foods

Article
Cocoa Nanoparticles to Improve the Physicochemical and
Functional Properties of Whey Protein-Based Films to
Extend the Shelf Life of Muffins
Sergio de Jesús Calva-Estrada 1 , Maribel Jimenez-Fernandez 2, *, Alba Adriana Vallejo-Cardona 3 ,
Gustavo Adolfo Castillo-Herrera 1 and Eugenia del Carmen Lugo-Cervantes 1, *

1 Unidad de Tecnología Alimentaria, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado
de Jalisco (CIATEJ) A.C., Camino Arenero 1227, El Bajío, Zapopan C.P. 45019, JAL, Mexico;
sce_tau@hotmail.com (S.d.J.C.-E.); gcastillo@ciatej.mx (G.A.C.-H.)
2 Centro de Investigación y Desarrollo en Alimentos, Universidad Veracruzana, Av. Doctor Luis Castelazo,
Industrial Las Animas, Xalapa Enríquez C.P. 91190, VER, Mexico
3 Consejo Nacional de Ciencia y Tecnología—Unidad de Biotecnología Médica y Farmacéutica,
Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CONACYT-CIATEJ) A.C.,
Av. Normalistas 800, Colinas de la Normal, Guadalajara C.P. 44270, JAL, Mexico; avallejo@ciatej.mx
* Correspondence: maribjimenez@uv.mx (M.J.-F.); elugo@ciatej.mx (E.d.C.L.-C.)
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Abstract: A novel nanocomposite whey protein-based film with nanoemulsified cocoa liquor (CL)
Citation: Calva-Estrada, S.d.J.; was prepared using one-stage microfluidization to evaluate the emulsion properties and the effect
Jimenez-Fernandez, M.; of CL on the film properties by response surface methodology (RSM). The results indicated that
Vallejo-Cardona, A.A.; the number of cycles by microfluidization had a significant effect (p < 0.05) on the particle size and
Castillo-Herrera, G.A.;
polydispersity of the nanoemulsion, with a polyphenol retention of approximately 83%. CL decreased
Lugo-Cervantes, E.d.C. Cocoa
the solubility (<21.87%) and water vapor permeability (WVP) (<1.57 g mm h 1 m 2 kPa 1 ) of the
Nanoparticles to Improve the
film. FTIR analysis indicated that CL modified the secondary protein structure of the whey protein
Physicochemical and Functional
and decreased the mechanical properties of the film. These results demonstrate that applying the
Properties of Whey Protein-Based
Films to Extend the Shelf Life of
film as a coating is feasible and effective to improve the shelf life of bakery products with a high
Muffins. Foods 2021, 10, 2672. moisture content. This nanocomposite film is easy to produce and has potential applications in the
https://doi.org/10.3390/ food industry.
foods10112672
Keywords: antioxidant capacity; bakery; nanocomposite film; polyphenol content; shelf life; water
Academic Editor: vapor permeability (WVP)
Ana Teresa Sanches-Silva

Received: 2 September 2021

Accepted: 30 October 2021 1. Introduction
Published: 3 November 2021
Emulsion-based delivery systems have potential applications, including the protection
and release of aroma molecules and polyphenols [1]. Oil-in-water (O/W) nanoemulsions
Publisher’s Note: MDPI stays neutral
are heterogeneous systems containing small oil droplets (between 50 and 500 nm) dispersed
with regard to jurisdictional claims in
within an aqueous phase that have attracted particular interest in the food industry for the
published maps and institutional affil-
iations.
delivery of bioactive lipophilic components due to their ease of fabrication, small particle
size, stability, and enhanced bioavailability of encapsulated compounds [2]. Microflu-
idization is a dynamic high-pressure shearing with excellent efficiency for the production
of nanoemulsions by forcing two immiscible liquids into an interaction chamber via mi-
crochannels of a special configuration generating high-velocity microstreams that reduce
Copyright: © 2021 by the authors.
the size of fat globules by the combined effect of cavitation, shear, and impact [3].
Licensee MDPI, Basel, Switzerland.
The incorporation of emulsifiers, such as proteins, into nanoemulsions is essential to
This article is an open access article
facilitate the formation of small droplets during homogenization after absorption at the
distributed under the terms and
conditions of the Creative Commons
interface, reducing the interfacial tension and protecting droplet aggregation by repulsive
Attribution (CC BY) license (https://
steric or electrostatic interactions [4]. Whey proteins are a natural emulsifier widely used
creativecommons.org/licenses/by/ in the formation and stability of O/W food emulsions [5]. These are highly available and
4.0/). economical dairy industry byproducts composed of a mixture of globular proteins (mainly

Foods 2021, 10, 2672. https://doi.org/10.3390/foods10112672 https://www.mdpi.com/journal/foods

Foods 2021, 10, 2672 2 of 17

-LG, ↵-LA, and BSA). After heat and microfluidization denaturation, its hydrophobicity
is increased, improving its capacity to form an interfacial layer around lipid droplets
due to the fast adsorption of whey protein molecules to the droplet surfaces within the
homogenizer and promoting its polymerization, constituting a fluid with film-forming
capacity that can be consolidated via cold gelation and/or dehydration [6,7].
The development of protein-based films has become the focus of research to produce
eco-friendly materials with potential applications as active food packaging, offering their
functionality as a vehicle for the incorporation of active agents, such as aromas and antioxi-
dant compounds [8]. Whey protein films are known for forming excellent barriers against
oxygen, lipids, and aromas and their interesting mechanical properties. However, their
hydrophilic nature and brittle behavior limit their applications as food packaging [9,10].
Recent studies have developed active nanocomposite whey protein films incorporating
nanoemulsions to improve their solubility and permeability and to give them antimicrobial
and/or antioxidant characteristics by adding bioactive compounds to the dispersed phase.
The formulations of these nanocomposite films have generally been developed through
two emulsification stages: the first stage involves the formation of the nanoemulsion, and
the second stage incorporates the nanoemulsion into the film formulation [11–13]. No
studies have shown the process combined in a single homogenization step to generate a
nanoemulsion with the ability to form a nanocomposite protein-based film.
Cocoa liquor is an important subproduct of cocoa beans (Theobroma cacao) produced
from dried, fermented, and roasted cocoa beans that are separated from their skins and
ground to form cocoa liquor, a product that is rich in polyphenols with antioxidant prop-
erties [14]. There is a growing interest in the incorporation of natural antioxidants into
active protein films with potential applications in the food industry [9]. However, cocoa
liquor has a high fat content (cocoa butter) that causes it to solidify at temperatures under
30 C [15], and its hydrophobic nature makes it incompatible with aqueous food systems,
requiring emulsification technologies, such as microfluidization, and the use of proteins as
emulsifying agents to diversify cocoa liquor applications in the food industry.
Therefore, the interest of this work was to optimize the process to obtain a nanocom-
posite film with cocoa liquor nanoemulsified by one-stage microfluidization, using whey
proteins as an emulsifying and film-forming agent, evaluating the effect of the microflu-
idization conditions on the physicochemical properties and antioxidant capacity of the
nanoemulsion, the effect of the cocoa liquor on the properties and microstructure of the film,
and its application as an edible coating to improve the shelf life of muffins as a model food.

2. Materials and Methods

2.1. Materials
The cocoa liquor (CL) sample was acquired from Oaxaca, Mexico. The CL presented a
46.4 ± 1.57% fat content, a 4.70 ± 0.90% moisture content, and a water activity value of
0.39 ± 0.04. Whey protein concentration 80 (WPC) was purchased from Vilher (Empresas
Vilher, S.A. de C.V., Guadalajara, Mexico).

2.2. Preparation of the Nanoemulsions

Nanoemulsions of CL were prepared with WPC as emulsifier. The protein was
solubilized in distilled water to obtain a concentration of 8% (w/w) and was mixed with
6% (w/w) glycerol (G). The protein solution was denatured in a Thermomix® vortex
operated at 400 rpm and 80 C for 30 min. Then, the CL was incorporated into the protein
solution and homogenized at 400 rpm at 50 C for 15 min. The pre-emulsion was fed into a
microfluidizer to obtain the nanoemulsions under different conditions: homogenization
pressure (20,000, 25,000, and 30,000 PSI or in units equivalent to 138.9, 172.4, and 206.8 MPa)
and the number of cycles (3, 5, and 7 cycles), varying the concentration of CL (0.5, 2.25, and
4%, w/w). Each emulsion was evaluated.
Foods 2021, 10, 2672 3 of 17

2.2.1. Characterization of the Nanoemulsions

The nanoemulsions were characterized in terms of their particle size (recorder as the
D(4,3) diameter), polydispersity index (PdI), and zeta potential (⇣-potential) using a Zeta-
sizer Nano ZS (Malvern Instruments, Worcestershire, UK) [2]. The total polyphenol content
(TPC) was measured by the Folin–Ciocalteu colorimetric method [16]. The TPC retention
(%) of the nanoemulsions (encapsulation efficiency) was calculated with Equation (1):

TPC retention = (TPCR ÷ TPC1 ) ⇥ 100 (1)

where TPCR is the TPC remaining within the droplets of the nanoemulsion, and TPC1 is
the content initially added to the emulsion. The antioxidant capacity was evaluated by the
DPPH radical inhibition method. All of the results obtained were analyzed using response
surface methodology.

Effect of the Microfluidization Parameters on the Emulsification of the Cocoa Liquor

Response surface methodology (RSM) was employed to study the effects of the in-
dependent variables: cocoa liquor concentration (x1 ), homogenization pressure (x2 ), and
the number of homogenization cycles (x3 ), on the particle size (y1 ), polydispersity index
(y2 ), ⇣-potential (y3 ), polyphenol retention (y4 ), and DPPH radical inhibition (y5 ) of the
nanoemulsions. The experiments were approached using a central composite design of 33 .
The experiments were generated by Design-Expert version 7.0.0 by Stat-Ease Inc. (Min-
neapolis, MN, USA). A total of 20 experiments were conducted, including the central
point, with five repetitions. The design matrix was created, and the results were statisti-
cally analyzed and then converted into a response surface. The designs were evaluated
separately based on the influence of the independent variables in the modeling of the
nanoemulsion-dependent variables. The experimental data were fitted to a second-order
polynomial equation as follows (Equation (2)):

y = b 0 + b 1 x1 + b 2 x2 + b 3 x3 + b 12 x1 x2 + b 13 x1 x3 + b 23 x2 x3 + b 11 x12 + b 22 x22 + b 33 x32 + b 123 x1 x2 x3

(2)
+ b 112 x12 x2 + b 113 x12 x3 + b 122 x1 x22

where y is the dependent response; x1 , x2 , and x3 are the levels of the independent vari-
ables; and b is the adjusted parameter in the model. The significance of the estimated
regression coefficient for each response variable was assessed using F-ratios at a probability
(p) of 0.05. The adequacy of the response surface models was determined using coefficient
of determination (R2 ) analysis. Only the significant terms (p < 0.05) were included in
the model.

2.3. Preparation of the Films

Films of the CL and WPC nanoemulsion were formulated. WPC (8% w/w) and G (used
as a plasticizer) were solubilized in distilled water. The protein solution was denatured
in a Thermomix® vortexer operated at 400 rpm and 80 C for 30 min. Then, the cocoa
liquor was incorporated into the protein-plasticizer solution and homogenized at 400 rpm
at 50 C for 15 min. The pre-emulsion was processed on the microfluidizer by 5 cycles at
25,000 PSI (172.4 MPa). The films were dried at 25 C and 50% relative humidity for 24 h
and then peeled off for testing. In the formulation, the concentration of cocoa liquor (0, 1,
and 2%, w/w) and plasticizer (5, 6, and 7%, w/w) of the total film-forming solution were
varied. Each edible film formulation was evaluated.

2.3.1. Film Characterization

The solubility of the films was evaluated according to the method of Kurt et al. [17].
The water vapor permeability (WVP) of the films was measured gravimetrically, and the
mechanical properties (tensile strength (TS, MPa) and elongation at break (EAB, %) of
the films were evaluated using a texture analyzer (TA XT plus, Stable Micro Systems,
Godalming, UK) [18]. The results were analyzed using RSM.
Foods 2021, 10, 2672 4 of 17

Effect of the Formulation on the Properties of the Films

RSM was generated to study the effects of the independent variables: cocoa liquor
concentration (x1 ), and plasticizer concentration (x2 ), on the solubility (y1 ), WVP (y2 ), TS
(y3 ), and EAB (y4 ) of the films. The experiments were approached using a central composite
design of 32 . A total of 13 experiments were conducted, including the central point, with
five repetitions. The response surface was obtained separately for each dependent variable.
The experimental data were fitted to a second-order polynomial equation (Equation (2)).
The significance of the response surface models was estimated (p < 0.05), and adequacy
(R2 ) was determined.

2.3.2. Microstructure Analysis by CLSM

The autofluorescence of the nanoemulsions was initially evaluated using a Cytation3
Cell Imaging Multi-Mode Reader (BioTek Instruments) to determine the conditions to visu-
alize their fluorescence by confocal laser scanning microscopy (CLSM). The images were
acquired using CLSM with a Leica confocal microscope (DM 5500 Q, Wetzlar, Germany)
equipped with a 63 ⇥ 1.3 oil objective using an excitation wavelength of 405 nm and an
amplitude of 420–600 nm.

2.3.3. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR analysis of the cocoa liquor and film samples was performed using Cary 630 Fourier
Transform InfraRed (FTIR) spectroscopy with an attenuated total reflectance (ATR) diamond
accessory (Agilent Technologies, Mississauga, Canada) at 500–4000 cm 1 wavelengths.

2.4. Coating Application on the Muffins

Two formulations were selected to evaluate their potential application as a coating
for muffins as a model food due to their high moisture content and compatibility. Freshly
baked samples were dipped into film solution blends (coating WPC: solution with 8% WPC
and 6% G; coating WPC/CL: solution with 8% WPC, 6% G, and 2% CL) for 5 s at room
temperature. The excess blend was allowed to drip. Then, the coated muffins were dried at
40 C for 15 min to solidify the coating.

Stability of the Muffins during Storage

The muffins were divided into 3 batches: control muffins (uncoated), WPC muffins
(with WPC coating without cocoa liquor), and WPC/CL muffins (with WPC coating and
nanoemulsified cocoa liquor). The weight loss and moisture, water activity, and texture
parameters of the muffins after they were stored for 5 days at 20 C and under different
controlled conditions of relative humidity (20, 50, and 80% RH) were measured. The
muffins were ground and then analyzed with an Aqualab Series 3 water activity meter
(Labcell Ltd., Basingstoke, Hants, UK) and dried at 105 C for 24 h to evaluate their moisture
content. The texture of the muffins was evaluated using a TA-XTplus texture analyzer
(Stable Micro Systems, Godalming, UK) according to the method of Bartolozzo et al. [19].
Hardness, springiness, and resilience parameters were obtained from the time–force curve.

2.5. Statistical Analysis

All experimental values included in the RSM represent the value of the mean of a tripli-
cate analysis (n = 3). The standard deviation (SD) from the mean of the experimental values
was also calculated. Data analysis was performed using XLSTAT 2019. 2.3 (Addinsoft,
Boston, MA, USA).

3. Results and Discussion

3.1. Effect of Microfluidization Parameters on the Emulsification of Cocoa Liquor
The effects of the microfluidization parameters and the cocoa liquor concentration on
the physicochemical properties and the antioxidant capacity of the nanoemulsion were
studied (Table 1).
Foods 2021, 10, 2672 5 of 17

Table 1. Central composite experimental design, presenting the effect of the independent variables on the physicochemical
properties and antioxidant capacity of the nanoemulsion. The total number of runs was generated by Design Expert version
7.0.0 by Stat-Ease Inc. (Minneapolis, MN, USA).

Independent Variable Response

Cocoa Homogenization Number of Polydispersity TPC DPPH•
Liquor Pressure Cycles Particle Size Index ⇣-Potential Retention Inhibition
Run (x1 ) (x2 ) (x3 ) (y1 ) (y2 ) (y3 ) (y4 ) (y5 )

% PSI (Equivalent d nm mV % %
in MPa)
1 4.00 25,000 (172.4) 5 232.00 0.42 22.30 98.73 28.38
2 5.00 20,000 (138.9) 7 209.50 0.36 22.30 98.54 3.39
3 0.50 30,000 (206.8) 3 210.26 0.38 21.60 93.05 5.44
4 2.25 25,000 (172.4) 5 217.33 0.39 22.70 96.51 15.01
5 2.25 25,000 (172.4) 5 212.13 0.37 22.90 95.12 13.50
6 0.50 25,000 (172.4) 5 212.13 0.33 22.83 93.99 8.04
7 2.25 30,000 (206.8) 5 213.10 0.40 22.83 95.36 12.61
8 2.25 25,000 (172.4) 5 213.38 0.38 21.90 97.20 14.32
9 4.00 30,000 (206.8) 7 251.43 0.55 20.03 78.50 35.12
10 4.00 30,000 (206.8) 3 261.76 0.45 20.30 83.03 34.29
11 2.25 25,000 (172.4) 5 219.24 0.39 23.30 97.45 14.20
12 0.50 20,000 (138.9) 3 227.86 0.40 22.10 97.76 1.62
13 2.25 25,000 (172.4) 5 221.33 0.39 22.76 96.51 13.83
14 2.25 20,000 (138.9) 5 226.43 0.45 22.90 97.13 11.24
15 0.50 30,000 (206.8) 7 206.76 0.36 22.33 94.19 5.10
16 2.25 25,000 (172.4) 5 214.43 0.40 22.63 94.95 13.83
17 2.25 25,000 (172.4) 7 215.96 0.42 22.10 90.09 13.90
18 4.00 20,000 (138.9) 7 225.70 0.38 22.23 87.85 29.38
19 2.25 25,000 (172.4) 3 213.36 0.42 23.63 99.64 12.28
20 4.00 20,000 (138.9) 3 258.66 0.41 20.16 89.39 34.79
x: represents the independent variables plotted on the x-axis; y: represents the response plotted on the y-axis. Response is the mean value
of three replicates. TPC = Total Polyphenol Content; DPPH• = 1-DiPhenyl-2-PicrylHydrazil radical; PSI: Pounds per Square Inch unit;
MPa: MegaPascal unit.

Figure 1 shows the RSM of the effect of the independent variables on the particle size,
polydispersity, total polyphenol content, and antioxidant capacity. Figure 1a,b shows the
effects of the independent variables on the particle size and PdI after microfluidization at
25,000 PSI (172.4 MPa). Both models were significant (p < 0.05), with R2 values of 0.957
and 0.846, respectively. The factors x1 (cocoa liquor content) and x2 (number of cycles)
had a strong effect on the particle size and the cocoa liquor concentration (x1 ) on the PdI.
This was not the case for factor x3 (homogenization pressure) for both dependent variables.
The final models, with the insignificant terms (p > 0.05) eliminated, were expressed by the
quadratic polynomial Equations (3) and (4):

Particle size = 424.48 11.73x1 30.45x3 + 0.007x1 x2 + 0.0047x2 x3 + 1.64x32 (3)

Polydispersity index = 1.45 0.04x1 + 0.00003x1 x2 + 0.00002x2 x3 (4)

The reduced droplet size of the emulsified fats can improve the mechanical properties
of protein-based films [13], but this depends on the operating pressure and the number
of cycles that determine the disruptive energy needed to break up the droplets [5]. In this
work, the particle size of the emulsions ranged from 206 to 261 nm with a monomodal
size distribution (PdI from 0.32 to 0.54) under all microfluidization conditions studied
(Figure 1a,b). These results indicate the efficient disruption of the cocoa liquor in small
droplets by microfluidization. The droplet diameter decreases with increasing homoge-
nization pressure for whey proteins [1]. The nonsignificant effect of the pressure on the
particle size may indicate that the pressures studied are located at the maximum disruptive
energy necessary for the formation of the droplets. The particle size decreased with a
greater number of cycles. A higher number of cycles ensures that the emulsion droplets
undergo a uniform disruptive force [2], increase the surface hydrophobicity of whey pro-
teins, and favor its ability to emulsify smaller fat globules [20]. The combined effect of the
previous denaturation by heating and the cycles of microfluidization could have increased
the adsorbed denatured whey proteins on the oil droplet surface, favoring smaller droplet
sizes [21]. A higher cocoa liquor concentration resulted in a larger droplet diameter under
Foods 2021, 10, 2672 6 of 17

constant homogenizing conditions. This was associated with insufficient protein molecules
available to cover the newly formed droplet surfaces when the oily phase increases, with an
induced aggregation of droplets and an increase in viscosity that diminished the efficiency
of droplet disruption, and/or with a higher rate of collision frequency of the droplets
formed [2,4]. The ⇣-potentials of the emulsions obtained under the conditions studied were
very similar, at 21.83 ± 1.8 mV at neutral pH, so its model was not included. These results
might indicate that the droplet interface composition was not altered. The ⇣-potential
values of the emulsions are related to the net negative charge of the whey protein-coated
droplets at neutral pH, in which the protein has more negatively charged carboxyl groups
Foods 2021, 10, x FOR PEER REVIEW 7 of 17
and constitutes a barrier to the droplets approaching electrostatic repulsion, resulting in
more stable emulsions [5].

Figure 1. Three-dimensional response surface plot for the interactive effect of independent variables
Figure
(cocoa1.liquor
Three-dimensional
concentrationresponse surface plot for
and homogenization the interactive
cycles) effect of
on: (a) particle independent
size; variablesindex;
(b) polydispersity
(cocoa liquor concentration and homogenization cycles) on: (a) particle size; (b) polydispersity in-
(c) polyphenols retention; and (d) DPPH radical inhibition of the nanoemulsion with 8% of whey
dex; (c) polyphenols retention; and (d) DPPH radical inhibition of the nanoemulsion with 8% of
protein
whey afterafter
protein microfluidization at 25,000
microfluidization PSIPSI
at 25,000 (172.4 MPa).
(172.4 MPa).

Figure 1c,d indicates the effect of the independent variables on the TPC retention and
The TPC retention ranged from 78 to 99%, and the DPPH radical inhibition ranged
from 1 to 35% at acapacity
the antioxidant of the of
concentration emulsions
100 mg ofafter microfluidization
nanoemulsion per mL.atHigher
25,000values
PSI (172.4
wereMPa).
2 values of 0.987 and 0.981, respectively.
The models were significant (p < 0.05) with R
observed for higher cocoa liquor contents. Higher TPC retention was observed for fewer
The cocoa liquor
homogenization concentration
cycles. Despite the(x 1 ) and homogenization
decrease cycles
in TPC, an effect of (x2 ) had a strong
microfluidization on theeffect
on TPC retention, while only the cocoa liquor concentration (x 1 ) determined
antioxidant capacity of the emulsion was not observed. Similar results were previously the DPPH
radical inhibition
observed by Karacam of et
theal.emulsion. Thebepressure
[3]. This may ofan
related to homogenization did not affect
increase in the temperature any of
that
favors the oxidation and/or polymerization of polyphenols, generating compounds with
greater antioxidant capacity [16].
The microfluidization conditions of five cycles at 25,000 PSI (172.4 MPa) were se-
lected that would obtain a smaller diameter and size distribution of the nanoemulsion that
can improve film properties, favoring a more homogeneous particle distribution through-
Foods 2021, 10, 2672 7 of 17

the dependent properties (p > 0.05). The final models for TPC retention and antioxidant
capacity are represented by Equations (5) and (6):

TPC retention (%)

= 294.3 109.8x1 + 9.14x3 2.81x1 x3 0.41x12 + 0.00003x22 0.79x32 0.001x12 x2 (5)
+0.69x12 x3 0.00002x1 x22
DPPH• inhibition (%) = 31.27 + 8.46x1 + 1.79x12 (6)
The TPC retention ranged from 78 to 99%, and the DPPH radical inhibition ranged
from 1 to 35% at a concentration of 100 mg of nanoemulsion per mL. Higher values were
observed for higher cocoa liquor contents. Higher TPC retention was observed for fewer
homogenization cycles. Despite the decrease in TPC, an effect of microfluidization on the
antioxidant capacity of the emulsion was not observed. Similar results were previously
observed by Karacam et al. [3]. This may be related to an increase in the temperature that
favors the oxidation and/or polymerization of polyphenols, generating compounds with
greater antioxidant capacity [16].
The microfluidization conditions of five cycles at 25,000 PSI (172.4 MPa) were selected
that would obtain a smaller diameter and size distribution of the nanoemulsion that can
improve film properties, favoring a more homogeneous particle distribution throughout
the polymer network [22] and preserve to a greater extent the cocoa liquor antioxidant
capacity to offer its greatest potential in the development of active packaging material.

3.2. Effect of the Formulation on the Properties of the Film

In the second stage, the effect of the formulation treated under the microfluidiza-
tion conditions previously selected for the nanocomposite film properties was evaluated
(Table 2).

Table 2. Central composite experimental design, presenting the effect of the independent variables on the film properties.
The total number of runs was generated by Design Expert version 7.0.0 by Stat-Ease Inc.

Independent Variable Response

Cocoa Liquor Plasticizer Solubility WVP TS EAB
Run (x1 ) (x2 ) (y1 ) (y2 ) (y3 ) (y4 )
% % % g mm h 1 m 2 Kpa 1 MPa %
1 1.00 6.00 35.28 1.67 0.94 24.00
2 1.00 7.00 35.82 1.65 1.17 11.71
3 2.00 5.00 21.87 1.57 2.69 8.06
4 2.00 7.00 33.70 2.72 1.94 7.43
5 0.00 6.00 34.88 3.97 1.82 22.76
6 1.00 5.00 34.07 2.80 1.96 8.74
7 1.00 6.00 32.97 2.46 0.86 16.16
8 2.00 6.00 20.99 1.27 1.10 11.54
9 1.00 6.00 36.13 1.60 1.13 25.53
10 1.00 6.00 34.15 2.08 0.93 20.17
11 0.00 5.00 38.94 3.21 3.71 27.23
12 1.00 6.00 33.39 1.49 0.97 17.02
13 0.00 7.00 44.36 4.21 2.15 14.11
x: represents the independent variables plotted on the x-axis; y: represents the response plotted on the y-axis. Response is the mean value
of three replicates. WPV: Water Vapor Permeability; TS: Tensile Strength; EAB: Elongation At Break.

Figure 2a shows the effect of the independent variables on the solubility of the films.
The model was significant (p < 0.05) and adequate (R2 = 0.927). The cocoa liquor concen-
tration (x1 ) had a strong effect on the solubility. The final model for the film solubility is
shown below:
Solubility (%) = 149.08 + 31.59x1 + 3.56x22 (7)
Foods 10,2672
2021,10,
Foods2021, x FOR PEER REVIEW 10 of 178 of 17

Three-dimensional
Figure2.2.Three-dimensional
Figure response
response surface
surface plot
plot forfor
thethe interactive
interactive effect
effect of independent
of independent variables
variables
(cocoaliquor
(cocoa liquorconcentration
concentrationandand plasticizer
plasticizer concentration)
concentration) on:
on: (a) (a) solubility;
solubility; (b) water(b)vapor
waterperme-
vapor per-
meability;
ability; (c) tensile
(c) tensile strength;
strength; and and (d) elongation
(d) elongation at break
at break of theofwhey
the whey protein-based
protein-based films films
with with
8% 8%
protein
proteincontent.
content.

3.3. Microstructure of the Emulsions

The film solubility ranged and
from Films
20.99 to 44.36 percent. The low solubility of the
filmsFor
wastheassociated
analysis of with protein denaturation,
the microstructure which introduces
of the nanoemulsions and films, strong interactions
two formula-
and disulfide
tions bonds
were selected betweenaprotein
comprising strands [13].
control (without cocoaThe films
liquor) andloaded
a second with
withcocoa liquor
incor-
showedcocoa
porated lowerliquor.
waterThe solubility
physical(pappearance
< 0.05), possibly
of both related to the formation
the nanoemulsions of a more
and selected
compact
films structure
are shown due to the
in Figures greater
S1 and molecular interactions
S2, respectively. of thethe
Figure 3a shows blended film and of
O/W emulsion by the
physical
cocoa effect of at
liquor/WPC thea nonpolar
5% dilution components
in the brightthat reduced
field, observingthetwo
interaction
types of of thestruc-
main hydroxyl
groups
tures, oneofcorresponding
the protein chains with theofwater
to the droplets molecules
the emulsion (marked[11–13]. Highly
with white water-soluble
arrows) and
other
films structures
can be used thataswere larger,
edible irregular,
coatings, and translucent,
whereas insoluble filmspossibly
can corresponding to
provide a protective
soluble aggregates of whey proteins (WPC) like a gel formed at neutral
function for products with a high moisture content [9]. This suggests that the obtained pH by the com-
bined effect of
WPC/cocoa heat films
liquor treatment
haveand microfluidization
potential applicationsby as shelf assembly
a coating, withvia hydrophobic
greater effectiveness
attractions and disulfide bond formation [7,21]. This was corroborated by
for products with a low moisture content. Pluta-Kubica et al. [22] reported the solubility analyzing the
fluorescence emission of the same sample at 415 nm (Figure 3b), observing
of furcellaran/whey protein films incorporated with antioxidant extracts (yerba mate small fluores-
cent
and particles
white tea)(shown
to bein green) corresponding
between 47% and 52%,tosuggesting
the emulsified
thatcocoa
cocoa liquor
liquordroplets,
can benot a great
the irregular and large structures that may be associated with protein aggregates formed
alternative for the incorporation of natural antioxidant compounds into whey protein-based
by an excess of nonadsorbed proteins in the continuous phase that are essential for
films with decreased water solubility.
Figure 2b shows the effect of independent variables on the permeability of the films.
The model was significant (p < 0.05) with an R2 of 0.942. The cocoa liquor concentration (x1 )
Foods 2021, 10, 2672 9 of 17

had a strong effect on the permeability of the films. The final model for WVP is represented
by Equation (8):
⇣ ⌘
WVP g mm h 1 m 2 kPa 1 = 7.34 + 30.74x1 5.94x12 + 1.11x12 x2 (8)

The WVP ranged from 1.27 to 4.21 g mm h 1 m 2 kPa 1 . An increase in WVP with
an increasing plasticizer amount was observed (Figure 2b). Plasticizers are hygroscopic
molecules that have an adverse effect on WVP due to increased molecular mobility and a
less dense protein network with a large free volume, permitting greater diffusion of water
through the film matrix [10]. Additionally, the results showed a lower permeability of
the films with increasing cocoa liquor content. This was probably due to the presence of
uniformly dispersed nanoinclusions in the polymer matrix leading to an impermeable
and nonporous polymeric structure [10], and by the generation of an interconnecting lipid
network within the protein matrix, which increased the hydrophobicity, reducing the
water absorption through the film [22]. The permeability of the films obtained is similar to
that reported by Talens and Krochta [23] in whey protein-based films incorporated with
beeswax or carnouba wax, but with lower permeability compared to the values reported in
whey proteins-based films incorporated with fatty acids (>4 g mm h 1 m 2 kPa 1 ) [24] or
with oil of oregano (>8.5 g mm h 1 m 2 kPa 1 ) [25].
Figure 2c,d shows the effect of the independent variables on the mechanical properties
of the films. The models for the tensile strength (TS) and elongation at break (EAB) were
significant (p < 0.05) with R2 values of 0.965 and 0.812, respectively. TS was strongly affected
by the cocoa liquor (x1 ) and plasticizer (x2 ) content. Only the cocoa liquor content (x1 )
affected the film elasticity. The final models for the mechanical properties are represented
by the following Equations (9) and (10):

Tensile strength (MPa) = 37.65 3.07x1 11.17x2 + 0.77x12 + 0.87x22 (9)

Elongation at break (%) = 178.64 84.28x1 6.77x22 (10)

The TS is required to maintain the structural integrity of films. A high TS indicates a
high mechanical resistance [6]. The TS of the films ranged from 0.86 to 3.71 MPa. Similar
values were previously reported in formulations of whey protein-based films [22]. These
results showed that the increase in plasticizer favored a less resistant film structure. A
similar phenomenon was reported by Sogut [10], associated with a compatibility that was
exceeded between the polymer and the plasticizer, causing destabilization and decreased
cohesion by reducing intermolecular forces along the polymer chain [6]. Cocoa liquor
reduces the strength of the film. Protein-based films loaded with nanoemulsions showed
a considerable reduction in TS attributed to the inability of lipids to form continuous
and cohesive matrices [11,22]. These results were similar to the effect observed in whey
proteins-based films with the incorporation of beeswax or carnauba (TS ⇡ 0.5–3.5 MPa) [23].
The EAB determines the flexibility of the film, which is desirable for easy handling [10].
The EAB was between 7.43% and 27.23%. The values were similar to those reported in
recent formulations of whey protein-based films [6,9]. A significant decrease in the EAB
of the films was observed after the incorporation of cocoa liquor. The addition of O/W
nanoemulsions to protein-based film formulations increased their elasticity through a
plasticizing effect. However, this effect is strongly dependent on the lipid characteristics
and its capacity to interact with the biopolymeric matrix [11,12]. The lipids of the cocoa
liquor are solid at room temperature. Therefore, these results may have been caused by the
reduced mobility of the protein chains due to the rigid structure conferred by cocoa butter
and/or caused by stronger interactions within the cocoa liquor components, such as the
polyphenolic compounds, with the protein chains that act as crosslinkers [9].
Foods 2021, 10, 2672 10 of 17

3.3. Microstructure of the Emulsions and Films

For the analysis of the microstructure of the nanoemulsions and films, two formula-
tions were selected comprising a control (without cocoa liquor) and a second with incorpo-
rated cocoa liquor. The physical appearance of both the nanoemulsions and selected films
are shown in Figures S1 and S2, respectively. Figure 3a shows the O/W emulsion of cocoa
liquor/WPC at a 5% dilution in the bright field, observing two types of main structures,
one corresponding to the droplets of the emulsion (marked with white arrows) and other
structures that were larger, irregular, and translucent, possibly corresponding to soluble
aggregates of whey proteins (WPC) like a gel formed at neutral pH by the combined effect
of heat treatment and microfluidization by shelf assembly via hydrophobic attractions
and disulfide bond formation [7,21]. This was corroborated by analyzing the fluorescence
emission of the same sample at 415 nm (Figure 3b), observing small fluorescent particles
(shown in green) corresponding to the emulsified cocoa liquor droplets, not the irregular
and large structures that may be associated with protein aggregates formed by an excess
of nonadsorbed proteins in the continuous phase that are essential for polymerization
and the consequent film formation. This acts as a “glue” between adsorbed protein lay-
ers of neighboring droplets, inducing aggregation of the emulsion droplets [26]. This
phenomenon is shown in Figure 3c,d, which shows the fluorescence micrograph of the
undiluted emulsions with a content of 1 and 2% cocoa liquor, respectively. The presence of
the emulsion droplets, and larger fluorescent emulsion droplet aggregates favored by a
higher amount and formation of hydrophobic interactions and thiol-disulfide interchanges
are observed, resulting in gelation between droplets by the combined effect of heating and
microfluidization [26].
Figure 3e–g shows micrographs of the whey protein/cocoa liquor films formed.
Figure 3e shows the WPC film without cocoa liquor that did not show autofluorescence,
followed by films with 1% and 2% emulsified cocoa liquor (Figure 3f,g, respectively). A
homogeneous distribution of the nanoemulsion can be highlighted throughout the poly-
merized protein matrix, showing in both cases some dark spaces possibly associated with
soluble protein aggregates of excess nonadsorbed protein that does not participate in the
emulsification of the cocoa liquor but is essential for the formation of the polymeric network
and/or is associated with the possible presence of pores.

3.4. FTIR Spectroscopy Analysis

The FTIR spectra of the films with and without cocoa liquor were slightly different,
indicating that the addition of cocoa liquor affected the film structure (Figure 4). The cocoa
liquor spectrum presented important signals at 2916, 2851, and 1740 cm 1 , which were
observed in all film samples with incorporated cocoa liquor. An increase in the intensity of
each signal with the increase in the content of the cocoa liquor in the film was observed. The
range between 3000 and 2800 cm 1 was assigned to the C–H stretching vibrations of alkane
groups in the film chains and emulsified fats [10,13,27]. The bands at 2916 and 2851 cm 1
can be assigned to the C-H aromatic ring of some molecules present in the cocoa liquor, such
as polyphenols [28], and the band at 1740 cm 1 may be associated with a C=O stretching
vibration likely caused by cocoa liquor triglycerides and carbonyl structures of aldehydes or
esters [8,29]. Other changes in important signals different from those for cocoa liquor were
observed. The intensity of the 1030 cm 1 band showed a slight increase with increasing
plasticizer in the film, which is attributed to the C–O stretching characteristic of glycerol [30].
All film samples showed a broad band located at 3000–3600 cm 1 , corresponding to the
stretching vibration of free and bound –OH and –NH groups of the protein matrix [8,10].
Between 1700 and 1500 cm 1 , high peaks were observed. The peptide bond is normally
represented by a C=O stretching vibration at the amide I band (1600–1700 cm 1 ) and the N–
H bending vibration and C–N stretching vibration at the amide II band (1500–1600 cm 1 ).
A different behavior was found in the amide I band among the film samples. The amide I
band is the most useful peak for infrared analysis of secondary protein structures (↵-helix,
-sheet, -turn, and random) of polypeptides or proteins [31]. Four peaks were observed
Foods 2021, 10, 2672 11 of 17

in the amide I region of films with emulsified cocoa liquor, located at 1622, 1634, 1649, and
1690 cm 1 (Figure 4). The films without cocoa liquor only presented the first three. These
four bands have been previously reported in several studies [32,33]. In all film samples, the
highest peak was observed at 1622 cm 1 . This signal is assigned to intermolecular -sheets
resulting from aggregations during network formation. The bands at 1634 also indicate
the formation of intramolecular -sheet structures, the bands at 1649 cm 1 correspond
to ↵-helices, and 1690 cm 1 corresponds to anti-parallel -sheets or -turns that were
only present in the films with higher cocoa liquor content regardless of the amount of
plasticizer. Therefore, the addition of cocoa liquor to the film formulation is accomplished
by modifying the secondary structure of whey proteins, favoring the unfolded proteins
(random coil) folded into ↵-helix and -turn structures more than the -sheet during the
rebuilding of the secondary structure in the emulsification and film formation process [32].
The major ↵-helix and -sheet structures formed with the incorporation of cocoa liquor
were corroborated with their respective spectral amide III regions arising at 1295 and
1220 cm 1 , respectively [27]. A shift of the band at 1621 cm 1 to 1627 cm 1 was observed
with the incorporation of the cocoa liquor in the films (Figure 4). The diminished position of
the band indicates a strengthening of the intermolecular hydrogen bonds between -sheets
and it is associated with the effect of the ↵-helix structure formed [33]. The molecular
protein conformation can be related to the mechanical properties of the films. These results
indicate that cocoa liquor favors -turns and -sheet folds that contribute to diminishing
the flexibility and tensile strength of the films, possibly because the more ordered -sheets
produce a stronger protein network through hydrogen bonding [31].

3.5. Coating Application on Muffins and Storage Study

Images of the appearance of the muffins with the different coatings studied are shown
in Figure S3, observing that the application of the coatings was primarily associated with
a brighter surface compared to the uncoated muffins. The effect of the coatings on the
quality properties of the muffins during their shelf life under the studied conditions is
shown in Figure 5. The kinetic parameters calculated from the charts (parameter vs. time)
are shown in Table 3. The results indicate that under conditions of relative humidity
(RH) of 50 and 20%, there was a significant decrease in the weight (Figure 5a), moisture
content (Figure 5b), and water activity (aw ) (Figure 5c) in the muffins. At 80% RH, the
properties of the muffins (coated and uncoated) did not change significantly during storage.
This can indicate that the relative humidity of 80% is close to the moisture content of the
product, and a higher difference between the moisture content of the product with respect
to that of the surrounding medium, observed at a lower relative humidity, favors a greater
transfer amount of moisture from the food to the outside environment, which leads to the
product losing weight. Under conditions of intermediate relative humidity (50%) and low
relative humidity (20%), a lower loss of humidity and aw was evident in the coated muffins
compared to the control. These results suggest that the coating on the surface of the muffins
hindered moisture loss and improved their water holding capacity during storage [34].
 sorbed protein layers of neighboring droplets, inducing aggregation of the emulsion drop-
lets [26]. This phenomenon is shown in Figure 3c,d, which shows the fluorescence micro-
graph of the undiluted emulsions with a content of 1 and 2% cocoa liquor, respectively.
Foods 2021,The presence of the emulsion droplets, and larger fluorescent emulsion droplet aggregates12 of 17
10, 2672
favored by a higher amount and formation of hydrophobic interactions and thiol-disul-
fide interchanges are observed, resulting in gelation between droplets by the combined
effect of heating and microfluidization [26].
Texture (hardness, resilience, and springiness) is one of the main characteristics of
bakery products, and the loss of the
Figure 3e–g shows micrographs of the whey protein/cocoa desired texture liquor
results in a shorter
films shelfFig-
formed. life. The
ure 3e shows the WPC film without cocoa liquor that did not show autofluorescence, fol- due
firmness or hardness of the muffins constitutes the most important texture parameter
to its strong correlation with the consumer’s perception of the freshness of the product [19].
lowed by films with 1% and 2% emulsified cocoa liquor (Figure 3f,g, respectively). A ho-
Therefore, the increase in hardness may be associated with the loss of moisture of the prod-
mogeneous distribution
uct andof itsthe nanoemulsion
freshness. Consideringcantheberesults
highlighted
obtainedthroughout the polymer-
under conditions of low relative
ized protein matrix, showing
humidity in both
(20%), it cancases some that
be observed darkthe
spaces
muffinspossibly
with theassociated
WPC/CL coatingwith proved
sol- to
be moreof
uble protein aggregates effective
excessrelative to the samples
nonadsorbed withthat
protein the WPC
doescoating and controlin
not participate during
the stor-
age, presenting a k value significantly lower for weight loss (
emulsification of the cocoa liquor but is essential for the formation of the polymeric net- 7.42 ± 0.44 (g/100 g)/day),
moisture ( 16.83 ± 0.52 (g/100 g)/day), and aw ( 0.080 ± 0.00 aw /day) and a smaller
work and/or is associated with the possible presence of pores.
increase in hardness (8.87 ± 1.45 N/day).

Figure 3. Microstructure
Figure of the (a) nanoemulsion
3. Microstructure with 8% Whey Protein
of the (a) nanoemulsion with 8%Concentrate (WPC), 6%
Whey Protein Glycerol (G), (WPC),
Concentrate and 2% cocoa
6%
liquor (CL) diluted 5% in the bright field; (b) nanoemulsion with 8% WPC, 6% G, and 2% CL diluted 5%; (c) nanoemulsion
Glycerol (G), and 2% cocoa liquor (CL) diluted 5% in the bright field; (b) nanoemulsion with 8%
with 8% WPC, 6% G, and 1% CL; (d) nanoemulsion with 8% WPC, 6% G, and 2% CL; (e) film with 8% WPC, 6% G without
WPC, 6% G, and 2% CL diluted 5%; (c) nanoemulsion with 8% WPC, 6% G, and 1% CL; (d)
CL; (f) film with 8% WPC, 6% G, and 1% CL; and (g) film with 8% WPC, 6% G, and 2% CL observed by fluorescence with a
nanoemulsion with 8% WPC, 6% G, and 2% CL; (e) film with 8% WPC, 6% G without CL; (f) film
Confocal Laser Scanning Microscope (CLSM). White arrows show emulsion droplets from an amplified field fraction. The
with 8% WPC, 6% G, and 1% CL; and (g) film with 8% WPC, 6% G, and 2% CL observed by fluores-
scale bar represents a length of 10 µm. The green regions represent the autofluorescence of the cocoa liquor polyphenols.
cence with a Confocal Laser Scanning Microscope (CLSM). White arrows show emulsion droplets
from an amplified field fraction. The scale bar represents a length of 10 µm. The green regions rep-
resent the autofluorescence of the cocoa liquor polyphenols.

3.4. FTIR Spectroscopy Analysis

The FTIR spectra of the films with and without cocoa liquor were slightly different,
indicating that the addition of cocoa liquor affected the film structure (Figure 4). The cocoa
FOR PEER REVIEW 13 of 17
Foods 2021, 10, 2672 13 of 17

Figure
Figure4. 4.Comparison
Comparison of FTIR
of absorbance spectra ofspectra
FTIR absorbance the cocoa
ofliquor and whey
the cocoa protein
liquor andfilms withprotein
whey differentfilms
cocoawith
liquor
(CL) (0, 1, and 2%) and glycerol (G) (5, 6, and 7%) contents, indicating the most relevant absorption signals, and the signals
different cocoa liquor (CL) (0, 1, and 2%) and glycerol (G) (5, 6, and 7%) contents, indicating the
associated with the different secondary conformations (↵-helix, -sheet, and -turn) in the amide I region (1700–1600 cm 1 )
most relevant absorption signals, and the signals associated with the different secondary confor-
of the proteins in the films.
mations (α-helix, β-sheet, and β-turn) in the amide I region (1700−1600 cm ) of the proteins in the
−1

films.

3.5. Coating Application on Muffins and Storage Study

Images of the appearance of the muffins with the different coatings studied are
shown in Figure S3, observing that the application of the coatings was primarily associ-
ated with a brighter surface compared to the uncoated muffins. The effect of the coatings
on the quality properties of the muffins during their shelf life under the studied conditions
is shown in Figure 5. The kinetic parameters calculated from the charts (parameter vs.
time) are shown in Table 3. The results indicate that under conditions of relative humidity
x FOR PEER REVIEW 15 of 17
Foods 2021, 10, 2672 14 of 17

Figure 5. Effect of the coatings on the stability parameters of the muffins stored at 20 C and different conditions of relative
humidity
Figure (20, 50, and
5. Effect of80%): (a) weight;on
the coatings (b)the
moisture content;
stability (c) water activity;
parameters and (d) hardness.
of the muffins stored at 20 °C and different
conditions of relative humidity (20, 50, and 80%): (a) weight; (b) moisture content; (c) water activity;
and (d) hardness.

4. Conclusions
The results showed that a greater number of homogenization cycles favored the hy-
drophobicity of the protein, decreasing the particle size and polydispersity of the emul-
sion. Microfluidization did not affect the ζ-potential and had a minimal impact on the
antioxidant capacity of the cocoa liquor, allowing for the retention of a high percentage of
polyphenolic compounds. Five microfluidization cycles with a pressure of 25,000 PSI
(172.4 MPa) were the conditions that favored better physicochemical properties in the
emulsion while also retaining its antioxidant capacity. The plasticizer content significantly
decreased the strength of the film, and the cocoa liquor content decreased its water solu-
bility and water vapor permeability. The latter was associated with modifications of the
Foods 2021, 10, 2672 15 of 17

Table 3. Kinetics parameters from the quality properties of the stored muffins at 20 C and different relative humidity (20,
50, and 80%) for 5 days.

Weight Loss Moisture Loss Water Activity Hardness

Sample RH (%)
r2 k (g/100 g)/day r2 k (g/100 g)/day r2 k aw /day r2 k N/day
Control 80 0.9106 1.86 ± 0.08 a 0.9632 0.72 ± 0.01 a 0.8834 0.002 ± 0.000 a 0.9945 0.27 ± 0.04 a
Coating WPC 80 0.9585 1.66 ± 0.09 a 0.8068 0.56 ± 0.03 b 0.9248 0.002 ± 0.000 a 0.9441 0.29 ± 0.02 a
Coating WPC/CL 80 0.9905 0.86 ± 0.01 b 0.8255 0.60 ± 0.02 b 0.9343 0.002 ± 0.000 a 0.8932 0.28 ± 0.04 a
Control 50 0.977 5.98 ± 0.19 a 0.9994 14.01 ± 0.11 a 0.9291 0.060 ± 0.00 a 0.9457 2.00 ± 0.42 a
Coating WPC 50 0.9757 6.01 ± 0.10 a 0.9919 12.66 ± 0.44 b 0.8913 0.054 ± 0.00 b 0.8683 1.75 ± 0.24 a
Coating WPC/CL 50 0.9924 5.84 ± 0.22 a 0.9957 12.63 ± 0.17 b 0.901 0.050 ± 0.00 c 0.9171 1.58 ± 0.16 a
Control 20 0.9139 9.30 ± 0.70 a 0.9525 21.51 ± 0.29 a 0.9961 0.095 ± 0.00 a 0.8638 15.35 ± 1.09 a
Coating WPC 20 0.9396 8.69 ± 0.48 a 0.9871 20.00 ± 0.19 b 0.9652 0.088 ± 0.00 b 0.8529 11.40 ± 0.53 b
Coating WPC/CL 20 0.9832 7.42 ± 0.44 b 0.994 16.83 ± 0.52 c 0.9235 0.080 ± 0.00 c 0.8075 8.87 ± 1.45 c
Control = uncoated muffins; Coating of Whey Protein Concentrate (WPC) = muffins with coating solution 8% WPC; Coating of WPC and
Cocoa Liquor (CL) (WPC/CL) = muffins with coating solution 8% WPC and 2% CL. RH = Relative Humidity; aw = water activity; k = rate
constant; N = Newtons. Data are expressed as means (n = 5) ± SD. Different letter superscripts within a column indicate statistically
significant differences (p < 0.05) per relative humidity condition.

4. Conclusions
The results showed that a greater number of homogenization cycles favored the
hydrophobicity of the protein, decreasing the particle size and polydispersity of the emul-
sion. Microfluidization did not affect the ⇣-potential and had a minimal impact on the
antioxidant capacity of the cocoa liquor, allowing for the retention of a high percentage
of polyphenolic compounds. Five microfluidization cycles with a pressure of 25,000 PSI
(172.4 MPa) were the conditions that favored better physicochemical properties in the
emulsion while also retaining its antioxidant capacity. The plasticizer content significantly
decreased the strength of the film, and the cocoa liquor content decreased its water sol-
ubility and water vapor permeability. The latter was associated with modifications of
the secondary protein structure influenced by the incorporation of cocoa liquor during
the emulsification and film formation process as evidenced by the FTIR analysis. The
results demonstrate the feasibility and effectiveness of applying WPC-based coatings to
extend the shelf life of bakery products with a high moisture content, such as muffins, and
the contribution of cocoa liquor nanoparticles in decreasing the permeability and loss of
weight and moisture of the product. This novel active nanocomposite whey protein-based
film with cocoa liquor nanoparticles may have potential applications in the food industry
because its characteristics can be mainly used as a coating to control moisture transfer and
prevent oxidation due to its antioxidant properties.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/foods10112672/s1, Figure S1: Appearance of the (A) protein solution with 8% WPC and
6% glycerol (G); and (B) nanoemulsion with 8% WPC, 6% G and 2% CL, Figure S2: Appearance of
the film obtained from (A) protein solution with 8% WPC and 6% glycerol (G); and (B) nanoemulsion
with 8% WPC, 6% G and 2% CL, Figure S3. Appearance of the muffins (A) uncoated (Control);
(B) Coating WPC (with coating 8% WPC and 6% glycerol; and (C) Coating WPC/CL (with coating
8% WPC, 6% glycerol, and 2% cocoa liquor).
Author Contributions: S.d.J.C.-E.: Conceptualization, Methodology, Investigation, Writing—Original
Draft. M.J.-F.: Conceptualization, Methodology, Writing—Review and Editing, Supervision.
A.A.V.-C.: Formal analysis, Validation, Investigation. G.A.C.-H.: Validation, Investigation. E.d.C.L.-C.:
Conceptualization, Resources, Writing—Review and Editing, Supervision. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the SAGARPA Conacyt grant (project number: 2017-02-291417).
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.
Foods 2021, 10, 2672 16 of 17

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