foods

Article
Phenolic Extraction of Moringa oleifera Leaves in DES:
Characterization of the Extracts and Their Application in
Methylcellulose Films for Food Packaging
Fazia Braham 1,2 , Luísa M. P. F. Amaral 2,3 , Krzysztof Biernacki 2 , Daniel O. Carvalho 3 , Luis F. Guido 3 ,
Júlia M. C. S. Magalhães 2 , Farid Zaidi 1 , Hiléia K. S. Souza 2,4, * and Maria P. Gonçalves 2

1 Département des Sciences Alimentaires, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia,
Route Targa Ouzemour, Bejaia 06000, Algeria
2 REQUIMTE/LAQV, Departamento de Engenharia Química, Faculdade de Engenharia,
Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
3 REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,
Rua do Campo Alegre 687, 4169-007 Porto, Portugal
4 IPC—Institute for Polymers and Composites/I3N, Department of Polymer Engineering, University of Minho,
Campus de Azurém, 4800-058 Guimarães, Portugal
* Correspondence: [email protected]

Abstract: In this work, a qualitative study of the phenolic content of Moringa oleifera leaves (MO),
extracted with deep eutectic solvents (DES) based on choline chloride (ChCl) with lactic acid (LA) or
glycerol (GLY), was performed by high-resolution mass spectrometry (HPLC-DAD-ESI-MSn ). The
two solvents (DES-LA and DES-GLY) extract similar classes of phenolics, and ten compounds were
identified. The antioxidant profile was also studied (TPC, TFC, DPPH, FRAP, ORAC, and ABTS).
Citation: Braham, F.; Amaral,
Both solvents show an efficient extraction of phenolic compounds and high antioxidant capacity was
L.M.P.F.; Biernacki, K.; Carvalho,
verified for the extracts. However, the DES-Gly have a higher capacity for polyphenolic extraction
D.O.; Guido, L.F.; Magalhães,
J.M.C.S.; Zaidi, F.; Souza, H.K.S.;
(TPC led to 38.409 ± 0.095 mg GAE.g−1 and 2.259 ± 0.023 mg QE.g−1 for TFC). Films based on
Gonçalves, M.P. Phenolic Extraction methylcellulose (MC) containing different amounts of DES or MO extracts, acting as plasticizers,
of Moringa oleifera Leaves in DES: were developed and characterized regarding their mechanical, optical, water vapor permeability, and
Characterization of the Extracts and microstructural properties. All films are uniform, clear, and transparent with smooth, homogeneous
Their Application in Methylcellulose surfaces. It was found that the presence of more than 10% of MO extract and/or DES provided more
Films for Food Packaging. Foods 2022, flexible films (Eb for MC 2%_DES 20% achieved 4.330 ± 0.27 %, and 8.15 ± 0.39 % for MC 2%_MO
11, 2641. https://doi.org/10.3390/ 20%) with less mechanical and barrier resistance. The ultimate objective of this study was to provide
foods11172641 information that could assist in the development of antimicrobial active methylcellulose films for
Academic Editor: Zhongxiang Fang sliced wheat bread packaging.

Received: 1 August 2022

Keywords: methylcellulose films; deep eutectic solvents; Moringa oleifera leaf extracts; water vapor
Accepted: 29 August 2022
permeability; mechanical and optical properties; HPLC-DAD-ESI-MSn analysis; antioxidant capacity
Published: 31 August 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations.
The growing demand of consumers for minimally processed foods, associated with
a swing away from the addition of synthetic preservatives used to prolong the microbial
and sensory life of bakery products, and the recent request for environmentally friendly
Copyright: © 2022 by the authors.
packaging, encouraged the search for innovative solutions to extend the shelf lives of
Licensee MDPI, Basel, Switzerland. these products. Natural extracts (i.e., polyphenol and oils) have been used as antimicrobial
This article is an open access article additives for these purpose. In particular, the use of Moringa oleifera to improve the efficiency
distributed under the terms and of food production systems, nutrition, and human health has attracted the interest of
conditions of the Creative Commons numerous researchers. Since its components, mainly in the leaves, consist of carotenoids,
Attribution (CC BY) license (https:// tannins, vitamins, flavonoids, and phenolic acids presenting potent antimicrobial and
creativecommons.org/licenses/by/ antifungal activities, it is considered one of the most useful plants in the world to be used
4.0/). in different applications [1–3].

Foods 2022, 11, 2641. https://doi.org/10.3390/foods11172641 https://www.mdpi.com/journal/foods

Foods 2022, 11, 2641 2 of 17

Traditionally, conventional organic solvents with liquid–liquid extraction or solid-

phase extraction were applied to extract phenolic compounds from plants. However,
from the viewpoint of green chemistry, most of the commonly used solvents are volatile,
flammable, and toxic liquids. Besides, these extraction methods have the major disadvan-
tage of solvent residue being present in the extracts. In this sense, an innovative category of
solvents such as Deep Eutectic Solvents (DES) has been used to overcome the disadvantages
of organic solvents. In recent years, sustainable “green” extraction procedures have been
published for the recovery of phenolic compounds from Moringa oleifera [4–6].
Edible and coating films derived from natural polymers have been used to protect
the quality and extend the shelf life of food products. In this sense, there are few studies
in the literature regarding the use of Moringa oleifera in polymer-based food packaging.
Lee et al. [7] recently developed an antimicrobial, antioxidative, and biodegradable packag-
ing material from puffer fish skin gelatin containing Moringa oleifera leaf extract. Núñez-
Gastélumn et al. [8] determined the physical–structural properties of biodegradable films
of chitosan and polycaprolactone (PCL) added with Moringa oleifera leaf extract. Tes-
fay & Magwaza [9] reported the potential of edible coating containing Moringa oleifera
leaf extracts to improve avocado fruit quality during the storage period. More recently,
Bhat et al. studied [10] the incorporation of Moringa oleifera leaf extract into a chitosan/guar
gum/poly(vinyl alcohol) matrix to produce active films. Also, triggerfish gelatin films
containing Moringa oleifera leaf were used to package ricotta cheese [11].
An emerging area of food technology is active packaging, where absorbing and/or
releasing compounds that are effective against the aging of bread and/or with antimicro-
bials, are incorporated into the packaging itself, thus preventing the growth of undesirable
microorganisms. The major changes that occur after baking are moisture redistribution,
starch retrogradation, increased firmness, and loss of aroma and flavor. Staling, in which
the starch begins to expel excess water returning to its crystalline form, is a spontaneous
and irreversible phenomenon, and firming can easily be controlled by active packaging [12].
Thus, the development of surface molds is the main reason for the shelf life reduction
of bakery products. The growth of microorganisms in bread-making products begins or
is more intense at the surface, which makes the use of edible films and coatings a good
choice for maintaining the quality of these products [13]. Agents may be incorporated
into the packaging that can directly interact with spoilage organisms or interact with the
environment within the packaging without the direct addition of chemical agents [14,15].
Cellulose is one of the most abundant agricultural biopolymer wastes produced on
the earth. Thus, this polysaccharide is useful for the creation of new approaches for various
commercial food applications [16–18]. For example, cellulose-based edible films have
good barrier properties (aroma, oxygen) and oil transfer similar to other hydrophilic films.
Moreover, strong and flexible water-soluble films were produced by cellulose derivatives,
such as methylcellulose and hydroxypropyl methylcellulose, and may offer a promising
alternative to transport natural antimicrobial agents [19]. In this regard, previous studies
using cellulose and its derivatives have shown that the microbial shelf life of bread can be
increased by active packaging. Vojdani & Torres [20,21] have examined the diffusion barrier
properties of methylcellulose and hydroxypropyl methylcellulose films with great potential
to be used as a substrate for the antimicrobial agent potassium sorbate. The addition of
fatty acids (e.g., lauric, palmitic, stearic, and arachidic acids) have been demonstrated to
diminish the diffusion of potassium sorbate in cellulose-based films [22]. More recently,
Otoni et al. [23] successfully produced methylcellulose films with the incorporation of
micro and nanoemulsions of clove bud and oregano essential oils that displayed antimi-
crobial activity against spoilage fungi in bakery products. Noshirvania et al. [24] studied
a nanocomposite film and coating, based on chitosan-carboxymethyl cellulose-oleic acid
incorporated with different concentrations of zinc oxide nanoparticles, as a high barrier
packaging material acting as a preservative (microbial and staling) on the surface of sliced
wheat bread.
Foods 2022, 11, 2641 3 of 17

This work aims to evaluate the in vitro effectiveness of bio-based films prepared from
methylcellulose with and without incorporating deep eutectic solvent (DES) and Moringa
oleifera leaf extracts (MO) and to ascertain its applicability in the design of active food
packaging systems for wheat sliced bread.

2. Materials and Methods

2.1. Materials
Calcium chloride ≥ 97.0%, (CAS: 10043-52-4), granular drying agent, and choline
chloride ≥ 99.0%, (CAS: 67-48-1), were obtained from Sigma-Aldrich (Darmstadt, Ger-
many), glycerol ≥ 99.5%, (CAS: 56-81-5) was purchased from Panreac, DL-lactic acid (≥90%)
from Sigma-Aldrich, Moringa oleifera leaves were harvested and treated as described previ-
ously [25], and MethocelTM (Methylcellulose, CAS: 9004-67-5, D.S. 25–33%), was purchased
from SpecialIngredients® . Methanol (purity 98%) was purchased from Panreac, and all the
other reagents used for antioxidant capacity were of analytical grade and purchased from
Sigma-Aldrich.

2.2. Methods
2.2.1. DES Preparation
The DES used in this study were prepared as previously described [26]. Briefly, choline
chloride (ChCl), previously dried at 70 ◦ C overnight in an oven and used as quaternary
ammonium salt (HBA), was mixed with an appropriate hydrogen bond donor (Lactic acid
(LA) and Glycerol (GLY)) at molar ratio 1:2.
A homogeneous liquid was formed by heating the mixture at 80 ◦ C under constant
stirring. After that the solution was left to cool at room temperature. The water content
for each DES (in triplicate) was determined by Karl Fischer titration using a Metrohm Karl
Fischer coulometer and it was found to be lower than 2% for all the DES.

2.2.2. Extraction of Moringa oleifera Leaves in DES

Phenolic compounds of Moringa oleifera leaves were extracted in deep eutectic solvents
DES (ChCl/GLY 1:2) or (ChCl/LA 1:2) following a previously published methodology by
Oomah et al. [27]. In brief, 1 g of Moringa oleifera leaves flour was mixed with 40 mL of DES
and extracted by continuous stirring at 900 rpm for 2 h. Then, the extract was centrifuged
for 30 min, at 11,000 g, and the supernatant was carefully removed and stored in the freezer
(−18 ◦ C).

2.2.3. Spectrophotometric Analysis of Moringa oleifera Leaf Extracts

Phenolic Compounds
The total phenolic content (TPC) was estimated by the Folin–Ciocalteu standard
method as previously reported by Skerget et al. [28]. Briefly, 2.5 mL of Folin–Ciocalteu
reagent (10% v/v) was added to 0.5 mL of diluted extract followed by 2 mL of sodium
carbonate (75 g·L−1 ) after 5 min. The samples were incubated at 50 ◦ C for 5 min then
cooled and the absorbance was measured at 760 nm. TPC was expressed as mg gallic acid
equivalent (GAE) per g of sample.
Total flavonoid content (TFC) was measured according to Djeridane et al. [29]. 1 mL of
2% AlCl3 .6H2 O (2 g in 100 mL methanol) solution was added to the same volume of diluted
extract, then vortexed and left for 15 min in the dark at room temperature. Afterwards, the
absorbance was monitored at 430 nm and TFC was expressed as mg quercetin equivalent
(QE) per g of sample. All the analyses were performed in triplicate.

Antioxidant Capacity
The determination of the antioxidant capacity of Moringa oleifera leaf extracts was
carried out following DPPH, ABTS, FRAP, and ORAC assays. All measurements were
performed in triplicate and expressed as mmol Trolox equivalents (TE) per g of sample,
except ABTS which was expressed as percentage of inhibition (PI).
Foods 2022, 11, 2641 4 of 17

A microplate reader (BioTek Instruments Inc., San Diego, CA USA) was used to
perform DPPH and FRAP assays according to the previously established protocol [30]. In
brief, 20 µL diluted extracts, blank (solvent), and standard were introduced into microplates
and 300 µL DPPH solution freshly prepared (10 mg in 250 mL of methanol) was added. The
samples were placed in the dark for 30 min at room temperature then the absorbance was
monitored at 515 nm. The ability of Moringa oleifera leaf extracts to reduce the complex of
Fe3+ -2,4,6-tris(2-pirydyl)-s-triazine into Fe2+ -2,4,6-tris(2-pirydyl)-s-triazine was determined
by adding 300 µL fresh FRAP reagent to 50 µL of diluted extracts and the absorbance was
read at 593 nm after 5 min.
The oxygen radical absorption capacity assay (ORAC) was determined according to
Huang et al. [31], with some modification as already detailed in our previous work [25]. The
ABTS radical scavenging capacity was measured following the method of Giao et al. [32],
in which a solution containing 7 mmol.L−1 ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt) and 2.45 mmol.L−1 potassium persulfate in equal volume
was allowed to react in the dark for 16 h at room temperature. This solution was diluted
until an absorbance of 0.700 ± 0.02 was reached at 735 nm. 20 µL of the extract was mixed
with 1.5 mL of the prepared solution and the absorbance was measured after 6 min at
735 nm. The absorbance of the solvent was subtracted from that of the sample in all tests.

2.2.4. HPLC-DAD-ESI-MSn Analysis

The phenolic profile of Moringa oleifera leaves extracted with deep eutectic solvent
(GLY and LA) was studied using the HPLC system (Thermo Electron Corporation, Waltham,
MA, USA) coupled with an ion-trap mass spectrometer and diode array detector (DAD),
equipped with a low-pressure quaternary pump with an autosampler and a diode array
detector (Finnigan Surveyor Plus, Thermo Fisher Scientific, Waltham, MA USA). The
qualitative analysis of the samples was carried out as described previously.

2.2.5. Films Preparation

For the determination of the proper concentration of methylcellulose, (MC), the flow
properties of different concentrations of MC were studied using 1–2 and 5% (dry basis) in
distilled water. A rheometer (TA Instrument, New Castle, DE, USA) was used to measure
the viscosity of these solutions. The solution that presented the best properties was MC 2%,
since for higher concentrations of MC the solutions have a very high viscosity.
The MC solution was prepared by first dispersing the MC powder with 1/3 of the
total required volume of distilled water that had been heated to above 70 ◦ C. For complete
solubilization, the remainder of the water was then added as cold water to lower the
temperature of the dispersion. Stirring continued until all particles had been thoroughly
wetted. The solution was then stored overnight at T = 4 ◦ C to remove all bubbles.
Three samples of about 1 g each of MC solutions were dried at T = 105 ◦ C and weighed
to ±0.0001 g for the determination of the MC dry mass.
Film-forming solutions preparation: DES (ChCl:glycerol, 1:2), or DES Moringa oleifera
extract (MO) were added as the plasticizer to the MC solution, and the composition of
various films used is given in Table 1.

Table 1. Composition of films. (wt% per dried MC amount).

Sample Methylcellulose % DES (ChCl:Gly, 1:2) % MO (Moringa oleifera Extracted in DES) %

MC 2% 2 - -
MC 2%-DES 10% 2 10 -
MC 2%-DES 20% 2 20 -
MC 2%-MO 10% 2 - 10
MC 2%-MO 20% 2 - 20
Foods 2022, 11, 2641 5 of 17

The films were produced in different plasticizer proportions in order to find the
formulation that resulted in films with better performance.
Films were made by the knife-coating technique (Sheen, model 1132N, Cambridge,
UK). Seventy grams of the solution was spread at 0.3 m·s−1 on acrylic plates using an 1132N
film applicator (Sheen Instruments, Cambridge, UK). The films were dried in a climate
chamber (Binder KMF 115) for 24 h (40 ◦ C and 60% relative humidity (R.H.)), removed
from the acrylic plates, and then conditioned at 25 ◦ C and 53% R.H. for at least 72 h before
performing their characterization.

2.3. Film Properties

2.3.1. Thickness Measurements
The thickness of the films was measured using a digital micrometer (Mitutoyo Co.,
Japan, model ID-F150). The average thickness was calculated from measurements taken at
ten different locations on each film sample.

2.3.2. Water Vapor Permeability (WVP)

The water vapor permeability (WVP) tests were determined gravimetrically as de-
scribed elsewhere, and according to ASTM Standard Test Methods for Water Vapor Trans-
mission of Materials (E 96-00) [33]. Briefly, the test films (80 mm in diameter) were sealed
on top of the permeation cells (Elcometer 5100/2 Payne Permeability Cup), containing
anhydrous calcium chloride (R.H. = 2%) and put in a desiccator at a constant temperature
of 21 ± 1 ◦ C and R.H. 100%. Air convection of approx. 0.3 m·s−1 was used to promote
water diffusion (using a fan) and to ensure uniform conditions throughout the chamber.
The cells were weighed periodically. The measured WVP of the films was determined
as follows:
∆m · x
WVP = , (1)
A · ∆t · ∆p
where WVP is the water vapor permeability (g·m−1 ·s−1 ·Pa−1 ) 1 ), ∆m is the weight gain (g),
x is the film thickness (m), and A is the area (0.003 m2 ) exposed for a time ∆t (s) to a partial
water vapor pressure ∆p (Pa).
For each type of film, WVP measurements were replicated three times for each batch
of films.

2.3.3. Mechanical Properties

Mechanical properties of the films (tensile strength, TS, elastic modulus, YM, and
elongation at break, Eb), were measured using a texture analyzer (TA.XT2, Stable Micro
Systems Ltd., Surrey, UK) equipped with adequate tensile test grips (A/TG model). Once
conditioned, films specimens with dimensions of 25 mm × 100 mm were mechanically
fractured; the applied test speed was 0.2 mm s−1 , while force (N) and deformation (%
strain) were recorded. At least seven replicates were used for each film formulation.

2.3.4. Scanning Electron Microscopy (SEM)

The scanning electron microscope FEI Quanta 400 FEG, under high vacuum (5 kV
voltage and ≈11.1 mm working distances—CEMUP—Centro de Materiais da Universidade
do Porto-PT) was used to study the morphology of the films. Before analyses, film samples
were rapidly cooled in liquid nitrogen, fractured, mounted on aluminum stubs, covered
with double-coated carbon conductive adhesive tabs, (Electron Microscopy Sciences, Hat-
field, PA, USA), and coated with a thin film of Au-Pd.

2.4. Bread Experiences

For many types of bread, the relatively high moisture content of the crumb is responsi-
ble for the appearance of mold, with the bread’s shelf life being around 4 to 8 days. Sensory
shelf life can be even shorter with some bread brands staling within 24 h although 6–10 days
is more common [34].
Foods 2022, 11, 2641 6 of 17

2.4.1. Evaluation of Antifungal Effectiveness of Methylcellulose Films in Sliced Bread

The wheat bread was purchased from a local bakery, cut into 20 mm thick slices, and
wrapped with methylcellulose film formulations: MC 2%; MC 2% _DES10%, and MC 2%
_MO10% (active packaging tests), sealed in plastic bags to avoid moisture loss, and left in
room temperature, 20 ± 2 ◦ C, to simulate usual commercialization conditions of bakery
products. Control packaging tests were performed in the same way without including the
MC film. Only if there is a delay in fungal growth, when compared to the control, is the
packaging considered effective. Tests were performed in duplicate.

2.4.2. Determination of Bread Firmness

The determination of the bread firmness was carried out using a TA-XT2 (Stable Micro
Systems) texture analyzer, equipped with a cylindrical probe of 6 mm (P/6). From the force
versus deformation curves, the maximum puncturing force, in N, was obtained. At least
8 measurements were made for each sliced bread.

2.4.3. Weight Loss Measurements

The weight loss of bread was determined gravimetrically and expressed as a percent-
age of initial bread weight.

2.5. Color Measurements

Color was measured with a Minolta colourimeter CR 300 (Tokyo, Japan), using a
white standard as a background to determine the CIELAB color parameters, lightness,
L* (0 [black] to 100 [white]), and chromaticity parameters a* (green [−60] to red [+60]) and
b* (blue [−60] to yellow [+60]). The opacity of the films was also measured using black
and white standards. For each sample, the test was performed in several zones, with a
minimum of 5 scans for every treatment, and an average value was obtained.

2.6. Statistical Analyses

Software package GraphPad Prism San Diego, CA, USA, version 5.00 for Windows,
was used for the experimental results evaluation. The mean differences of the film proper-
ties were examined by an ANOVA with Bonferroni’s post hoc-test and were considered
significant at p < 0.05.

3. Results and Discussion

3.1. Spectrophotometric Analysis of Moringa oleifera Leaf Extract
Deep eutectic solvents offer advantages in the extraction process including low price,
chemical inertness with water, ease of preparation, biodegradability, and so on [35].
In addition, their compatibility with food industries allows them to be more appro-
priate than organic solvents. In this context, two types of DES were tested in the ex-
traction of antioxidants from Moringa oleifera leaves. As illustrated in Table 2, both of
ChCl/GLY and ChCl/LA extracts show an efficient extraction of polyphenols and ex-
hibit a high antioxidant capacity where values of TPC and TFC attained 38.409 ± 0.095,
28.314 ± 0.146 mg GAE.g−1 , and 2.259 ± 0.023, 1.595 ± 0.023 mg QE.g−1 , respectively, also
the antioxidant capacity led to 0.361 ± 0.017, 0.469 ± 0.003 mmol TE·g−1 for FRAP and
1.200 ± 0.066, 2.107 ± 0.003 mmol TE·g−1 for ORAC assay. Statistical data show signifi-
cant differences between the two solvents, therefore, ChCl/GLY extract presents a higher
amount of TPC and TFC, and similarly the highest antiradical potential against DPPH and
ABTS (0.294 ± 0.010 mmol TE·g−1 and 92.614 ± 0.085%). On the other hand, ChCl/LA
extract shows more ability to reduce iron and scavenge peroxyl radicals, and the obtained
results are in accordance with a previous study [36], denoting that the ChCl/LA extract
exhibits the highest FRAP value and the lowest ABTS value compared to the other DES
extract. In addition, the antioxidant power of an extract can be influenced by several factors,
including the polarity and solubility of the extracted ingredients, structure (number and
Foods 2022, 11, 2641 7 of 17

the position of hydroxyl groups), the free radicals’ nature and their interaction mechanisms
with other moieties, and the solvent type [37].

Table 2. Phenolic compounds (TPC and TFC) and antioxidant capacity (DPPH, FRAP, ABTS, and
ORAC).

Samples TPC TFC DPPH FRAP ORAC ABTS

(mg GAE·g−1 ) (mg QE·g−1 ) (mmol TE·g−1 ) (mmol TE·g−1 ) (mmol TE·g−1 ) (PI)
ChCl/LA1:2 28.314 ± 0.146 b 1.595 ± 0.023 b 0.176 ± 0.022 b 0.469 ± 0.003 a 2.107 ± 0.003 a 55.529 ± 0.267 b
ChCl/GLY1:2 38.409 ± 0.095 a 2.259 ± 0.023 a 0.294 ± 0.010 a 0.361 ± 0.017 b 1.200 ± 0.066 b 92.614 ± 0.085 a
Results are expressed as mean ± SD per gram of sample. Different superscript letters in the same column indicate
significant differences at p < 0.05.

3.2. HPLC-DAD-MSn Profiling of Phenolics from Moringa oleifera Leaf Extracts

The qualitative analysis of the phenolic composition from extracts of Moringa oleifera
leaves obtained using DES with lactic acid (LA) and glycerol (GLY) has been carried out
using HPLC-DAD-ESI-MSn in negative ionization mode. The chromatograms of both
extracts recorded at 280 nm are shown in Figure 1. Similar phenolic compounds were
extracted with DES-LA and DES-GLY, however, the variation found in terms of antioxidant
capacity can be explained by the difference in the relative concentration of extracted
compounds. They were tentatively identified by MS data together with the interpretation
of MSn and UV spectra in comparison with those found in the literature. The identities,
Foods 2022, 11, x FOR PEER REVIEWretention times, and observed molecular and fragment ions of individual compounds
9 of 18 are
presented in Table 3. Ten phenolic compounds were tentatively identified (identified as
peaks 1–10).

Figure 1. HPLC-UV
Figure HPLC-UVchromatograms
chromatogramsofofM.M.oleifera extracts
oleifera prepared
extracts with
prepared DES
with (with
DES LA LA
(with andand
GLY)
GLY)
detected at
detected at 280
280 nm.
nm. Peaks
Peaksare
aretentatively
tentativelyidentified
identifiedininTable
Table2.2.

Table 3. Retention times, absorption maxima, and characteristic ions of tentatively identified
compounds in M. oleifera extracts by HPLC-ESI-DAD-MSn.

Rt λmax [M-H]- MSnfragments

Peak Identification
(min) (nm) (m/z) (m/z)
1 24.6 325 353 191, 179, 135 3-Caffeoylquinic acid
2 33.1 310 337 191, 173, 163 p-Coumaroylquinicacid
Foods 2022, 11, 2641 8 of 17

Table 3. Retention times, absorption maxima, and characteristic ions of tentatively identified com-
pounds in M. oleifera extracts by HPLC-ESI-DAD-MSn .

Rt λmax [M-H]- MSn fragments

Peak Identification
(min) (nm) (m/z) (m/z)
1 24.6 325 353 191, 179, 135 3-Caffeoylquinic acid
2 33.1 310 337 191, 173, 163 p-Coumaroylquinicacid
3* 40.5 325 353 191, 179, 135 5-Caffeoylquinic acid
4 52.4 271, 334 593 575, 503, 473, 383, 353 Apigenin 6,8-di-C-glucoside
5 62.5 268, 337 431 341, 311, 283 Vitexin/isovitexin
6 65.5 271, 337 431 341, 311, 283 Vitexin/isovitexin
7* 69.1 256, 290sh, 355 609 301, 179, 151 Rutin
8 72.8 265, 290sh, 350 447 327, 285 Kaempferol-3-O-glucoside
9 73.1 253, 290sh, 355 593 285 Kaempferol-3-O-glucoside-7-rhamnoside
10 76.5 264, 289sh, 346 533 489, 447, 285 Kaempferol-3-O-malonylglucoside
* Compounds were identified by comparison with reference standards.

Two structural isomers of caffeoylquinic acid (CQA) were identified in Moringa oleifera
leaf extracts (peaks 1 and 3 with retention times of 25 and 40 min). These peaks exhibited a
characteristic intense molecular ion with m/z 353 in negative ion mode and a characteristic
absorption maximum at 325 nm (Table 2), characteristic from CQA. CQA yielded a fragment
ion at m/z 191, which produced an MS/MS spectrum identical to that of quinic acid (m/z
85, 127, 173). Therefore, m/z 191 is due to quinic acid ion resulting from cleavage of the
C-O bond of the ester linkage. The ion at m/z 179 was identified at the caffeic acid portion
of chlorogenic acid [38,39]. The ion at m/z 179 loses a carbon dioxide moiety to produce the
product ion observed at m/z 135. Peaks 1 and 3 fragment into m/z 135, 179, and 191 ions.
Compound 1, one of the most intense peaks on the chromatogram of both extracts, was
tentatively attributed to 3-CQA, by giving the characteristic negative ion fragments of
m/z 191 and 179, which has also been previously identified as one of the main phenolic
compounds in hydroalcoholic and hydroacetonic extracts of Moringa oleifera leaves [25].
Peak 3 was identified as 5-CQA by comparison with the reference pure standard, with an
MS/MS base peak m/z 191 and a low-intensity MS/MS ion at m/z 179 [39]. The 4-CQA
can be differentiated from the other isomers by having a base peak at m/z 173 [39,40]. No
similar fragmentation pattern was found in the leaves extract, so the presence of 4-CQA
was excluded in these extracts.
Peak 2 (Rt 33 min) showed a pseudomolecular ion at m/z 337 and was tentatively
identified as p-coumarylquinic acid. As previously described, the precursor ion was
fragmented in product ions corresponding to quinic acid (m/z 191, [quinic acid–H]-; and
m/z 173 [quinic acid–H–H2 O]-), p-coumaric acid (m/z 163, [p-coumaric acid–H]-), and
p-coumaric acid residue (m/z 119, [p-coumaric acid–H–CO2 ]-) [41].
Compound 4 was identified as the di-C-glucosyl flavone Apigenin 6,8-di-C-glucoside
(vicenin-2). The compound showed a deprotonated molecule at m/z 593, and a characteris-
tic fragmentation pattern with fragment ions at m/z 575 [(M-H)-18], m/z 503 [(M-H)-90],
m/z 473 [(M-H)-120], m/z 383 [(M-H)-90-120], and m/z 353 [(M-H)-120-120]. The vicenin-2
was recently reported in Moringa oleifera leaves [25]. Peak 5 and 6 exhibited a pseudo-
molecular ion at m/z 431. The MS/MS spectra gave fragment ions at m/z 341, 311, and
283, consistent with the presence of vitexin or isovitexin, as previously reported [25]. Peak
7 was identified as Rutin by comparison with the reference standard, and is one of the
main phenolic compounds present in the extracts. The mass of the parent ion is 609 and its
fragmentation pattern revealed a base peak ion at m/z 301, corresponding to the deproto-
nated aglycone. Peaks 8, 9, and 10, very abundant in Moringa oleifera DES extracts, were
tentatively identified as kaempferol glycoside derivatives. Peak 8, with a retention time
of 72 min and a characteristic absorption maximum at 350 nm, produced a deprotonated
molecular ion peak at m/z 447 and yielded fragment ions at m/z 327 [(M-H)-120] and m/z
285 [(M-H)-162], consistent with the loss of a glucosyl reside and the generation of the char-
Foods 2022, 11, 2641 9 of 17

acteristic aglycone fragment, respectively. Other characteristic fragment ions at m/z 255 and
227 were also observed (Table 1). The compound was therefore identified as kaempferol-
3-O-glucoside, as previously described [42,43], and also identified in Moringa oleifera leaf
extracts [44]. Peak 9, the most intense peak identified in the extracts at 280 nm, exhibits a
deprotonated molecule at m/z 593. According to the information previously reported by
Ibrahim et al. [42] this peak may be attributed to kaempferol-3-O-glucoside-7-rahmonisde.
The fragmentation patterns with a characteristic fragment at m/z 285 is consistent with the
loss of glycosidic residues (rhamnosyl, 146 amu; and glucosyl, 162 amu) [42]. Peak 10 was
tentatively identified as kaempferol-3-O-malonylglucoside according to the mass spectrum
with a deprotonated molecule at m/z 533 and characteristic absorption maximum at 264
and 346 nm. The molecular ion fragmentation yielded ions with m/z 489 and 447 related
to kaempferol glucoside after the loss of the CO2 ([M-H]-44) and subsequent malonyl
moiety ([M-H]-86). The presence of the ion at m/z 285 confirms the presence of aglycone
kaempferol after losing a hexose moiety ([M-H]-162).

3.3. Color Parameters and Optical Properties of Films

The effect of DES-GLY or MO extract (extracted in DES-Gly) incorporation on the
color parameters of the MC films is shown in Table 4. All the films are uniform, clear,
and transparent with high lightness (high Hunter L-value of 93). A decrease in lightness
(p < 0.05) was observed in films with 20% DES or 20% MO extract. The chromatic coordinate
parameters increased (red-green (a*)) or decreased (blue-yellow (b*)), significantly by
increasing the plasticizer content (20% for DES or any MO content) in relation to the control
film (MC 2%). In addition, MC 2% films loaded with 20% DES or 20% MO extract showed
higher ∆E* than MC control. Moreover, the opacity values of films plasticized with 10%
DES or 10% MO extract were similar to the control film and higher for 20% MO extract.
The increase in the opacity for the MC 2%_MO 20% film can be mainly attributed to the
presence of Moringa compounds in the formulation.

Table 4. Color parameters and opacity of the different Methylcellulose films (Opacity, L*, a*, b* and
∆E*).

Material Opacity (%) L* a* b* ∆E*

MC 2% 0.64 ± 0.07 a 93.66 ± 0.20 a 0.25 ± 0.04 a 2.48 ± 0.18 a 2.62 ± 0.09 a
MC 2%_DES 10% 0.62 ± 0.06 b 93.67 ± 0.06 a 0.25 ± 0.03 a 2.46 ± 0.18 a 2.66 ± 0.04 a
MC 2%_DES 20% 0.48 ± 0.03 c 93.33 ± 0.14 b 0.58 ± 0.09 b 2.02 ± 0.06 b 2.99 ± 0.14 b
MC 2%_MO 10% 0.53 ± 0.02 d 93.53 ± 0.14 a 0.40 ± 0.02 2.14 ± 0.19 c 2.78 ± 0.14 a
MC 2%_MO 20% 0.97 ± 0.06 e 93.25 ± 0.08 b 0.53 ± 0.08 b 2.06 ± 0.13 b,c 3.04 ± 0.07 b
Same letters in the same column indicate that values are not significantly different (p > 0.05).

3.4. Thickness, Mechanical Properties, and Water Vapor Permeability of MC Films

Thickness, d, around 0.019 mm was obtained for 2% MC films, while no significant
differences (p > 0.05) were observed for the addition of MO extract or DESs plasticizers.
Similar behavior was observed for the tensile strength, TS, and no significant differences
(p > 0.05) were found in the rigidity when 10% plasticizer was added to the film formulation.
However, the intermolecular bonds between the MC chains are reduced (TS reduction
(p < 0.05)) when 20% DES or any MO content is added to the formulation, leading to a
weaker (less resistant) film. The elasticity, Eb, of the 2% MC films with the addition of DES
(10% or 20%) or MO extract (10%) as plasticizing agents showed no significant difference
(p > 0.05) in relation to the 2% MC films with no plasticizer added. The addition of more
MO extract (20%) led to a remarkable plasticizing effect providing an almost 3.5-fold
increase in Eb (p < 0.05) when compared to the control film. Regarding the YM values, it
was observed that the addition of any plasticizer seems to destabilize the stable structure
of the MC network, leading to a decrease (p < 0.001) in the YM values. In other words,
the incorporation of DES or MO extract led to more flexible films, the effect being more
pronounced in films plasticized with 20% MO extract, as can be confirmed by the Eb values.
Foods 2022, 11, 2641 10 of 17

The tensile strength (TS) and Eb values reported in the literature for pure MC films
produced by the casting method were higher than our results [45–49]. The differences
are probably due to physicochemical characteristics of the methylcellulose used, film
production conditions, and different thicknesses obtained. In the last two decades, deep
eutectic solvents have been tested as plasticizing agents for polysaccharide films [50].
Choline-based deep eutectic solvents have been found to efficiently plasticize pectin [26],
starch [51], guar gum [52], or chitosan films [53,54]. However, to the authors’ knowledge, to
date, no data on the mechanical properties of DES-plasticized MC films have been reported,
therefore, no comparison with the literature can be made. According to the literature, the
addition of different plasticizers (malic acid, sorbitol, PEG400, or glycerol) decreases TS and
increases Eb values in MC films [48,49], which is in agreement with the results presented in
the Table 5.

Table 5. Film thickness (d) and mechanical properties (tensile strength, TS; elongation at break, Eb ;
and elastic modulus, YM) and water vapor permeability (WVP) for the methylcellulose films.

WVP·10−10 /
Material d/(mm) TS/(MPa) Eb/(%) YM/(MPa) (g·m−1 ·s−1 ·Pa−1 )
MC 2% 0.020 ± 0.001 a 24.30 ± 1.7 a 2.455 ± 0.31 a 32.87 ± 1.0 1.40 ± 0.002
MC 2%_DES 10% 0.018 ± 0.002 a 23.97 ± 1.7 a 3.290 ± 0.35 a,b,c 28.65 ± 0.95 1.70 ± 0.028 a
MC 2%_DES 20% 0.019 ± 0.002 a 20.84 ± 1.6 b 4.330 ± 0.27 b 22.26 ± 1.3 1.71 ± 0.074 a
MC 2%_MO 10% 0.021 ± 0.002 a 21.74 ± 1.3 c 3.850 ± 0.79 c 20.5 ± 0.47 a 2.02 ± 0.039 b
MC 2%_MO 20% 0.019 ± 0.001 a 21.72 ± 0.71 b,c 8.15 ± 0.39 18.02 ± 2.8 a 2.00 ± 0.026 b
Same letters in the same column indicate that values are not significantly different (p > 0.05).

Regarding the barrier properties, the water vapor permeability (WVP) values were
statistically dependent on the DES or MO extract content (p < 0.05) An increase in WVP
(hence a decrease in barrier properties) was observed for all MC films containing plasticizer,
with the greatest increase being observed for MC films with 10% or 20% MO extract (Table 5).
However, the increase of DES or MO extract contents on the MC-film formulations do not
affect the WVP values. The decrease in the water vapor barrier properties of the plasticized
samples is mainly due to the hydrophilic nature of the DESs and MO extract components.
Similar behavior was observed with Khorasan wheat starch films containing MO
extract, the phenolic compounds in the extract affected the interactions between starch
and water molecules and caused the increase in WVP [55]. In other studies, the addition
of PEGs with several MWs to the MC matrix resulted in an increase in the WVP of the
films [48] while the incorporation of resveratrol, a lipophilic compound, tended to reduce
the WVP of MC films [49].

3.5. Morphologic Analysis of Films

Figure 2 shows SEM micrographs of the surface of the films. The 2% MC film, without
plasticizer, was characterized by having a smooth, homogeneous surface (Figure 2a) and
compact cross-section (Figure 3a). In corroboration with the TS results, 10% of DES does
not affect the surface structure or cross-section of the film. However, the MC films con-
taining 20% DES and MO extract (10% and 20%) showed a relatively loose cross-sectional
structure, and some surface irregularities (increased porosity) are observed in the films
(Figures 2c–e and 3c–e), which could illustrate the decrease in the strength of the films
measured in the mechanical tests carried out. The microstructure of the films observed
by SEM was consistent with the results of TS, YM, and WVP (Table 4). In this sense, the
2% pure MC film presented the highest values of TS and YM and the lowest permeability,
which corroborates its relatively dense and homogeneous image observed by SEM.
Foods 2022, 11, x FOR PEER REVIEW 12 of 18
Foods2022,
Foods 2022,11,
11,2641
x FOR PEER REVIEW 12
11 of 18
of 17

Figure 2. SEM
Figure 2. SEM micrographs
micrographsofofallallstudied
studied film
film samples
samples (magnification
(magnification 10,000×,
10,000 ×, 5.005.00
kV).kV). (a) Pure
(a) Pure MC
Figure 2. SEM micrographs of all studied film samples (magnification 10,000×, 5.00 kV). (a) Pure
MC
2%, 2%,
(b) (b)
MC MC 2%_DES
2%_DES 10%,10%,
(c) (c)
MC MC 2%_DES
2%_DES 20%, 20%,
(d) (d)
MC MC 2%_MO
2%_MO 10%, 10%,
(e) (e) MC2%_MO
MC2%_MO 20%. 20%.
MC 2%, (b) MC 2%_DES 10%, (c) MC 2%_DES 20%, (d) MC 2%_MO 10%, (e) MC2%_MO 20%.

Figure 3.
Figure SEM images
3. SEM images of
of cryo-fractured
cryo-fractured cross
cross section
section (magnification
(magnification 5000 ×, 10.00
5000×, 10.00 kV).
kV). (a)
(a) Pure
Pure MC
MC
Figure 3. SEM images of cryo-fractured cross section (magnification 5000×, 10.00 kV).20%.
(a) Pure MC
2%, (b) MC 2%_DES 10%, (c) MC 2%_DES 20%, (d) MC 2%_MO 10%, (e) MC 2%_MO 2%_MO 20%.
2%, (b) MC 2%_DES 10%, (c) MC 2%_DES 20%, (d) MC 2%_MO 10%, (e) MC 2%_MO 20%.
 3.6. Effectiveness of MC Films on Sliced Wheat Bread
The shelf life of sliced wheat bread has been investigated. As previously observed,
the MC_10% DES film presented properties close to the control film. For this reason, it
was selected for further tests performed on a food model. The MC 2%_ MO 10% film was
Foods 2022, 11, 2641 used for comparison. Accordingly, several samples of sliced wheat bread wrapped12with of 17
MC films were compared with the control (unwrapped sliced wheat bread). The samples
were evaluated for the following characteristics: texture, color, visual appearance, and
moldCross
growth, during
sections ofathe
period of 11-days. MC films observed by SEM are represented in
cryo-fractured
Figure Figure
3. 4 shows the growth of mold on slices of bread exposed to a humid
environment, after 6, 7 and 11 days of storage, at room temperature, 20 ± 2 °C. It is well
3.6.
knownEffectiveness
that the of MC Films
growth on Sliced
of fungi Wheatcan
in foods Bread
be affected by a number of chemical and
physical
The parameters (e.g., temperature,
shelf life of sliced wheat bread haswaterbeenactivity, nutrient
investigated. Asstatus, etc.). observed, the
previously
MC_10% As the DESbread
filmformulation used appeared
presented properties close to
tocontain no preservatives,
the control film. For this fungal
reason,growth
it was
was easily
selected fordetected in the
further tests control bread
performed on a (Figure 4). Regarding
food model. The MC 2%_ the formulations,
MO 10% film MC was films
used
for
seem comparison.
to delay fungal Accordingly, several samples
growth, regardless of the of sliced
type wheat
of film bread
used. wrapped
A possible with MC
explanation
films
couldwere
be the compared with
fact that the theabsorbs
film controlsome
(unwrapped sliced
of the water onwheat bread).
the surface ofThe samples
the bread were
making
evaluated
the medium forless
thefavorable
followingtocharacteristics:
mold growth, whichtexture, is color,
in fullvisual appearance,
agreement with WVP andresults
mold
growth,
(Table 5).during a period of 11-days.
Figure 4 shows the
These findings aregrowth of moldwith
in agreement on slices of bread
previous exposed
studies to a which
[56,57], humid reported
environment,
that
after 6, 7 and 11 days of storage, at room temperature, 20 ± 2 ◦ C. It is well known that the
water barrier properties and water vapor sorption by the package had an effect on
growth
moisture of accumulation
fungi in foods and can be affected growth,
microbial by a numberplayingof chemical and physical
an important role inparameters
extending
(e.g.,
breadtemperature,
shelf life. water activity, nutrient status, etc.).

Figure 4.
Figure 4. Growth
Growthof of
fungi naturally
fungi present
naturally in wheat
present bread:
in wheat (a,e,i) (a,e,i)
bread: controlcontrol
(unwrapped), (b,f,j)
(unwrapped),
wrapped with MC 2% film, (c,g,k) wrapped with MC 2%_DES 10% film, (d,h,l) wrapped
(b,f,j) wrapped with MC 2% film, (c,g,k) wrapped with MC 2%_DES 10% film, (d,h,l) wrapped with MC
2%_MO 10% film. (a–d, after 6 days; e–h, after 7 days; i–l, after 11 days).
with MC 2%_MO 10% film. (a–d, after 6 days; e–h, after 7 days; i–l, after 11 days).

As the bread formulation used appeared to contain no preservatives, fungal growth

was easily detected in the control bread (Figure 4). Regarding the formulations, MC films
seem to delay fungal growth, regardless of the type of film used. A possible explanation
could be the fact that the film absorbs some of the water on the surface of the bread making
the medium less favorable to mold growth, which is in full agreement with WVP results
(Table 5).
Foods 2022, 11, 2641 13 of 17

These findings are in agreement with previous studies [56,57], which reported that
water barrier properties and water vapor sorption by the package had an effect on moisture
accumulation and microbial growth, playing an important role in extending bread shelf life.
Firmness (N) values for sliced bread during storage are shown in Table 6. Firmness is
one of the key factors in determining bread quality and, in addition to starch retrogradation,
distribution of water plays an important role by increasing the moisture content, causing
less firmness of the bread [58].

Table 6. Firmness of sliced bread.

Bread (N·mm−1 )
Day
Control MC MC_DES MC_MO
0 1.03 ± 0.05 a

1 0.97 ± 0.04 a 0.96 ± 0.08 0.91 ± 0.06 1.34 ± 0.03 a

4 1.59 ± 0.12 1.88 ± 0.08 a 1.44 ± 0.05 1.35 ± 0.11 a
7 1.94 ± 0.04 b 1.94 ± 0.05 a 1.58 ± 0.08 2.16 ± 0.10
11 1.99 ± 0.02 b 1.95 ± 0.10 a 2.14 ± 0.11 2.51 ± 0.04
Same letters in the same column indicate that values are not significantly different (p > 0.05).

The increase in bread crumb firmness is visible for all samples over time, reaching the
highest value for bread with MC_MO. The control sample showed no significant difference
in the parameter after 1 day of exposure to the environment. Also, for the first day, the
results indicate a higher firmness for bread with MC_MO when compared to the other
samples. The migration of moisture from the crumb to the MC_MO film could be the
phenomenon responsible for the increase in bread firmness. In addition, after 4 days,
the MC 2% film seems to protect the samples, since the bread firmness parameter is not
significantly affected compared to the others (MC_DES and MC_MO).
The results of the weight loss of bread slices, measured on days 1, 4, 7, and 11, are
shown in Table 7. It appears that weight loss is more pronounced for the control, while a
slightly better overall result was obtained for the bread wrapped in the MC and MC_DES
films. On the other hand, the bread wrapped in the MC_MO film showed the greatest
weight loss of all samples on day 11.

Table 7. Weight loss of sliced bread (%).

Day
Sample
1 4 7 11
Control 0.48 0.66 0.86 1.35
MC 2% 0.00 0.04 0.35 0.68
MC 2% _DES 10% 0.18 0.39 0.40 0.66
MC 2% _MO 10% 0.17 0.52 0.69 2.01

Bread crumb color changes on days 0, 1, 4, 7 and 11 of storage are collected in Tables
S1–S4. The color of the breads (control and packaged) was affected by storage, which is
consistent with the results reported in the literature [59].
It was observed that the bread crumb color showed a significant increase (p < 0.05)
in redness (a* value) and yellowness (b* value) but a decrease in L* value after 11 days
of storage. The decrease (p < 0.05) in the value of L indicates the intensification in the
development of the darker color of the bread crumb, compatible with an outcome of
Maillard reaction and also associated with the fungal growth in the bread. The increase in
a* values could be explained by the loss of moisture during storage.
Minolta color a*/b* values decreased with increasing time storage. The values varied
less until the 4th day of storage, with results between −0.1 and −0.12 being obtained for
the control sample and the one wrapped in 2% MC film; lower values were observed for
samples wrapped in 2% MC films with plasticizer values up to the 7th day also negative,
Foods 2022, 11, 2641 14 of 17

but positive values were found after 11 days of storage. Interestingly, on day 11, the
Minolta a*/b* color value is similar for all samples except the one wrapped in MC 2%_DES
10% film.

4. Conclusions
In this work, methylcellulose-based active films were prepared with DES and MO
extracts at different concentrations to study the effect on the physical properties of the
polymer and the possible application as active packaging for sliced wheat bread. At
the highest MO concentration, films showed some changes in their physicochemical and
microstructural properties. The presence of phenolic compounds affected the interactions
between polysaccharides and water molecules and caused the increase in WVP of the films,
but did not affect the tensile strength, TS. From the results, it is clear that the incorporation of
DES and MO extract influenced attributes such as firmness, water permeability and visual
microbial growth in bread. Overall, the films have demonstrated some effectiveness against
mold growth, so these active cellulose-based films could be an approach to increasing the
microbial shelf life of bread without directly adding chemical preservatives to the baking
formula. No conclusions have been drawn as to whether films can slow staling in bread.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods11172641/s1, Table S1: Color parameters for bread (un-
wrapped control); Table S2: Color parameters for bread wrapped in MC 2% film; Table S3: Color
parameters for bread wrapped in MC 2% _DES 10% film; Table S4: Color parameters for bread
wrapped in MC 2% _MO 10% film.
Author Contributions: F.B.: investigation; data curation; writing—original draft; L.M.P.F.A.: in-
vestigation; data curation; writing—original draft; K.B.: investigation; data curation; visualization;
D.O.C.: investigation; data curation; writing—original draft; L.F.G.: review; editing; and validation;
J.M.C.S.M.: conceptualization; methodology; writing—review and editing; supervision; F.Z.: con-
ceptualization; writing—review and editing; supervision and validation; H.K.S.S.: methodology;
investigation; formal analysis data curation; writing—original draft; writing—review and editing;
M.P.G.: conceptualization; resources; writing—review and editing; supervision, funding acquisition
and validation. All authors have read and agreed to the published version of the manuscript.
Funding: Projects UIDB/50006/2020 and UIDP/50006/2020, funded by FCT/MCTES through
national funds.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material.
Acknowledgments: Hiléia Souza thanks FCT for PTDC/BII-BIO/5626/2020 project. L. Amaral
thanks funding from FCT/MEC (CEECIND/03202/2018). Fazia Braham thanks University A.Mira
Bejaia for awarding an internship grant.
Conflicts of Interest: The authors declare no conflict of interest.

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