
CH 1.183 0.937 0.217 0.996 sponding to the amide II band (–C ═ O vibration and N–H bending vi­
WPI-6 1.095 0.986 0.137 0.998 bration corresponding to the NH+ 3 protonated amide group), ~1408
CH/WPI 1.697 0.978 0.089 0.996
cm− 1 (–OH and –CH vibrations) and 1033 cm− 1 (C–O stretching vibra­
tion). The intensity of the characteristic amide I band (at 1655 cm− 1
with smooth surface without phase segregation and absences of cracks attributed to –C ═ O vibration) was very low, which indicate interaction
and grooves, which are desired characteristics for edible films applica­ between the plasticizer glycerol with CH molecules (Matet, Heuzey,
tions in food products. Pollet, Ajji, & Avérous, 2013). Similar spectral had been described
previously for CH films (Galvis-Sánchez, Castro, Biernacki, Gonçalves, &
3.3. FTIR analysis Souza, 2018).
Similar FTIR spectra were observed for WPI-6 and WPI-7 films,
FTIR analyses were used to evaluate the differences in the functional namely a broad band in the range from ~3737 to 3000 cm− 1 due to the
groups of the produced films and to investigate possible interactions vibrations of O–H and N–H groups, ~1628 cm− 1 corresponding to amide
between the components of composite CH/WPI film. The FTIR profile I band (–C ═ O vibration) and ~1535 cm− 1 due to amide II band (–C ═ O
obtained for CH, WPI-6, WPI-7 and CH/WPI films are shown in Fig. 6. and C–N stretching). The band at 1033 cm− 1 (C–O stretching vibration)
The absorption intensities of all samples were normalized with respect to results as the influence of the plasticizer glycerol in the both films, and
the intensity of the highest band in order to evaluate the differences the peak obtained at 1240 cm− 1 is an indicator of alfa-helix strands of

Fig. 5. Scanning electron microscopy (SEM) imaging of films surface composed by A) CH 3% (w/w), B) WPI-6 prepared at 5% (w/w) and C) CH/WPI mass ratio of
0.1:1 (w/w); and films cross-section for the D) CH 3% (w/w), E) WPI-6 prepared at 5% (w/w) and F) CH/WPI coacervate with mas ratio 0.1:1 (w/w). Amplifications
of x10000. The accelerating voltage was 5 kV and working distance 15 mm in all cases.

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

Fig. 6. FTIR spectra of films composed by CH 3% (w/w), WPI-6 and WPI-7 both prepared at 5% (w/w), and CH/WPI coacervate with mass ratio of 0.1:1 (w/w).

the proteins (Mohammadi, Mirabzadeh, Shahvalizadeh, & Hamisheh­ groups (COO− ) of WPI and amino groups (NH+ 3 ) of CH. However, the
kar, 2020). WPI-7 film showed high peaky intensity at 1400 cm− 1 low peak intensity obtained at 1400 cm− 1 for CH/WPI film indicate that
indicating that the carboxyl group vibrational band with ionized groups not all proteins present in solution was capable to interact with CH
(COO− ) capable to interact with amino groups (NH+ 3 ) of CH. Similar during the process of complex coacervation. This result corroborates
results were reported in other studies (Böcker, Wubshet, Lindberg, & with the finding reported by Tavares and Noreña (2019), that demon­
Afseth, 2017; Singh, Suri, Tiwary, & Rana, 2012). However, the peak of strated complex of CH/WPI at ratio of 0.1:1 (w/w) with coacervate yield
1400 cm− 1 was practically not visible for WPI-7 film, which may suggest ranged from ~60 to 70% for complex coacervation between WPI and CH
conformational changes and reorganization of its denatured globular with degree of deacetylation of 83, 94 and 96%.
proteins. WPI-7 film also showed higher band intensity than WPI-6 film The intensity of the broad band at around ~3737 to 3000 cm− 1 for
at 1628 cm− 1 due to the formation of intermolecular β-sheet structure CH/WPI film was higher when compared with obtained for WPI-6 and
promoted by the denaturation of its proteins (Farrokhi, Badii, Ehsani, & CH films, indicating enhanced of hydrogen bonding in the WPI/CH film.
Hashemi, 2019). According to van de Weert, Haris, Hennink, and Similar results were reported by Huang, Sun, Xiao, and Yang (2012) for
Crommelin (2001) the amide I band is originated from intermolecular complex coacervation of soybean protein isolate and chitosan. Aliza­
antiparallel β-sheet formation, and usually the band in the region of deh-Sani, Khezerlou, and Ehsani (2018) showed that the available peak
1620–1630 cm− 1 is much more intense. at 3400 cm− 1 became wider when the cellulose nanofiber was incor­
For CH/WPI film, the characteristic FTIR spectrum of WPI are porated into WPI composite film. These results indicate that the
dominated in combination with the spectra of CH due to the formation hydrogen bonding and electrostatic interactions were involved in the
of a composite film with concentration of WPI higher than CH. However, formation of the complex coacervate of CH/WPI.
a decreased in the intensity of the band at 1535 cm− 1 is observed for CH/
WPI film when compared with WPI-6 film, probably caused by the 3.3.1. DSC analysis
electrostatic interaction between the positive amino groups (NH+ 3 ) of CH The thermal properties of films are important factors for industrial
and negative carboxyl groups (–COO− ) of WPI. This chance in the applications, as packaging materials are often heated during the pro­
carbonyl-amide region suggest existence of electrostatic interaction duction, storage and processing of foods (Fernandes et al., 2020). DSC
between the amino groups (NH+ 3 ) and carboxyl groups (–COO ) (Espi­
−
analyses for CH, WPI-6, WPI-7 and CH/WPI films were performed to
nosa-Andrews et al., 2010). understand their thermal behavior. Fig. 7 shows the DSC curves of CH,
CH/WPI film presented decrease in the peaky intensity at 1400 cm− 1 WPI-6, WPI-7 and CH/WPI films. The thermograms of the all films
in comparison of WPI-6 films, indicating interactions between ionized showed endothermic peaks, representing the typical behaviour of this

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L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

kind of systems. Endothermic thermograms of CH films exhibited one the thermal stabilities of the composite CH/WPI film. Further, in full
wide main peak (142 ◦ C) that could be related with the α-relaxation of agreement with chitosan degradation, the small peak tail at tempera­
chitosan chain and one phase transition temperature process at tem­ tures nearly at 231 ◦ C may indicate that the chitosan polymer is starting
perature of 233 ◦ C, that could be related with start of chitosan’ structure to degrade.
degradation. Similar results were reported by Peng, Wang, Shi, Chen,
and Zhang (2020) for chitosan films plasticized with glycerol. The au­ 4. Conclusion
thors reported that the endothermic peak at 162 ◦ C corresponds to the
α-relaxation of chitosan chain and the phase transition temperature, The formulation of films containing two biopolymers under coacer­
detected at 271.2 ◦ C, was associated with decomposition of the poly­ vation conditions improved the characteristics of the films due to the
saccharide backbone. The difference in the value of the endothermic synergistic effects between the biopolymers. Cryo-SEM analysis proved
main peak and phase transition between the results can be associated that a small amount of chitosan in the polymeric matrix of the protein
with films formulations methodology, films conditioning, plasticizers improved its structure, increasing the cohesion of the biopolymers and
and others important parameters such as molecular weight and deace­ consequent homogeneity. The organization, mesh size, arrangement,
tylation degree of chitosan used in both works. porous structure formation and composition of the biopolymers in the
The WPI-7 film also showed three endothermic peaks at 112, 188 and polymer matrix influence the physical and chemical properties of edible
215 ◦ C. The shape of the obtained calorimetric curve is in good agree­ films and coatings. All the films presented suitable morphological as­
ment with previously reported ones (Hernandez-Izquierdo, Reid, pects, transparency and formation of a homogeneous structure with
McHugh, Berrios, & Krochta, 2008). Moreover, the last endothermic well-defined interactions between biopolymers. The composite films
peak was similar to those reported by Kim and Ustunol (2001), that produced by complex coacervation reveled different properties that can
indicate the degradation of whey proteins at 241 ◦ C. Similarly, for WPI-6 be interesting for food applications, including a stronger network
film, the thermal changes occurs in three stages at 111, 160 and 196 ◦ C. structure, higher tensile strength, lower water vapor permeability and
Interesting, the last endothermic peak was detected at a lower temper­ lower solubility in comparison to the mono-component WPI and chito­
ature than that one observed for WPI-7 (215 ◦ C), mainly due to the san films. In this context, considering coacervation as an additional
previously denaturation of its whey proteins components by heat available technique for film formation allows a wider range of WVP and
treatment. mechanical properties that can be tuned to cover different applications
The DSC thermogram of the CH/WPI film was very similar to WPI-6 in the food preservation and packaging technology.
film, which is in accordance with the coacervate composition, though
slightly shifted into higher temperatures, indicating eventually higher CRediT authorship contribution statement
thermal stability. Two thermal events with endothermic main peaks at
111 and 208 ◦ C were observed. The second endotherm peak for CH/WPI Loleny Tavares: Conceptualization, Methodology, Investigation,
was less depth and shifted tip centre, indicating the formation of new Formal analysis, Data curation, Writing - original draft, Writing - review
network structures due to the hydrogen bond and electrostatic in­ & editing. Hiléia K.S. Souza: Conceptualization, Methodology, Inves­
teractions between negatively charged whey proteins and positively tigation, Formal analysis, Data curation, Writing - original draft, Writing
charged CH. This indicated that the incorporation of a small amount of - review & editing. Maria Pilar Gonçalves: Conceptualization, Meth­
CH to form complex coacervate with WPI had a significant influence on odology, Investigation, Data curation, Formal analysis, Writing -

Fig. 7. DSC thermograms of films composed by CH 3% (w/w), WPI-6 and WPI-7 both prepared at 5% (w/w), and CH/WPI coacervate with mass ratio of 0.1:1 (w/w).

10
L. Tavares et al. Food Hydrocolloids 113 (2021) 106471

original draft, Writing - review & editing, Supervision. Cristina M.R. Eghbal, N., Yarmand, M. S., Mousavi, M., Degraeve, P., Oulahal, N., & Gharsallaoui, A.
(2016). Complex coacervation for the development of composite edible films based
Rocha: Conceptualization, Methodology, Investigation, Data curation,
on LM pectin and sodium caseinate. Carbohydrate Polymers, 151, 947–956.
Formal analysis, Writing - original draft, Writing - review & editing, Espinosa-Andrews, H., Sandoval-Castilla, O., Vázquez-Torres, H., Vernon-Carter, E. J., &
Supervision. Lobato-Calleros, C. (2010). Determination of the gum Arabic–chitosan interactions
by Fourier transform infrared spectroscopy and characterization of the
microstructure and rheological features of their coacervates. Carbohydrate Polymers,
79(3), 541–546.
Declaration of competing interest Farrokhi, F., Badii, F., Ehsani, M. R., & Hashemi, M. (2019). Functional and thermal
properties of nanofibrillated whey protein isolate as functions of denaturation
temperature and solution pH. Colloids and Surfaces A: Physicochemical and Engineering
The authors declare that they have no known competing financial Aspects, 583, 124002.
interests or personal relationships that could have appeared to influence Fernandes, L. M., Guimarães, J. T., Silva, R., Rocha, R. S., Coutinho, N. M.,
the work reported in this paper. Balthazar, C. F., et al. (2020). Whey protein films added with galactooligosaccharide
and xylooligosaccharide. Food Hydrocolloids, 104, 105755.
Ferreira, A. R., Alves, V. D., & Coelhoso, I. M. (2016). Polysaccharide-based membranes
Acknowledgements in food packaging applications. Membranes, 6(2), 22.
Ferreira, C. O., Nunes, C. A., Delgadillo, I., & Lopes-da-Silva, J. A. (2009).
Characterization of chitosan–whey protein films at acid pH. Food Research
This work was supported by the Portuguese Foundation for Science International, 42(7), 807–813.
and Technology (FCT) under the scope of the strategic funding of UID/ Galus, S., & Lenart, A. (2013). Development and characterization of composite edible
BIO/04469/2020 and UID/QUI/50006/2020 units; the work was also films based on sodium alginate and pectin. Journal of Food Engineering, 115(4),
459–465.
supported by BioTecNorte operation (NORTE-01-0145-FEDER-000004) Galvis-Sánchez, A. C., Castro, M. C. R., Biernacki, K., Gonçalves, M. P., & Souza, H. K. S.
funded by the European Regional Development Fund under the scope of (2018). Natural deep eutectic solvents as green plasticizers for chitosan
Norte 2020 - Programa Operacional Regional do Norte. thermoplastic production with controlled/desired mechanical and barrier properties.
Food Hydrocolloids, 82, 478–489.
Gounga, M. E., Xu, S. Y., & Wang, Z. (2007). Whey protein isolate-based edible films as
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