polymers
Article
Nanocellulose from Cocoa Shell in Pickering Emulsions of
Cocoa Butter in Water: Effect of Isolation and Concentration on
Its Stability and Rheological Properties
Catalina Gómez Hoyos 1, * , Luis David Botero 1 , Andrea Flórez-Caro 1 , Jorge Andrés Velásquez-Cock 1
and Robin Zuluaga 2
Citation: Gómez Hoyos, C.; Botero,
L.D.; Flórez-Caro, A.; Velásquez-
Cock, J.A.; Zuluaga, R. Nanocellulose
from Cocoa Shell in Pickering
Emulsions of Cocoa Butter in Water:
Effect of Isolation and Concentration
on Its Stability and Rheological
Properties. Polymers 2023, 15, 4157.
https://doi.org/10.3390/
polym15204157
Academic Editors: Cristina Mihaela
Nicolescu and Marius Bumbac
Received: 17 August 2023
Revised: 13 October 2023
Accepted: 15 October 2023
Published: 19 October 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Polymers 2023, 15, 4157. https://doi.org/10.3390/polym15204157
1 Programa de Ingeniería en Nanotecnología, Universidad Pontificia Bolivariana, Circular 1 N_ 70-01,
Medellín 050031, Colombia; luisd.botero@upb.edu.co (L.D.B.); andrea.florezc@upb.edu.co (A.F.-C.);
jorgeandres.velasquez@upb.edu.co (J.A.V.-C.)
2 Facultad de Ingeniería Agroindustrial, Universidad Pontificia Bolivariana, Circular 1 N_ 70-01,
Medellín 050031, Colombia; robin.zuluaga@upb.edu.co
* Correspondence: catalina.gomezh@upb.edu.co
Abstract: There is a growing interest in developing new strategies to completely or partially replace
cocoa butter in food and cosmetic products due to its cost and health effects. One of these alternatives
is to develop stable emulsions of cocoa butter in water. However, incorporating cocoa butter is chal-
lenging as it solidifies and forms crystals, destabilizing the emulsion through arrested coalescence.
Prevention against this destabilization mechanism is significantly lower than against coalescence.
In this research, the rheological properties of nanocellulose from cocoa shell, a by-product of the
chocolate industry, were controlled through isolation treatments to produce nanocellulose with a
higher degree of polymerization (DP) and a stronger three-dimensional network. This nanocellulose
was used at concentrations of 0.7 and 1.0 wt %, to develop cocoa butter in-water Pickering emulsion
using a high shear mixing technique. The emulsions remained stable for more than 15 days. Nanocel-
lulose was characterized using attenuated total reflection–Fourier transform infrared spectroscopy
(ATR–FTIR), hot water and organic extractives, atomic force microscopy (AFM), degree of poly-
merization (DP), and rheological analysis. Subsequently, the emulsions were characterized on days
1 and 15 after their preparation through photographs to assess their physical stability. Fluorescent
and electronic microscopy, as well as rheological analysis, were used to understand the physical
properties of emulsions.
Keywords: cellulose nanofibers; cocoa shell; cocoa fat; cocoa fat-in-water Pickering emulsions;
arrested coalescence
1. Introduction
The potential applications of nanocellulose in the food and cosmetic industries have
been identified since its development in the 1980s. From that point on, it has been used
in oil and fat stabilization in a wide range of food and cosmetic products [1] and as a
functional ingredient. In vivo tests have demonstrated that CNFs (cellulose nanofibrils)
interact with glucose, limiting its diffusion [2], increasing fecal volume, absorbing harmful
substances, and lowering cholesterol [3]. Nowadays, nanocellulose is not approved by
the FDA (Food and Drug Administration) or EFSA (European Food Safety Authority);
however, recent research suggests that it is a safe food contact material, and cytotoxic or
genotoxic effects have not been identified [4,5]. There is currently a request to the FDA to
include nanocellulose-based wood sources in the GRAS (Generally Recognized as Safe)
list [6].
Nanocellulose has traditionally been isolated from wood and agro-industrial by-
products through a multistep sequence comprising chemical or enzymatic stages, culminat-
ing in a homogenization treatment. However, the exploitation of wood-derived sources
https://www.mdpi.com/journal/polymers
Polymers 2023, 15, 4157 2 of 21

may not be convenient in tropical regions, as it could promote the degradation of native
forests [7]. Therefore, agricultural by-products, such as cocoa shells, emerge as an interest-
ing and readily available option, adding value and reducing the amount of waste produced
by the agroindustry. For instance, cocoa shells represent 12 wt % of cocoa by-products and
are removed either before or after roasting the cocoa nibs. In 2017, Colombia generated
approximately 6.816 t of this by-product [8], which has primarily been used in animal food
formulation, as fuel for boilers, and in fertilizer manufacturing [9].
In a previous study, the authors isolated a suspension of nanocellulose from cocoa
shells using a four-step alkaline sequence followed by a grinding treatment. The final sus-
pension consisted of a combination of nanocellulose, cocoa butter, and hemicelluloses, with
potential applications in the food industry [10]. The presence of non-cellulosic components,
such as cocoa butter, makes nanocellulose from cocoa shell an ingredient for developing oil-
in-water (O/W) Pickering emulsions (PEs). In PEs, dispersed particles with sizes ranging
from just a few nanometers to tens of micrometers participate in stabilizing a multi-phase
structure by providing a physical barrier to emulsion droplet coalescence [11]. The physical
properties of nanocelluloses, such as their high aspect ratio, wettability, and gel rheology,
combined with their characteristics as functional ingredients, make this biopolymer a suit-
able stabilizer for food PEs. The use of nanocellulose as a stabilizer in PEs has been widely
studied, with a clear indication that cellulose concentration, as well as morphological and
surface properties, could be controlled to stabilize different emulsions. These emulsions are
usually prepared with model non-polar phases such as dodecane [12] and with commercial
liquid oils like sunflower [13], canola oil [14], and corn oil [15], among others.
On the other hand, developing PEs with crystalline fats, for instance, cocoa butter, is
also relevant for the formulation of foods. Incorporating this kind of fat into emulsions is
challenging, as it solidifies during its processing and/or storage and forms crystals that
may protrude through the interface, leading to partial coalescence [16], a process in which
two or more crystalline droplets merge to form a single irregularly shaped aggregate [17].
Stability against partial coalescence is usually lower than against coalescence [18]. They
can differ by a factor of 106 or even more for similar emulsions with or without crystals in
the globules [18]. PEs with liquid or crystalline dispersed phases are relevant for creating
foods with compositions and microstructures that can be fine-tuned to obtain the desired
quality attributes in the final products [17,18].
Physicochemical and sensory attributes of emulsion-based food products are strongly
influenced by droplet properties, such as droplet morphology, interactions, and rheological
behavior [17]. There is a great deal of research attempting to link these properties in
PEs [19–22]. Microscopic and rheological measurements can also be used to monitor
changes in the structural organization and interactions of the droplets in emulsions. For
instance, observing changes in microstructure and measuring changes in the viscosity or
elastic modulus of an emulsion over time can be used to track changes in the emulsion’s
physical properties and stability [23].
Literature about cocoa butter in water emulsions is dedicated to studying the crys-
tallization process of cocoa butter in the emulsion [24,25]. The growing chocolate market
and the significant increase in demand for cocoa butter, coupled with the limited source of
cocoa butter, drive up its cost and the cost of food and cosmetic products that use it. On the
other hand, consumption of chocolate is related to some health risks associated with its
saturated fatty acid content (around 60%). A high-saturated fatty acid diet has been found
to be related to the occurrence of high blood pressure and insulin resistance, among other
issues [26,27]. Therefore, there is a growing interest in developing substitutes to partially
or fully replace cocoa butter in food and cosmetic products, and these substitutes include
stable emulsions. However, to the authors’ knowledge, the literature is lacking reports that
evaluate the physical stability, microstructure, and rheological behavior of cocoa butter PEs,
especially with products derived from biological sources such as nanocellulose.
While previous research on nanocellulose in PEs has been developed for dispersed
phases that remain liquid or partially solidify, such as n-hexadecane [28], coconut oil [22],
Polymers 2023, 15, 4157 3 of 21

and olive oil [29], among others, they have focused on the morphology of the nanocellulose
incorporated and used wood as the main cellulosic source, neglecting the influence of
the isolation procedure for cellulose or alternative sources of cellulose, for instance, cocoa
shells. This research is more relevant considering in the literature the use of cellulose
nanofibrils to decrease oil or fat content in edible formulations, which helps to reduce the
caloric value of food products while functioning as a dietary fiber, suggesting its possible
use as a functional food ingredient with added health benefits [30–32].
For the above-mentioned objectives, an exploration of alternative sources of nanocellu-
lose was undertaken, aiming to reduce cocoa butter consumption by evaluating the effects
of nanocellulose concentration and its rheological properties in the preparation of stable
O/W Pickering emulsions using cocoa butter as the dispersed phase. The development
of stable O/W cocoa butter Pickering emulsions is intended to diminish the cocoa butter
content in food and cosmetic formulations.
To achieve these objectives, two different chemical treatments were employed to
isolate nanocellulose from cocoa shells. Differences in nanocellulose resulting from the
isolation treatments were analyzed using attenuated total reflection–Fourier transform
infrared spectroscopy (ATR–FTIR), extractive determination, atomic force microscopy
(AFM), degree of polymerization (DP), and rheological analysis. Additionally, the physical
stability of the emulsions formulated with the two types of nanocellulose was assessed
through visual observation as well as fluorescence and scanning electron microscopy to
investigate the morphology of the formulated Pickering emulsions. Finally, rheological
analysis was conducted to examine the viscoelastic properties of both nanocellulose and
Pickering emulsions.

2. Materials and Methods

Cocoa shells and cocoa butter, extracted from the main varieties of cocoa beans (Theo-
broma cacao L.) cultivated in Colombia, were supplied by Compañía Nacional de Chocolates
(Medellín, Colombia) after the roasting process. Potassium hydroxide, hydrochloric acid,
and sodium chlorite analytical grade, were purchased from Merck KGaA (Darmstadt,
Germany).

2.1. Isolation of Nanocellulose

Cocoa shell (CS) was chemically treated according to two different methods. As
described in Figure 1, C1 was isolated following a two-step treatment, and C2 was isolated
using a four-step treatment developed by [10,33]. Briefly, in the case of both C1 and C2
samples, ground cocoa husk underwent an initial alkaline hydrolysis treatment with a
5 wt % KOH solution at room temperature for 14 h to remove a portion of the lignin and
hemicellulose [10,33]. For the C2 sample, this process was followed by delignification using
chlorine dioxide, produced by acidifying NaClO2 to pH 5. Afterward, a second alkaline
treatment with a 5 wt % KOH solution was carried out for 14 h. Finally, the material
underwent a reaction with 1 wt % HCl for 2 h at 80 ◦ C [10,33]. After each step, the insoluble
residue was extensively washed with distilled water until the pH was neutral. Finally, a
2 wt % suspension of the cellulosic material obtained from C1 and C2 was passed 30 times
through a grinder (Masuko Sangyo, Supermasscolloider, Kawaguchi, Japan) following the
G30 procedure developed by [34].
The nanocellulose suspensions C1 and C2 with concentrations of 0.1 wt % were filtered
under vacuum through a 0.2 µm nylon membrane. The resulting films were oven-dried at
40 ◦ C for 72 h and stored for further analysis.
Polymers 2023,
Polymers 15, x
2023, 15, 4157
FOR PEER REVIEW 4 4ofof 22
21

Figure 1. Scheme illustrating the process of isolation and preparation of nanocelluloses and their
Figure 1. Scheme illustrating the process of isolation and preparation of nanocelluloses and their
Pickering emulsions. C1 0.7, C1 1.0, C2 0.7, and C2 1.0 represent the CNFs isolated through treatments
Pickering
C emulsions. C 0.7, C1 1.0, C2the
1 and C2, with 0.7 and 11.0 indicating
0.7,CNF
andconcentrations.
C2 1.0 representPEC
the 1CNFs isolated
0.7, PEC through treatments
1 1.0, PEC2 0.7, and PEC2
C1 denote
1.0 and C2 ,the
with 0.7 andemulsions
Pickering 1.0 indicating the CNF
stabilized concentrations.
by CNFs, PEC
with 0.7 and 1 0.7,
1.0 PEC1 1.0,
specifying thePEC
CNF2 0.7, and
concen-
PEC2 1.0used
trations denote thePE
in the Pickering emulsions stabilized by CNFs, with 0.7 and 1.0 specifying the CNF
formulation.
concentrations used in the PE formulation.
The nanocellulose suspensions C1 and C2 with concentrations of 0.1 wt % were fil-
2.2. Emulsions Formulation
tered under vacuum through a 0.2 μm nylon membrane. The resulting films were oven-
driedCocoa butter
at 40 °C in h
for 72 water Pickering
and stored for emulsions (4:1) were prepared using a method based
further analysis.
on previous works [29,35,36]. Cocoa butter was melted for one hour at 110 ◦ C to remove
all crystal
2.2. memory
Emulsions [24]. Subsequently, as illustrated in Figure 1, nanocellulose suspension,
Formulation
either C1 or C2 at two different concentrations (0.7 wt % and 1.0 wt %) [22,36], was added
Cocoa butter in water Pickering emulsions (4:1) were prepared using a method based
to the molten cocoa butter at room temperature (25 ◦ C). Finally, the two-phase system
on previous works [29,35,36]. Cocoa butter was melted for one hour at 110 °C to remove
was processed through a high shear mixing (HSM) or high-speed rotor-stator system
all crystal memory [24]. Subsequently, as illustrated in Figure 1, nanocellulose suspension,
(Ultraturrax T50, IKA, Cologne, Germany, impeller model S25N-25F) at 25,000 rpm for
either C1 or C2 at two different concentrations (0.7 wt % and 1.0 wt %) [22,36], was added
180 s. HSM is based on a rotor-stator system, where the rotor can rotate at 25,000 rpm and is
to the molten cocoa butter at room temperature (25 °C). Finally, the two-phase system was
enclosed in a stator with a gap between them ranging from 100 to 3000 µm. This equipment
processed through a high shear mixing (HSM) or high-speed rotor-stator system (Ultra-
applies a high shear force, which disperses fat droplets in the water phase. After HSM,
turrax T50, IKA,
the samples wereCologne,
labeled PEC Germany, impeller model S25N-25F) at 25,000 rpm for 180 s.
1 0.7, PEC1 1.0, PEC2 0.7, and PEC2 1.0. They were kept in a
HSM is based on a rotor-stator system,
container for transportation. Table 1 describes wherethe
thesample
rotor can rotate
labels at 25,000 rpm and is
for PEs.
enclosed in a stator with a gap between them ranging from 100 to 3000 μm. This equip-
ment applies
Table 1. a high
Description shear for
of labels force,
PEs.which disperses fat droplets in the water phase. After
HSM, the samples were labeled PEC1 0.7, PEC1 1.0, PEC2 0.7, and PEC2 1.0. They were kept
in a container for transportation. Table 1 describes the sample
Sample labels for PEs.
Description
PEC1 0.7 PEs stabilized with 0.7 wt % of C1 CNFs
Table 1. Description
PEC1of1.0
labels for PEs. PEs stabilized with 1.0 wt % of C1 CNFs
PEC2 0.7 PEs with 0.7 wt % of C2 CNFs
SamplePEC 1.0 Description
PEs stabilized with 1.0 wt % of C2 CNFs
2
PEC1 0.7 PEs stabilized with 0.7 wt % of C1 CNFs
2.3. Nanocellulose Characterization PEs stabilized with 1.0 wt % of C1 CNFs
PEC 1 1.0

PEC2 0.7 Total Reflection–Fourier

2.3.1. Attenuated PEs with 0.7 Infrared
Transform wt % of C 2 CNFs
Spectroscopy (ATR–FTIR)
PEC2 1.0 PEs stabilized with 1.0 wt % of C2 CNFs
ATR-FTIR spectroscopy analysis of C1 and C2 was performed with an FTIR spectrom-
eter (Thermo Scientific Nicolet iS50, Waltham, MA, USA) with a single-reflection ATR and
type-IIA diamond crystal mounted on tungsten carbide. The diamond ATR had a sampling
area of approximately 0.5 mm2 , and a consistent reproducible pressure was applied to
Polymers 2023, 15, 4157 5 of 21

every sample. The FTIR spectra were collected for 64 scans at a 4 cm−1 resolution. Each
spectrum represents the average of three spectra, and all spectra were corrected using the
advanced ATR correction of OMNIC 9, a software developed by Thermo Fisher ScientificTM ,
to eliminate the diamond crystal effect and ease the comparison with transmission spectra.

2.3.2. Nanocellulose Extractives

Aqueous extractives in hot water and organic extractives in ethanol/toluene were
determined for C1 and C2 using the TAPPI (Technical Association of the Pulp and Paper
Industry, Atlanta, GA, USA) method T207 cm-99 (1999) and TAPPI method T204 cm-97
(2007), respectively. Soluble extractives were calculated as the difference between the
weights of the dried samples before and after the extraction process.

2.3.3. Atomic Force Microscopy (AFM)

The morphology of nanocellulose fibers in C1 and C2 was evaluated by AFM. Samples
were imaged in tapping mode with a Flex AFM (Nanosurf, Liestal, Switzerland) equipped
with a multimode head and operated with a resonance frequency of 183 kHz. It utilized
a cantilever of 125 mm in length and a tip radius of 5–10 nm. Nanocellulose samples
were diluted in distilled water and sonicated using an Elma Elmansonic P ultrasonic bath
sonicator operating at 37 Hz and 70% power for 15 min at room temperature to achieve
a good dispersion of the nanocellulose without introducing significant morphological
changes. Subsequently, a fine layer of the sample was deposited on a mica substrate. Before
the morphological analysis, samples were stored in a vacuum desiccator for 3 days.

2.3.4. Degree of Polymerization (DP)

The degree of polymerization (DP) of C1 and C2 was determined according to ISO (In-
ternational Organization for Standardization, Geneva, Switzerland) 5351-2010 [37]. Dried
samples were torn into fine pieces and were introduced into an amber flask with 5 copper
rods and 25 mL of distilled water and stirred. After the dispersion of the specimens, 25 mL
of the CED solution (1 mol L−1 ) was added. A flow of nitrogen was used to expel the
air inside the flasks. Samples were mixed until the cellulose had completely dissolved.
The solutions were then introduced in a controlled-temperature bath and measured in a
calibrated capillary viscometer (Lagge, LV0003) at 25 ◦ C [34]. The time required to elute the
solution was measured. From this information, the DP of the samples was calculated ac-
cording to the Staudinger-Mark-Houwink equation for a cellulose-CED solution, expressed
in Equation (1).
[η ] = KDP DPα (1)
[η ] is the intrinsic viscosity of the solution (units mL/g), DP is the degree of polymerization
of the cellulosic material (dimensionless), and KDP (units mL/g) and α (dimensionless)
are experimental constants defined by Marx Figini [38] for a cellulose-CED solution. These
constants depend on the DP of the cellulosic material and are indicated elsewhere.

2.3.5. Rheological Analysis

Rheological measurements of nanocellulose were conducted using a Discovery HR-2®
rheometer (T.A. Instruments, New Castle, DE, USA) equipped with a 40 mm plate-plate
geometry and a fixed gap between the plates of 0.5 mm [39]. All measurements were
performed at 20 ◦ C, following an equilibration time of 10 min [22,40]. A solvent trap
was used to prevent water evaporation [41]. Amplitude viscoelastic measurements were
performed using a frequency of 6.28 rad/s and an amplitude sweep between 0.5 Pa and
1000 Pa. A frequency sweep was carried out from 6.28 to 62.8 rads/s at a constant shear
stress within the linear viscoelastic region of each sample. The values of the energy storage
(G0 ) and loss moduli (G00 ) were recorded. Flow curves were obtained by measuring viscosity
during logarithmic increases in shear stress from 0.1 to 100 rad/s.
Polymers 2023, 15, 4157 6 of 21

2.4. Emulsion Characterization

2.4.1. Physical Stability
The stability of all the emulsions was evaluated by visual observation with pho-
tographs taken 1 and 15 days after preparation.

2.4.2. Fluorescent Microscopy

The distribution of fat and cellulosic phases in nanocellulose and Pickering emul-
sions was examined by fluorescence microscopy (Zeiss Axio Observer, Zeiss, Oberkochen,
Germany) with 10× and 40× objectives. In the sample preparation, 1 mL of nanocellulose
was stained with 8 µL of Nile Red (for the oil phase) and then with 6 µL of Calcofluor White
(for the nanocellulose); 4 µL of the prepared samples were used for this study [42]. Samples
were covered using a glass coverslip (18 × 18 × 2 mm3 ), and the borders were protected
with nail polish to fix the borders of the coverslip and prevent evaporation [42,43].

2.4.3. Scanning Electron Microscopy

The morphological features of the Pickering emulsions were examined by scanning
electron microscopy (SEM) with a JEOL JCM-6000 (Akishima, Japan) scanning electron
microscope at an acceleration voltage of 15 kV. Before the analysis, a drop of each sample
was placed on carbon tape and evaporated at room temperature. Subsequently, the samples
were coated with gold.

2.4.4. Rheological Analysis

Amplitude viscoelastic measurements and frequency sweeps were conducted on
emulsions following the same procedure used in nanocellulose, described in Section 2.3.5.

2.4.5. Statistical Analysis

Data for this study were obtained through a series of experiments aimed at charac-
terizing Pickering emulsions. The primary variables of interest included isolation, treat-
ment, and concentration. Experiments were conducted in triplicate to ensure data repro-
ducibility. All data preprocessing procedures were conducted using software specific to
each technique.
Descriptive statistics were employed to provide a summary of the collected data,
including measures such as mean and standard deviation. To aid in the interpretation of
results, graphical representations were created using Origin. Scatter plots, box plots, and
bar charts were utilized to visualize trends and relationships within the data.

3. Results and Discussion

3.1. Physicochemical Characterization of CNFs: C1 and C2
CNFs isolated from cocoa shells will undergo evaluation for the development of
cocoa butter-in-water Pickering emulsions. These emulsions stabilize as the CNFs create
a physical barrier and a network, acting as an obstacle between contiguous droplets and
preventing droplet coalescence [44]. Factors influencing the stability of such emulsions
include the morphology, size, and shape of the CNFs, as well as their chemical composition,
which can affect the surface charge and interactions. The morphology and chemical
composition of CNFs are closely linked to their isolation process [44]. Hence, an assessment
was conducted on the chemical, physical, and morphological characteristics of both CNFs,
C1 and C2 , to comprehend the impact of the isolation process on the physical barrier and
network responsible for stabilizing the Pickering emulsion.
Surface hydroxyl groups in CNFs make them hydrophilic polymers capable of sta-
bilizing oil-in-water (O/W) emulsions [44]. ATR-FTIR is a commonly used technique for
studying these groups and their interactions with both cellulosic and non-cellulosic com-
ponents present in CNFs [34]. The ATR–FTIR spectra of C1 and C2 films are presented in
Figure 2a,b. They exhibit the characteristic peaks of cellulose at 3335 cm−1 and 3285 cm−1 ,
assigned to hydrogen bonds, and at 711 cm−1 , assigned to Iβ cellulose [45]. The spectra
 tion of nanocellulose from cocoa shells [10].
Hemicelluloses are one of the most difficult components to remove from cell walls
owing to their strong interaction with cellulose and lignin [33,34,48]. It could be physically
interwoven into the cell wall through the formation of hydrogen bonds, physically bond
Polymers 2023, 15, 4157 to cellulose [33,48], or form covalent complexes with lignin by ester bonds [49]. Hemicel- 7 of 21
luloses were liberated from cocoa shells by alkaline extraction [33], which can cleave ester
bonds between lignin and hemicelluloses in the cell walls, freeing a fraction of the lignin
present [49]. It has
also evidenced thebeen demonstrated
presence that thecomponents,
of non-cellulosic extraction of such
hemicellulose from a partially
as hemicelluloses, lignin,
delignified material results in a more efficient process [50], as is the
and fat, as observed by the peaks present at 2953, 1462, and 1377 cm , related case
− 1 of the C2 to
sample,
cocoa
which includes two
butter structures alkaline
[46,47], whilereactions with
the bands at 51743,
wt %1590,
KOH for 14
1510, andh,1235
one cm
before and
−1 are the sec-
related to
ond one hemicelluloses
residual after delignification with NaClO
and lignin 2 (as presented in Figure 1), while C1 includes
[45]. These results agree with previous works on the
only one of
isolation reaction with 5 wt
nanocellulose % KOH
from cocoafor 14 h,[10].
shells before delignification.

Figure 2. ATR–FTIR
ATR–FTIR spectra
spectra of the C
of the C1 and C2 films. _E represents the sample after removing their
Figure 2. 1 and C2 films. _E represents the sample after removing their
extractives: (a) ATR-FTIR spectrum between 3800 and 2700 cm−−11; (b) ATR-FTIR spectrum between
extractives: (a) ATR-FTIR spectrum between 3800 and 2700 cm ; (b) ATR-FTIR spectrum between
1800 and 400 cm− −1.
1800 and 400 cm 1 .
As mentioned, other
Hemicelluloses are onenon-cellulosic
of the mostcomponents were identified
difficult components in thefrom
to remove FTIRcell
spectra,
walls
such as cocoa butter from the cocoa shell. Both cellulosic samples C 1 and C2 exhibit several
owing to their strong interaction with cellulose and lignin [33,34,48]. It could be physically
vibrations
interwovenrelated
into thetocell
functional groups
wall through theofformation
the main of components of this physically
hydrogen bonds, fat, such asbond
bandsto
associated with palmitic and capric fatty acids (2953 cm −1 2922 cm−1, 2853 cm−1, 1462 cm−1,
cellulose [33,48], or form covalent complexes with lignin by ester bonds [49]. Hemicellu-
1377
losescm
−1) [10,46,47,51]. The absorbances of these vibrations are lower in C2, as the extrac-
were liberated from cocoa shells by alkaline extraction [33], which can cleave ester
tion of cell
bonds between walllignin
polymers, e.g., pectic polysaccharides
and hemicelluloses andfreeing
in the cell walls, hemicellulose,
a fractionincreases the
of the lignin
extraction of oil or fat from the lignocellulosic material [52–54]. The presence
present [49]. It has been demonstrated that the extraction of hemicellulose from a partially of vegetable
fat was alsomaterial
delignified evidenced by fluorescent
results microscopy
in a more efficient (Figure
process [50],S1as of
is the supplementary
the case infor-
of the C2 sample,
mation). Despite the hydrophilic character of cellulose, the fluorescence
which includes two alkaline reactions with 5 wt % KOH for 14 h, one before and the second images show the
one after delignification with NaClO2 (as presented in Figure 1), while C1 includes only
one reaction with 5 wt % KOH for 14 h, before delignification.
As mentioned, other non-cellulosic components were identified in the FTIR spec-
tra, such as cocoa butter from the cocoa shell. Both cellulosic samples C1 and C2 ex-
hibit several vibrations related to functional groups of the main components of this fat,
such as bands associated with palmitic and capric fatty acids (2953 cm−1 , 2922 cm−1 ,
2853 cm−1 , 1462 cm−1 , 1377 cm−1 ) [10,46,47,51]. The absorbances of these vibrations
are lower in C2 , as the extraction of cell wall polymers, e.g., pectic polysaccharides
and hemicellulose, increases the extraction of oil or fat from the lignocellulosic mate-
rial [52–54]. The presence of vegetable fat was also evidenced by fluorescent microscopy
(Figure S1 of the Supplementary Information). Despite the hydrophilic character of cellu-
lose, the fluorescence images show the coexistence of fat particles (red) and cellulosic
material (blue) in a homogeneous suspension, with no evidence of phase separation despite
the nature of both components [10].
The presence of non-cellulosic components in CS, C1 , and C2 was also verified by
soluble extractive tests in hot water and organic solvents presented in Table 2. Results
show that CS and C1 have a higher percentage of both aqueous and organic extractives.
The organic solvent-extractable material in the samples is composed of fatty acids, resin
acids, waxes, and tannins [55]. For this reason, absorbances at 2953, 2922, and 2853 cm−1
associated with asymmetric stretching of aliphatic –CH3 and symmetric stretching of
aliphatic –CH2 in fatty acids are significantly reduced in CS, C1 , and C2 samples after
Polymers 2023, 15, 4157 8 of 21

extraction with organic and aqueous solvents (CS_E, C1 _E, and C2 _E in the FTIR-ATR
spectrum, Figure 2). The hot water-soluble part of the untreated material consists primarily
of low-molecular-weight molecules, such as carbohydrates, inorganic salts, polyphenols,
sugars, and other water-soluble components [56].

Table 2. Properties of CNFs. Water, organic, and total extractables of CNFs. Polymerization degree (DP).

Water Organic Total Extraction

Sample DP
Extractables (%) Extractables (%) (%)
Cocoa shell 25.52 ± 2.74 27.68 ± 2.96 53.20 ± 2.74 N/A
C1 10.74 ± 1.66 11.80 ± 1.9 22.53 ± 3.53 1098.52 ± 1.10
C2 4.86 ± 0.14 5.66 ± 0.22 10.53 ± 0.32 554.31 ± 1.67

Finally, changes after the different isolation treatments were evaluated by comparing
three regions in the FTIR-ATR spectrum, from 3600 to 3000 cm−1 , which are associated
with the OH stretching frequencies of cellulose, correlating with the hydrogen bonding
pattern [57–59]. This vibration range exhibits poor resolution. However, mathematical
processing such as the deconvolution of spectra obtained in FTIR spectrometers can be
used to enhance the resolution [60]. Figure S2 of the Supplementary Information displays
the deconvolution of all FTIR spectra between 3000–3650 cm−1 . The height of the peaks at
3405 cm−1 , 3305 cm−1 , and 3285 cm−1 , which related to the O(2)H· · · O(5) intermolecular
bonding [59,61], O(6)H· · · O(3) intermolecular bonding [45], and OH stretching intermolec-
ular hydrogen bonds in the 101 plane, respectively, increased in C1 with respect to C2, which
is associated with a higher number of intermolecular hydrogen bonds in C1 . Changes in
hydrogen bonding associated with different isolation processes have been reported by
other authors [34]. Table 3 presents the absorbances and descriptions of the vibrations
related to the hydrogen bonds.

Table 3. Characteristic bands in ATR–FTIR spectra of cocoa shell nanocellulose and their assignments
according to the literature.

Wavenumber Absorbances (a.u.)

Description References
(cm−1 ) C1 C2
O(2)H· · · O(5) intermolecular
3405 1.1510 0.7503 [62]
bonding
3335 1.5017 0.8003 Intramolecular contribution
OH stretching (intramolecular
3350 1.2120 0.8033 [61]
hydrogen bonds)
O(6)H· · · O(3) intermolecular
3285 1.1282 0.7271 [61]
bonding
OH stretching (intermolecular
3305 1.1565 0.7534 hydrogenbonds in the [61,62]
101 plane)

Previous studies on banana rachis and cocoa husk have proposed the use of an alkaline
four sequence to isolate C2 CNFs [10,33], as described in Figure 1. This sequence is based
on the cleavage of β-aryl-O-4 linkages in lignin by alkaline media, exposing new zones
for the following treatment [63], the degradation of phenolic and non-phenolic structures
in lignin by chloride dioxide [64]. After the elimination of lignin, a secondary alkaline
treatment can react with muconic acids produced during delignification while using the
freshly exposed surfaces to cleave the hemicellulose-cellulose bonds present, reducing the
presence of hemicelluloses in the final structure [65]. Finally, a demineralization treatment
with HCl at a low concentration was used; the acid concentration allowed a reduction
 c present the morphology of C1 at different scales. AFM images showed individual nano-
fibers with diameters around 30.5 ± 12.5 nm and the presence of fiber bundles and sub-
fibrillated cellulosic material with diameters ranging between 275 and 380 nm. In addi-
tion, Figure 3d–f shows the morphology of C2. It is evident that there are separate individ-
Polymers 2023, 15, 4157 ual nanofibers with diameters around 26.9 ± 8.2 nm and fiber bundles with diameters 9 of 21
ranging between 98 and 225 nm. AFM images reveal the presence of bundles in both sam-
ples C1 and C2, marked with white arrows in Figure 3b,c and Figure 3e,f, respectively. In
summary, AFM revealed
of the cellulosic hydrolysisthat C1 Therefore,
[33]. shows an entangled network
the influence with a alkaline
of a second lower degree of fi-
treatment,
brillation, which is associated with its isolation treatment [34]. C CNFs also
accompanied by an HCl treatment in C2 , favored the removal of non-cellulosic components,
1 showed the
presence ofahemicellulose,
resulting in greater numberwhich is related
of individual to aand
fibers lower degree
smaller of fibrillation
diameters after theand bundles
mechanical
with larger[33,66,67],
treatment diametersas[68].
can be observed in the AFM images presented in Figure 3.

Figure 3. AFM images of nanocellulose: (a–c) C1 and (d–f) C2 . White arrows indicate the presence of
bundles. White arrows indicate the CNFs bundles.

Consequently, the isolation treatment influences the chemical composition of cellulose

and its fibrillation [68]. Figure 3a–f shows that both samples C1 and C2 were nanofibril-
lated and presented an entangled network of cellulosic material. The formation of this
three-dimensional entangled network is relevant in PE formulation because it favors the
irreversible adsorption of nanocelluloses at the oil-water interface, increasing the steric
hindrance between the droplets and inhibiting the free movement of the droplets, resulting
in a lower coalescence rate [69].
For this reason, differences between the entangled networks formed by C1 and C2
should be comprehended with the aim of understanding the properties of PEs. Figure 3a–c
present the morphology of C1 at different scales. AFM images showed individual nanofibers
with diameters around 30.5 ± 12.5 nm and the presence of fiber bundles and sub-fibrillated
cellulosic material with diameters ranging between 275 and 380 nm. In addition, Figure 3d–f
shows the morphology of C2 . It is evident that there are separate individual nanofibers with
diameters around 26.9 ± 8.2 nm and fiber bundles with diameters ranging between 98 and
225 nm. AFM images reveal the presence of bundles in both samples C1 and C2 , marked
with white arrows in Figure 3b,c and Figure 3e,f, respectively. In summary, AFM revealed
that C1 shows an entangled network with a lower degree of fibrillation, which is associated
with its isolation treatment [34]. C1 CNFs also showed the presence of hemicellulose, which
is related to a lower degree of fibrillation and bundles with larger diameters [68].
On the other hand, the isolation process influences the length of CNFs [34,70]. Zuluaga
et al. evidenced that a reaction with HCl 2 M longitudinally cuts the cellulose microfibrils.
This is typical of CNFs treated with strong acids that preferentially degrade the disordered
regions along the microfibrils [33]. The length and polymerization degree of CNFs are
interconnected parameters that cannot be changed independently easily [68]. A higher
Polymers 2023, 15, 4157 10 of 21

polymerization degree (DP) is directly related to longer fibers [39,41,43], and fiber length
is a parameter that directly affects the stability of PEs, allowing the formation of a three-
dimensional network that would enclose the fat droplets [71]. Also, long fiber aggregates
form an entangled network, increasing the viscosity of the formed suspension compared to
fibers with a lower aspect ratio [41,72]. Aiming to improve the understanding and explain
the behavior of CNFs as a Pickering emulsifier, the DP of the nanocellulose obtained by the
different treatments was assessed.
In CNFs, the DP is measured through its intrinsic viscosity, which is a characteristic of
macromolecules that is directly related to their ability to disturb the flow and indirectly
relates to the chain length. The relation between intrinsic viscosity and polymerization
degree for cellulose was established in the 20th century by different authors [38], and the
constants used by Marx-Figini were used to determine the DP reported in Table 1 [38]. The
DP obtained for C1 and C2 was 1098.52 ± 1.10 and 554.31 ± 1.67, respectively, which are
within the range reported in the literature [34,73–75]. The DP of a cellulosic polymer can
be around 10,000 in wood cellulose, but after its degradation and purification process, the
DP is reduced to about 300–1700 [73]. In cellulose from agroindustrial by-products like
banana rachis, DP was reported between 1012 and 612, depending on the homogenization
process [34]. Another aspect that influences the DP is the presence of non-cellulosic compo-
nents. It was reported that a higher concentration of non-cellulosic components is related to
a higher DP, as was observed for commercial cellulose [74] and cotton cellulose [75]. Finally,
HCl used to remove mineral traces in C2 [33] also reduces the polymerization degree, as it
degrades the short, disordered, non-crystalline regions in cellulose [75,76]. Therefore, the
higher DP observed for C1 is clearly associated with a higher presence of non-cellulosic
components and a lack of mild acid hydrolysis present in the C2 sample.
As previously noted, the assembly of long fiber aggregates can create a complex three-
dimensional network, resulting in increased suspension viscosity when contrasted with
fibers possessing a smaller aspect ratio [41,72]. Consequently, an increase in the degree of
polymerization (DP) and intrinsic viscosity (Table 2) may be linked to changes in their shear
flow behavior, as shown in Figure 4a, for both samples. To assess the rheological properties
Polymers 2023, 15, x FOR PEER REVIEW 11 of 22
of the cellulosic suspensions, evaluations were conducted at two different concentrations:
0.7 and 1.0 wt % for both C1 and C2 . Shear flow and oscillatory rheology results are
presented in Figure 4a,b.

(a) (b)
Figure
Figure 4.
4. Rheological
Rheological characterization
characterization of
of CNFs
CNFs isolated
isolated from
from cocoa
cocoa husks
husks C1 and C
C1 and C2:: (a)
(a) viscosity
viscosity
2
curves
curves and
and (b)
(b) frequency
frequency sweep,
sweep, with
with G′
G0 represented
represented by
by filled
filled symbols
symbols and
and G″
G00 by
by empty
empty symbols.
symbols.

The viscoelastic characterization of C1 and C2 shows the behavior of G′ and G″ in the

linear viscoelastic region in Figure 4b. They are associated with the strength of the entan-
gled network. It can be observed that G′ and G″ depend on the type of sample used,
whether it is C1 or C2, and their respective concentrations. Comparing G′ and G″ for C1
and C2 at the same concentration, it is observed that both modules are one order of mag-
Polymers 2023, 15, 4157 11 of 21

The shear flow behavior of the cellulosic samples is depicted in Figure 4a. The
apparent viscosity of C1 and C2 decreased with increasing shear rate; this behavior is typical
of non-Newtonian fluids such as shear-thinning fluids, which is the expected behavior
for suspensions or fibers since these tend to orient themselves and partly disentangle
due to the hydrodynamic forces exerted over them, resulting in a lower viscosity as the
shear rate increases [77]. The floc breakage corresponds to the three regions observed in
Figure 4a. In the first region, there is a marked shear-thinning behavior, with a decrease
in sample viscosity at higher shear rates. It is followed by a plateau around 10 s−1 , where
the formation of a more entangled network reduces the floc breakage and maintains the
viscosity at a stable value. Finally, the shear rate is high enough to fracture this structure,
and a further reduction in the viscosity occurs as the entangled network releases the
individual fibers [72].
The viscoelastic characterization of C1 and C2 shows the behavior of G0 and G00 in
the linear viscoelastic region in Figure 4b. They are associated with the strength of the
entangled network. It can be observed that G0 and G00 depend on the type of sample used,
whether it is C1 or C2 , and their respective concentrations. Comparing G0 and G00 for C1 and
C2 at the same concentration, it is observed that both modules are one order of magnitude
higher for C1 compared to C2 , probably because of differences in fibrillation degree, as
observed by AFM. It has been reported that fibrillation influences network strength; higher
fibrillation of CNFs will result in lower network strength and lower values for G0 and G00 ,
as is the case with C2 [41]. Saarinen et al. showed, using a plate-plate geometry, that the
storage modulus of mechanically disintegrated cellulose suspensions decreases when the
level of fibrillation is increased in a microfluidizer [78].
On the other hand, fiber length plays a role in viscoelastic properties. Longer fibers are
expected to have higher G0 and G00 , as was the case with C1 , whose higher DP was related
to longer fibers. Pääkkö et al. reported that at a concentration of 2% w/w of nanocellulose,
it leads to G0 ≈ 103 Pa, whereas 2 wt % of modified rodlike cellulose crystallites leads
to G0 ≈ 101 Pa [79]. The higher elastic modulus is explained by longer fibrils, and fibril
aggregates in CNFs can form an inherently entangled network structure [79]. In addition,
more flocs composed of longer and thicker fibers increase the elastic contribution [78].
For this reason, the viscoelastic properties of nanocellulose were evaluated. Linear
viscoelastic properties of C1 0.7 wt %, C1 1.0 wt %, and C2 1.0 wt %, exhibit gel-like
properties (G0 > G00 ), as has been reported for different nanocellulose suspensions. The
storage modulus G0 was higher than the loss modulus G00 in a range of frequencies without
a crossover. However, C2 0.7 wt %, exhibits gel-like behavior at lower frequencies. When
the angular frequency increases beyond 40 rad/s, it exhibits a cross-over point at which
the system shows equal contributions from both elastic and viscous components. After
that point, higher frequencies weaken the structure, and C2 behaves as a viscose fluid,
where G00 > G0 [41,72,79–86]. In C1 and C2 , as the concentration of nanocellulose increases
from 0.7 to 1.0 wt %, a stronger fibrous network is formed, resulting in an increase in the
dynamic moduli G0 and G00 . Finally, no statistical differences were observed between G0
and G00 for C1 at 0.7 wt % and C2 at 1 wt %. This means that at those concentrations, both
samples form a network with the same rheological characteristics.
AFM, fluorescence microscopy, and rheological analysis of CNFs indicated that C1
and C2 form a strong three-dimensional network [22] with a gel-like structure that limits
the flow of cocoa butter droplets through the water phase [87], as evidenced in fluorescence
microscopy. However, as observed through the characterization of both types of CNFs,
there are differences in fibrillation morphology, DP, and rheological properties between
both types of nanocellulose, which could influence their emulsifying capacities [71]. To
assess the extent of this contribution, a qualitative stability assay was performed.
 PEC1 1.0, indicating that there was no emulsion breakage.
To the authors’ knowledge, the development of a stable cocoa butter-in-water Pick-
ering emulsion has not been reported in the literature and is an interesting topic for the
cosmetic industry, where cocoa butter is used as a moisturizer and emollient in product
Polymers 2023, 15, 4157 formulations [88]. The formation of a stable suspension of cellulose and cocoa butter12isofan 21
interesting phenomenon because oil-in-water (o/w) emulsions, where the oil droplets
have at least partly crystallized but the continuous aqueous phase remains liquid, are fre-
quently less physically
3.2. Physicochemical stable than the
Characterization same systems
of Pickering where
Emulsions the oil droplets
Stabilized are liquid [89].
with Different
According to the
Concentrations literature,
of CNFs: PEC1 crystallization
0.7, PEC1 1.0, PECof the
2 fat
0.7, structures
and PEC 2 in
1.0 cocoa butter-in-water
emulsions occurs during
The photographs of their storage and
the physical causes
stability testdestabilization
for cocoa butter [24,25].
suspensions are pre-
The
sented inmechanism
Figure 5a–c.ofPEC physical
2 0.7 instability
creamed on for
day crystalline
0 (dashed or partially
rectangles in crystalline
Figure 5a).lipids in
Cream-
o/w emulsions
ing was is arrested
not evident in PEC coalescence
2 1.0; [89],
however, which
the arises
emulsion when
was notthe crystals
stable of
over fat
time, penetrate
and the
the interphase
formation layer and, following
of fat-coalesced droplets aofcollision
around between droplets, penetrating
several millimeters the layer
can be observed of
after
a15second droplet, allowing lipid-to-lipid contact [18,90]. The liquid oil
days of storage (white arrows in Figure 5c). During the assessed period, separation of flows out from both
droplets to reinforce
cocoa butter the link,forbut
was not evident PEsthe fat crystals
prepared with do the not
CNFsformthatspherical droplets;
exhibit higher they
viscosity
maintain the shape
and a stronger of the individual
three-dimensional droplets,
network in as can be
linear observed properties,
viscoelastic in Figure 5c,PEC where some
1 0.7 and
non-spherical particles
PEC1 1.0, indicating thatcan be observed
there on the surface
was no emulsion of the glass container.
breakage.

Figure 5. Photographs of cocoa butter Pickering emulsions: (a) visual appearance of PEC1 0.7, PEC1
Figure 5. Photographs of cocoa butter Pickering emulsions: (a) visual appearance of PEC1 0.7, PEC1
1.0, PEC2 0.7, and PEC2 1.0 samples at days 0 and 15 of storage; (b) visual appearance of PEC 2 1.0 at
1.0, PEC 0.7, and PEC2 1.0 samples at days 0 and 15 of storage; (b) visual appearance of PEC2 1.0 at
day 0 of 2storage without evident creaming; (c) visual appearance of PEC2 1.0 at day 15 of storage,
day 0 of storage without evident creaming; (c) visual appearance of PEC2 1.0 at day 15 of storage,
showing fat-coalesced droplets pointed out by white arrows in the image, indicating emulsion
destabilization. PEC2 0.7 creamed on day 0 as indicated by dashed rectangles in Figure 5a.

To the authors’ knowledge, the development of a stable cocoa butter-in-water Pick-

ering emulsion has not been reported in the literature and is an interesting topic for the
cosmetic industry, where cocoa butter is used as a moisturizer and emollient in product
formulations [88]. The formation of a stable suspension of cellulose and cocoa butter is an
interesting phenomenon because oil-in-water (o/w) emulsions, where the oil droplets have
at least partly crystallized but the continuous aqueous phase remains liquid, are frequently
less physically stable than the same systems where the oil droplets are liquid [89]. Accord-
ing to the literature, crystallization of the fat structures in cocoa butter-in-water emulsions
occurs during their storage and causes destabilization [24,25].
The mechanism of physical instability for crystalline or partially crystalline lipids in
o/w emulsions is arrested coalescence [89], which arises when the crystals of fat penetrate
the interphase layer and, following a collision between droplets, penetrating the layer
of a second droplet, allowing lipid-to-lipid contact [18,90]. The liquid oil flows out from
both droplets to reinforce the link, but the fat crystals do not form spherical droplets; they
maintain the shape of the individual droplets, as can be observed in Figure 5c, where some
non-spherical particles can be observed on the surface of the glass container.
To observe the changes exerted on the microstructure of the emulsion at different
times and to identify them, a fluorescence microscopy analysis was performed as described
by Bai et al. in the sample at days 0 and 15, after processing them, and they are depicted in
Figure 6 [43]. Oil droplets are dyed with Nile red and are observed as red objects, while
Calcofluor white was used to dye the cellulosic structures and are seen as blue objects in
 showing fat-coalesced droplets pointed out by white arrows in the image, indicating emulsion
destabilization. PEC2 0.7 creamed on day 0 as indicated by dashed rectangles in Figure 5a.

To observe the changes exerted on the microstructure of the emulsion at different

times and to identify them, a fluorescence microscopy analysis was performed as de-
Polymers 2023, 15, 4157 scribed by Bai et al. in the sample at days 0 and 15, after processing them, and they are
13 of 21
depicted in Figure 6 [43]. Oil droplets are dyed with Nile red and are observed as red
objects, while Calcofluor white was used to dye the cellulosic structures and are seen as
blueimage.
the objectsSamples
in the image. Samples
formulated withformulated
C2 (PEC2 0.7with
andCPEC2 (PEC 2 0.7 and PEC2 1.0) did not
2 1.0) did not show satisfactory
stability before 15 days, as droplet agglomerates were observablewere
show satisfactory stability before 15 days, as droplet agglomerates observable
to the to the
naked eye, as
naked eye, as marked with white arrows in Figure 5c. As their size was larger
marked with white arrows in Figure 5c. As their size was larger than the microscope focus,than the
microscope
these imagesfocus,
were these images were
not included not included
in Figure 6. in Figure 6.

Figure 6. Fluorescent microscopy images of PEC1 0.7, PEC1 1.0, PEC2 0.7, and PEC2 1.0 samples at
Figure Fluorescent
(a,c,e,f)6.day microscopy
0 and (b,d) images of
day 15 of storage. OilPEC 1 0.7,
phase PEC1 1.0,
is shown PECand
in red, 2 0.7,the
and PEC2 1.0
cellulose is samples
shown inat
(a,c,e,f)
blue. The day 0 and
white (b,d) indicate
arrows day 15 ofthe
storage. Oilofphase
presence is shown in red, and the cellulose is shown in
fat particles.
blue. The white arrows indicate the presence of fat particles.
Fluorescence microscopy images evidence that cocoa butter droplets in the emulsion
can beFluorescence
divided intomicroscopy imagesofevidence
a fine dispersion thatembedded
oil droplets cocoa butterin droplets in the
a cellulosic emulsion
network and
can be divided into a fine dispersion of oil droplets embedded in a cellulosic network
a rough distribution of bigger coalesced fat droplets. The formation of nonspherical coa-
and a rough distribution of bigger coalesced fat droplets. The formation of nonspherical
lesced particles associated with arrested coalescence is evident in all emulsion samples
coalesced particles associated with arrested coalescence is evident in all emulsion samples
since Day 0, as indicated by the arrows in Figure 6a–d. In addition, as observed in the
since Day 0, as indicated by the arrows in Figure 6a–d. In addition, as observed in the
physical stability test presented in Figure 5, PEC1 0.7 and PEC1 1.0 did not evidence exten-
physical stability test presented in Figure 5, PEC1 0.7 and PEC1 1.0 did not evidence
sive droplet destabilization during the assessed period (day 0 to day 15), because most of
extensive droplet destabilization during the assessed period (day 0 to day 15), because
the cocoa butter is entrapped in the flocs formed by the CNFs network. Despite the same
most of the cocoa butter is entrapped in the flocs formed by the CNFs network. Despite the
concentrations being used to formulate the emulsions with C2 CNFs, it is evident that in
same concentrations being used to formulate the emulsions with C2 CNFs, it is evident
this case, the CNF network (blue) does not cover each cocoa butter droplet (red) and there-
that in this case, the CNF network (blue) does not cover each cocoa butter droplet (red)
fore does not form a dense barrier of CNFs to prevent coalescence [91]. Other authors have
and therefore does not form a dense barrier of CNFs to prevent coalescence [91]. Other
reportedhave
authors that reported
this concentration depends on the
that this concentration different
depends onphysicochemical characteristics
the different physicochemical
of the cellulosic material as well as on the oil and aqueous phases
characteristics of the cellulosic material as well as on the oil and aqueous phases [42], as observed in
[42], as
emulsions obtained with nanocellulose [22].
observed in emulsions obtained with nanocellulose [22].
To further
To further investigate
investigate thethe surface
surface coverage,
coverage, fat fat droplets
droplets were
were characterized
characterized by by SEM
SEM
after water
after waterevaporation
evaporationatatroom roomtemperature,
temperature, as as
seenseen in Figure
in Figure 7. SEM
7. SEM images
images revealed
revealed that
that nanofibrillated C 1 and C2 formed a covering layer via interfacial adsorption (marked
nanofibrillated C1 and C2 formed a covering layer via interfacial adsorption (marked with
with arrows
arrows in Figure
in Figure 7), as7), as reported
reported by other
by other authors
authors [71]. [71]. However,
However, according
according to Man-
to Manning
ning et al., when fat crystals grow under physical restriction, a smooth
et al., when fat crystals grow under physical restriction, a smooth surface morphology surface morphol-
ogy
is is observed,
observed, but but without
without suchsuch restriction,
restriction, crystalsgrow
crystals growmultidirectionally
multidirectionally(as (as marked
marked
with dashed
with dashed circles
circlesininFigure
Figure7).7).
In In
thisthis
case, nanocellulose
case, nanocellulosein cocoa butter
in cocoa acts asacts
butter theasphys-
the
ical barrier
physical thatthat
barrier restricts crystal
restricts growth.
crystal growth. SEMSEM revealed
revealedthat
thatall
allemulsions
emulsionspresent
present zones
zones
where multidirectional crystals can be observed, with a morphology very similar to that
observed by Manning & Dimick (1985), marked by dashed circles in Figure 7 [92]. This
kind of morphology is more pronounced in PEC2 0.7 and PEC2 1.0 than in PEC1 0.7 and
PEC1 1.0 emulsions, which confirms that in those samples, CNFs did not cover each cocoa
butter droplet.
 where multidirectional crystals can be observed, with a morphology very similar to that
observed by Manning & Dimick (1985), marked by dashed circles in Figure 7 [92]. This
Polymers 2023, 15, 4157
kind of morphology is more pronounced in PEC2 0.7 and PEC2 1.0 than in PEC1 0.7 and
14 of 21
PEC1 1.0 emulsions, which confirms that in those samples, CNFs did not cover each cocoa
butter droplet.

Figure 7. SEM images of dehydrated cocoa butter water emulsions stabilized by C1 and C2 at 0.7 wt
Figure 7. SEM images of dehydrated cocoa butter water emulsions stabilized by C1 and C2 at 0.7 wt %,
%, 1.0 wt % taken at (a–d) 500× and (e–h) 100×. Dashed circles indicate multidirectional crystal
1.0 wt % and
growth, takenwhite
at (a–d) 500×indicate
arrows and (e–h)the ×. Dashed circles indicate
100three-dimensional networkmultidirectional
formed by CNFs crystal growth,
around fat
and white
droplets arrows
after indicatemixing.
high-shear the three-dimensional network formed by CNFs around fat droplets after
high-shear mixing.
The rheological properties of the emulsion are powerful techniques to complement
The rheological
the interpretation properties
of physical of the and
stability emulsion are powerful
interfacial techniques
interaction to complement
between cellulosic fibers
the interpretation of physical stability and interfacial interaction between cellulosic
and cocoa butter droplets observed in microscopy. Linear viscoelastic properties were fibers
as-
and cocoa butter droplets observed in microscopy. Linear viscoelastic properties were
sessed for the stable emulsions 1 and 15 days after processing (Figure 8). Results for the
assessed for the stable emulsions 1 and 15 days after processing (Figure 8). Results for
viscoelastic properties of the emulsion allowed us to establish that PEC1 0.7 and PEC1 1.0
the viscoelastic properties of the emulsion allowed us to establish that PEC1 0.7 and PEC1
can be classified as gel-like emulsions, as the storage modulus G′ showed a higher value
1.0 can be classified as gel-like emulsions, as the storage modulus G0 showed a higher
than the loss modulus G″, and00 G′ is independent of frequency [22,93,94]. The elastic be-
value than the loss modulus G , and G0 is independent of frequency [22,93,94]. The 15
Polymers 2023, 15, x FOR PEER REVIEW elastic
of 22
havior dominates over the viscous one for the evaluated PEs, as the value of G′0 is greater
behavior dominates over the viscous one for the evaluated PEs, as the value of G is greater
than G″00 through the evaluated frequency range [22,93].
than G through the evaluated frequency range [22,93].

Figure8.
Figure 8. Rheology
Rheology tests
tests in
inPEs.
PEs. Frequency
Frequency sweep,
sweep,with
withGG′0 represented
representedby
byfilled
filledsymbols
symbolsand 00 by
andGG″ by
emptysymbols.
empty symbols.

The independence of G′ from frequency is characteristic of an elastic behavior where

the material’s ability to store and return energy does not vary with the rate of deformation.
It is noticeable that the structure of gels has been described as a network of interacting
rheological units, with G′ depending on the strength and number of interactions [95]. The
Polymers 2023, 15, 4157 15 of 21

The independence of G0 from frequency is characteristic of an elastic behavior where

the material’s ability to store and return energy does not vary with the rate of deformation.
It is noticeable that the structure of gels has been described as a network of interacting
rheological units, with G0 depending on the strength and number of interactions [95].
The formation of a gel network has been associated with enhanced creaming stability in
casein-stabilized systems [96], while emulsion gels with high mechanical properties, such
as storage modulus, exhibit higher chemical stability in their dispersed phase, suggesting
a physical role in the stability of these structures [97]. Also, due to their firmness and
stability, gels can provide a desirable texture and mouthfeel to food products. They can
add creaminess, thickness, and smoothness to various foodstuffs [23].
As observed in microscopic (Figure 5) and SEM images (Figure 7), CNFs formed a three-
dimensional network around droplets after high shear mixing, which plays a prominent
role in stability and rheological behavior [98]. As observed in Figure 7a–d, nanocellulose
was absorbed over the surface of the oil droplets to form a covering layer after the high
shear mixing processing, resulting in a stable droplet-fiber 3D network structure. This
network structure contributes to the elasticity of the emulsion. CNF networks can deform
elastically when subjected to stress and then return to their original shape when the stress
is removed.
PEC1 emulsions stabilized by nanocellulose with different dosages showed a signif-
icant difference in elastic modulus. For example, at a shear rate of 10 rad/s, the G0 of
PEC1 0.7 and PEC1 1.0 were 0.0750 MPa and 0.0546 MPa, respectively, indicating that the
emulsion stabilized by a higher CNF concentration exhibited stronger gel behavior. This
increment in G0 at higher concentrations of CNFs is consistent with previous reports, where
CNFs from agro-industrial by-products were used to stabilize PEs.
Velásquez-Cock et al. extracted CNFs from banana rachis following the same proce-
dure as that used for C2 and evaluated the effect of CNF concentration on the rheological
properties of coconut or palm oil-in-water-based Pickering emulsions [22]. They reported
an increase in G0 values around three orders of magnitude when CNF concentration in-
creased from 0.15 wt % to 0.7 wt %. Another study added CNFs from banana rachis,
isolated using C2 methodology, to Curcuma longa suspensions and reported similar results.
Increasing the CNF concentration from 0.1 wt % of CNFs to 0.9 wt % enhanced G0 by nearly
3 orders of magnitude and improved the stability over 30 days [93]. Banana rachis and
cocoa husk are both agro-industrial by-products. Despite using the same CNF isolation
methodology, the resulting CNFs exhibit different physicochemical characteristics, mainly
due to variations in their physicochemical properties [10,33]. These differences necessitated
different concentrations for stabilizing PEs, as is the case for C1 and C2 CNFs.
Finally, the observed increase in G0 and G00 after storage corresponds to an enhance-
ment of gel strength in CNFs [99]. As mentioned, the crystallization of cocoa butter-in-water
emulsions occurs during emulsion storage and destabilizes the suspension by enhancing
particle coalescence [24,25]. Cocoa butter is a complex fat known to crystallize in six differ-
ent polymorphs [100]. These various forms of cocoa butter crystallization can significantly
impact the properties of emulsions formulated with this ingredient. For example, the
different crystalline forms have distinct melting points, with the α polymorph being less
stable than the β form, which typically has a higher melting point [101]. Therefore, if cocoa
butter in an emulsion undergoes a phase transition from a more stable to a less stable form
during storage or processing, it can lead to undesirable changes in texture, appearance, and
product deterioration [101]. Furthermore, the crystallization of solid fats plays a pivotal
role in the partial coalescence of droplets in oil-in-water (O/W) emulsions [101]. This
type of destabilization is of particular interest in food applications and is a prerequisite
for producing whipped cream and ice cream [101,102]. Additionally, the crystallization
behavior of these fats in emulsions is intricate and subject to various influences, such
as droplet size, collision dynamics, additives, emulsifiers, crystallization medium, and
thermal fluctuations during storage [101,103]. It has also been reported that shear forces
Polymers 2023, 15, 4157 16 of 21

can impact the crystallization of fats in emulsions and promote the formation of higher
polymorphs [104].
In the cases of PEC1 0.7 and PEC1 1.0, cocoa butter crystallization did not destabilize
the emulsion. To the author’s knowledge, there are no literature reports of stable cocoa
butter in water emulsions. Hindle et al. developed cocoa butter in water emulsions
using 0.8% v/v Tween 20 or 1.0 wt % caseinate, but this research was focused on droplet
collision-mediated heterogeneous nucleation and its effect on the crystallization kinetics of
cocoa butter [24,25]. Therefore, the findings of this work are significant from a scientific
perspective and have implications for various applications, including food, cosmetics, and
pharmaceutical formulation. Some potential directions and applications stemming from
this research are as follows:
• The use of cocoa butter in water Pickering emulsions in the food industry offers
several key advantages: reduced caloric density of products [105], the potential for
incorporating active ingredients to enhance nutritional value [106], and lowered
production costs due to reduced raw material usage [100]. However, further research
is required in this field, particularly concerning the crystallization of cocoa butter with
the integration of new active compounds.
• The formulation of innovative aqueous-based products, such as nano-delivery systems
and edible coatings, can be developed using stable cocoa butter-in-water emulsions.
This approach leverages the antioxidant, moisturizing, and barrier properties of cocoa
butter, which is widely utilized in the food, pharmaceutical, and cosmetic indus-
tries [107].

4. Conclusions
In this research, an isolation treatment was used to control the rheological properties
of nanocellulose from cocoa shells. Subsequently, the influence of the rheological behavior
of nanocellulose on the stabilization of cocoa butter in a water Pickering emulsion was
evaluated. The ATR-FTIR spectra, in conjunction with the extractives in hot water and
organic solvents, indicated the partial elimination of non-cellulosic components for both
samples. However, a higher presence of non-cellulosic components was observed in C1 , in
addition to a higher number of hydrogen bonds. This difference in chemical composition
is attributed to the influence of a second alkaline treatment accompanied by a treatment
with HCl in C2 . These reactions also favored the breaking of hydrogen bonds. In addition,
according to AFM and rheological analysis, both samples form a strong entangled network
of nanofibers with a gel-like structure that limits the flow of the cocoa butter droplets in
the aqueous phase. However, the second alkaline and HCl reaction promotes a higher
degree of fibrillation of the material, resulting in nanocellulose with smaller diameters,
more individually separated, and a lower degree of polymerization. This results in lower
network strength, as evidenced by lower G0 and G00 values and lower apparent viscosity
in C2 .
Thus, C1 presented higher DP and a stronger entangled network, which improved
the stabilization of the cocoa butter in water-based Pickering emulsions, as analyzed by
physical stability visualization and rheological analysis. C1 showed an enhanced 3D
droplet-fiber network structure. Fat crystallization caused arrested coalescence but did not
destabilize the PEC1 0.7 and PEC1 1.0 emulsions, even during 15 days of storage. Emulsions
developed in this research offer the possibility of reducing the content of cocoa butter in
food and cosmetic formulations.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/polym15204157/s1, Figure S1. Fluorescent microscopy images of
CNFs; Figure S2. ATR-FTIR spectra deconvolution between 3700 and 3000 cm−1 .
Polymers 2023, 15, 4157 17 of 21

Author Contributions: C.G.H., L.D.B., A.F.-C. and J.A.V.-C. planned and executed the experiments.
C.G.H. wrote the manuscript text, and R.Z. prepared the figures and the discussions about nanocellu-
lose characterization. All authors reviewed the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the Research Center for Investigation Development (CIDI) at
the Universidad Pontificia Bolivariana (Project No. 48C-01/21-49).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The raw data for the FTIR spectra and rheological analysis is available
as Supporting Information.
Acknowledgments: The authors acknowledge the financial support from the Research Center for In-
vestigation Development (CIDI) at the Universidad Pontificia Bolivariana (Project No. 48C-01/21-49).
Conflicts of Interest: We declare that we have no financial or personal relationships with other
people or organizations that can inappropriately influence our work, and there is no professional or
other personal interest of any nature or kind in any product, service, and/or company that could be
construed as influencing the position presented.

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