International Journal of Biological Macromolecules 233 (2023) 123478

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Microencapsulation of marjoram essential oil as a food additive using

sodium alginate and whey protein isolate
Karen Elbert Leal Mazza a, André Mesquita Magalhães Costa a, Janine Passos Lima da Silva b,
Daniela Sales Alviano c, Humberto Ribeiro Bizzo a, b, Renata Valeriano Tonon a, b, *
a
Programa de Pós-Graduação em Ciência de Alimentos, Universidade Federal do Rio de Janeiro, Instituto de Química, Brazil
b
Embrapa Agroindústria de Alimentos, Rio de Janeiro, RJ, Brazil
c
Laboratório de Estruturas de Superfície de Microrganismos, Universidade Federal do Rio de Janeiro, Instituto de Microbiologia, Brazil

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

Keywords: Encapsulation techniques are generally used to preserve the volatile compounds of essential oils. This study
Origanum majorana aimed to evaluate the influence of process variables on the microencapsulation of marjoram essential oil (MEO)
Alginate beads (Origanum majorana L.) by ionic gelation. The effect of sodium alginate concentration (0.5–2 g/100 mL),
Whey protein isolate
emulsifier concentration (0.5–2 g/100 mL whey protein isolate (WPI)), and cationic bath concentration
Encapsulation efficiency
(0.05–0.3 mol/L CaCl2) on the emulsions and beads properties were investigated, according to a rotatable central
Morphology
Emulsion properties composite design. MEO chemical composition and antimicrobial activity were assessed. Emulsions were char­
acterized for droplet size and viscosity, while the particles were analyzed for encapsulation efficiency, size and
circularity, and morphology. High concentrations of alginate and WPI intensified the porous structure of the
beads, reducing droplet mean diameter and encapsulation efficiency. High alginate concentrations also increased
emulsion viscosity, affecting positively beads' circularity. The intermediate concentration of sodium alginate
(1.25 g/100 mL), WPI (1.25 g/100 mL), and CaCl2 (0.175 mol/L) were selected as the most appropriate con­
ditions to produce beads with satisfactory circularity and high encapsulation efficiency.

1. Introduction Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus

The use of preservatives by the food industry is a common practice,
aiming to increase products' shelf life. Recently, due to a greater con­
sumer awareness related to health and environmental impacts, the
search for natural and sustainable food additives has considerably
increased. In this context, essential oils with antimicrobial activity
emerge as potential natural preservatives, satisfying consumers' criteria.
Essential oils are obtained by steam or dry distillation from distinct
parts of the plant, or by cold pressing from Citrus fruits, after water
removal by physical methods, i.e., without the use of solvents [1].
Essential oils are a rich source of compounds with antimicrobial prop­
erties, such as terpinen-4-ol, carvacrol, thymol, linalool, and terpineol
[2,3].
The essential oil of marjoram (Origanum majorana L., family Lam­
iaceae) has been recently investigated due to its marked antimicrobial
activity against a wide range of microorganisms, such as Staphylococcus
aureus, Staphylococcus epidermitis, Streptococcus A, Enterococcus faecalis,
mirabilis, Shigella dysenteria, Salmonella Enteritidis, Salmonella Typhimu­
rium, Enterobacter aerogenes and Listeria monocytogenes [4–8]. Marjoram
essential oil composition is divided into two main chemotypes: alcoholic
and phenolic monoterpenoids. The main compounds of alcoholic mon­
oterpenoids are terpinen-4-ol alone or associated with cis-sabinene hy­
drate, while thymol and carvacrol are the major compounds in the
phenolic monoterpenoids chemotype [9].
Essential oils' active compounds can be degraded by oxygen, heat,
and light, besides being highly volatile, hampering their direct appli­
cation in food [10]. Moreover, essential oils can negatively affect the
food sensory characteristics, due to the high concentrations often
required to have the intended activity [11–13]. Therefore, the stabili­
zation of essential oils before food application is recommended.
Microencapsulation is a “packing” technique in which an active
ingredient is coated by an encapsulating material, promoting its pro­
tection [14]. Essential oil microencapsulation can be used to mask or
preserve flavors, aromas, and active components, avoiding their

* Corresponding author at: Embrapa Agroindústria de Alimentos, Av. das Américas, 29501- Guaratiba, CEP 23020-470 Rio de Janeiro, RJ, Brazil.
E-mail addresses: janine.passos@embrapa.br (J.P.L. da Silva), danialviano@micro.ufrj.br (D.S. Alviano), humberto.bizzo@embrapa.br (H.R. Bizzo), renata.
tonon@embrapa.br (R.V. Tonon).

https://doi.org/10.1016/j.ijbiomac.2023.123478
Received 28 October 2022; Received in revised form 18 January 2023; Accepted 25 January 2023
Available online 1 February 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
K.E.L. Mazza et al.
volatilization and degradation. In this context, this technology could
extend the shelf-life and promote the insertion of a predominantly hy­
drophobic ingredient in a water-rich environment, thus favoring its use
by the food industries [15].
Ionic gelation is an appealing technique to encapsulate marjoram
essential oil, since the process is usually carried out at low temperatures,
reducing essential oils volatilization, and does not require complex
equipment, being a low-cost method. It is a simple and versatile tech­
nology, which comprises the crosslinking of an anionic polymer with
polyvalent cations, forming insoluble gels, called spheres, particles, or
beads. The most frequently used gels are those of Ca-alginate [16,17].
Alginate is a biocompatible, biodegradable, and safe biopolymer ob­
tained from brown algae and some bacterial strains, which production
has minimal impact on the environment [18].
The main process variables influencing the ionic gelation process are:
viscosity and concentration of alginate solution, emulsifier type, and
concentration, polymer:oil ratio, pH, flow rate, the concentration of
CaCl2 in the gelation bath and curing time [16,19]. These variables can
directly affect the gel formation and the encapsulation efficiency, as well
as the beads' stability and size. In the case of beads produced from
emulsions, the concentration of the emulsifier is also an important
variable to be considered, since it is directly related to droplet size
[16,20]. Recently, natural emulsifiers such as proteins have been
preferred by consumers over synthetic ones. In this sense, whey protein
isolate (WPI) has been successfully used in the emulsification of
resveratrol, α-tocopherol, and basil essential oil emulsions [20,21].
Ionic gelation has been used to encapsulate Satureja hortensis, clove,
thyme and cinnamon essential oils, showing that the beads successfully
loaded essential oils and reduced the evaporation rate [22,23]. How­
ever, to the best of our knowledge, the effects of ionic gelation process
variables on Origanum majorana L. essential oil beads properties have
not been investigated yet.
In this context, this work aimed to evaluate the influence of process
variables (sodium alginate, whey protein isolate, and cationic bath
concentrations) on the physicochemical and morphological properties
(encapsulation efficiency, size, circularity, and microstructure) of mar­
joram essential oil beads microencapsulated by ionic gelation.
2. Material and methods
2.1. Chemicals
Commercial marjoram essential oil, hydrodistilled from the leaves of
Egyptian marjoram (Origanum majorana), was acquired from Ferquima®
(Vargem Grande Paulista, São Paulo, Brazil). Sodium alginate P.A.
(Dinâmica®, Diadema, São Paulo, Brazil), with M/G ratio of 45/55, was
used as encapsulating polymer, and whey protein isolate (WPI) (Ali­
bra®, Campinas, São Paulo, Brazil) was used as an emulsifier. Dihy­
drated calcium chloride P.A. (Vetec Química Fina®, Duque de Caxias,
Rio de Janeiro, Brasil) was used for the cationic bath. Methyl octanoate
(99,8 % purity) (Sigma Aldrich®, Darmstadt, Germany) was used as an
internal standard for gas chromatography analysis.
2.2. Preparation of emulsions
Prior to microencapsulation, oil-in-water (O/W) emulsions were
prepared. Sodium alginate and WPI were dissolved, separately, in
distilled water, with manual stirring, 24 h before preparing the emul­
sions, for complete hydration. The concentrations of both solutions
varied from 0.5 to 2 g/100 mL, according to the experimental design
(item 2.4.1). Marjoram essential oil concentration was fixed at 2 g/100
g, in relation to the total solution mass [12].
To prepare the emulsions, the essential oil was added to the mixture
of alginate and WPI solutions (80:20, w/w) [24] and homogenized in an
Ultra-Turrax (IKA T8 Basic, Wilmington, USA) at 16,000 rpm for 5 min.
2
International Journal of Biological Macromolecules 233 (2023) 123478
2.3. Characterization of emulsions
2.3.1. Droplet size distribution
Droplet size distribution was measured by laser diffraction, using the
SDC - Microtrac S3500 equipment (Microtrac, Montgomery Ville, USA),
with distilled water as the dispersion medium. A small amount of each
emulsion, in triplicate, was dispersed after homogenization and sub­
jected to three readings of droplet size distribution.
The droplets polydispersity was evaluated by the particle size scat­
tering index (Span), defined as Eq. (1) [25]:
Span = (D90 − D10 )/D50 (1)
where D10, D50 and D90 are the diameters in 10, 50 and 90 % of cu­
mulative volumes.
The mean droplet size was expressed as the average Sauter diameter
(D [2, 3], Eq. (2)).
(∑ )/( ∑ )
D [2, 3] = ni di 3 ni di 2 (2)
where: di = droplet diameter; ni = number of droplets with diameter di.
2.3.2. Rheological behavior
Emulsions rheological behavior was evaluated by the determination
of steady-shear flow curves (shear stress × shear rate), in a MARS II
rheometer (Thermo Scientific, Karlsruhe, Germany) using a titanium
plate-plate geometry with a diameter of 60 mm (PP60Ti), temperature
controller UTC-Mars II (Thermo Scientific, Karlsruhe, Germany) at 25 ◦ C
and gap of 0.2 mm. Three flow ramps (up, down, and up-cycles) were
obtained in a range of shear stress corresponding to shear rates from 0 to
300 s− 1. Experimental data were adjusted to rheological models (New­
tonian, Casson, Bingham, Ostwald de Waele, and Herschel-Bulkley)
using the software RheoWin Data Manager version 4,750,002 (Thermo
Scientific, Karlsruhe, Germany).
2.4. Microencapsulation by ionic gelation
Microencapsulation by ionic gelation was carried out by extru­
sion–dripping of the emulsions described in item 2.2, as stated in
Benavides et al. [12] with some modification, using a 22G gauge syringe
(0.70 mm in diameter and 25 mm in length) coupled to a vacuum sys­
tem, and a CaCl2 bath with concentrations varying from 0.05 to 0.3 mol/
L, according to the experimental design (item 2.4.1). The distance be­
tween the tip of the syringe needle and the CaCl2 solution was 15 cm.
The resulting beads remained under magnetic stirring in the CaCl2 so­
lution for 30 min. Afterward, they were separated from the solution
using a sieve (0.3 mm opening diameter), washed with distilled water to
remove CaCl2 excess and tissue paper was used to absorb the water
excess on the beads' surface [26].
2.4.1. Experimental design - RCCD
The effect of process conditions on the beads' physical-chemical
properties was analyzed according to a rotatable central composite
design (RCCD) 23 with triplicate at the central point. The independent
variables were: i) alginate concentration (0.5 to 2 g/100 mL); ii) WPI
(0.5 to 2 g/100 mL); and iii) CaCl2 concentration (0.05 to 0.3 mol/L),
totalizing 17 tests (Table 3). The evaluated responses were: encapsula­
tion efficiency, area, and circularity of beads.
The following polynomial equation was fitted to data (Eq. (3)):
y =β0 + β1 x1 + β2 x2 + β3 x3 + β11 x1 2 + β22 x2 2 + β33 x3 2 + β12 x1 x2 + β13 x1 x3
+ β23 x2 x3
(3)
where βn are constant regression coefficients, y is the response (encap­
sulation efficiency, area, and circularity), and x1, x2 and x3 are the coded
independent variables (sodium alginate, WPI, and CaCl2 concentration).
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Table 1
Compounds identified in the marjoram essential oil (MEO) and in the marjoram essential oil recovered from the beads (mean values ± SD).
Peak Compounda LRI expb LRI litb Typec Content in MEO (mg/g) Content in encapsulated MEO (mg/g)d

1 alpha-thujene 925 924 MT 7.05 ± 0.06 5.66 ± 0.02

2 alpha-pinene 932 932 MT 8.84 ± 0.08 7.08 ± 0.03
3 camphene 947 946 MT 0.33 ± 0.02 0.30 ± 0.01
4 sabinene 972 969 MT 61.84 ± 0.42 56.16 ± 0.03
5 beta-pinene 976 974 MT 5.15 ± 0.05 4.74 ± 0.01
6 myrcene 991 988 MT 20.04 ± 0.08 18.67 ± 0.01
7 alpha-phellandrene 1005 1002 MT 4.28 ± 0.03 3.75 ± 0.01
8 alpha-terpinene 1016 1014 MT 104.43 ± 0.59 90.14 ± 0.18
9 p-cymene 1023 1022 MT 23.42 ± 0.12 36.75 ± 0.22
10 beta-phellandrene 1027 1025 MT 41.73 ± 0.24 40.87 ± 0.01
11 1,8-cineole 1031 1026 OM 1.80 ± 0.02 1.17 ± 0.01
12 trans-beta-ocimene 1036 1032 MT 0.30 ± 0.04 0.42 ± 0.00
13 cis-beta-ocimene 1046 1044 MT 0.47 ± 0.03 0.77 ± 0.00
14 gamma-terpinene 1058 1054 MT 181.61 ± 0.93 172.09 ± 0.13
15 cis-sabinene-hydrate 1066 1065 OM 26.97 ± 0.10 15.57 ± 0.01
16 terpinolene 1088 1086 MT 39.25 ± 0.17 38.66 ± 0.03
17 trans-sabinene-hydrate 1100 1098 OM 53.34 ± 0.26 26.88 ± 0.02
18 linalool 1101 1099 OM 13.40 ± 0.52 18.09 ± 0.01
19 p-cis-ment-2-en-1-ol 1121 1118 OM 15.26 ± 0.02 10.14 ± 0.00
20 p-trans-ment-2-en-1-ol 1138 1136 OM 12.99 ± 0.01 8.95 ± 0.00
21 terpinen-4-ol 1177 1174 OM 403.11 ± 0.89 262.52 ± 0.06
22 alpha-terpineol 1190 1186 OM 39.59 ± 0.05 25.63 ± 0.18
23 cis-piperitol 1194 1195 OM 4.75 ± 0.01 4.34 ± 0.19
24 trans-piperitol 1207 1207 OM 8.65 ± 0.01 7.73 ± 0.09
25 linalyl-acetate 1255 1254 OM 27.40 ± 0.02 17.38 ± 0.02
26 trans-caryophyllene 1414 1417 ST 23.56 ± 0.02 20.63 ± 0.01
27 alpha-humulene 1448 1452 ST 1.03 ± 0.09 0.88 ± 0.00
28 bicyclogermacrene 1491 1500 ST 8.49 ± 0.01 7.23 ± 0.01
29 spathulenol 1573 1577 OS 0.74 ± 0.03 0.67 ± 0.02
30 caryophyllene oxide 1578 1582 OS 1.46 ± 0.06 1.48 ± 0.03
a
Compounds listed in order of elution from a HP-5MS capillary column.
b
LRI exp.: experimental linear retention indices/LRI lit: linear retention indices from the literature [28] and the program NIST MS search 2.0.
c
MT – monoterpenoid; OM – oxygenated monoterpenoid; ST – sesquiterpene; OS – oxygenated sesquiterpenoid.
d
Content of beads prepared with 1.25 g/100 mL alginate, 1.25 g/100 mL WPI, and 0.175 mol/L CaCl2.

2.5. Characterization of the essential oil and the beads produced by ionic
gelation
2.5.1. Chemical characterization
The composition of free commercial marjoram essential oil and the
essential oil recovered from the beads (1 μL of the samples) were
analyzed by gas chromatography coupled to mass spectrometry
(GC–MS) and gas chromatography with flame ionization detector (GC-
FID). The beads chosen to be compared with the free essential oil were
those prepared with the intermediate concentrations of the studied
factors: 1.25 g/100 mL sodium alginate, 1.25 g/100 mL WPI, and 0.175
mol/L CaCl2.
To recover the essential oil from the beads, the extraction was per­
formed by hydrodistillation of the samples (approximately 100 g) for 3 h
in a Clevenger apparatus [27]. The encapsulated samples were
completely dissolved by a sodium citrate solution (10 g/100 mL) before
analysis.
The GC–MS was performed on a 7890A gas chromatograph coupled
to a 5975C mass spectrometer (Agilent, USA), equipped with an HP-5MS
capillary column (30 m × 250 μm × 0, 25 μm). For mass spectrometry
analysis, a selective mass detector in electronic ionization mode (70 eV)
was used. Analyses were performed in full scan acquisition mode, at 2.0
scans/s. The oven program was as follows: the run started at 60 ◦ C,
increased until 240 ◦ C at a 3 ◦ C/min rate, and was held for 10 min. The
temperature of the injector was 250 ◦ C and the split ratio was 20:1. The
flow of carrier gas (helium) was 1.0 mL/min.
Data were collected by ChemStation GC–MS software package
(version B.07.00 SP2; Agilent Technologies Inc., Santa Clara, Califor­
nia). The identification of the compounds was based on mass spectra
databases, Wiley 6th edition, NIST MS search 2.0, with the mass spectra
of the literature, and on the linear retention indices (LRI) [28]. The LRI
of the compounds was calculated based on the retention time of the
compounds and a homologous series of n-alkanes (C7-C26), injected in
the same conditions as the samples [29]. Afterward, it was compared
with the LRI described by Adams [28].
The compounds were quantified by GC-FID, using an Agilent 7890B
system. Compounds were separated in the same column and nearly
identical analytical conditions as described in the MS qualitative anal­
ysis. The detector temperature was kept at 280 ◦ C, and the carrier gas
flow (hydrogen) was 1.5 mL/min. Marjoram essential oil compounds
quantification was determined by internal standardization. A solution of
methyl octanoate (~400 μg/mL) in dichloromethane was added to 1 μL
of the free EO or the EO recovered from the beads, and the compounds'
areas were corrected by applying predicted response factors, as in a
previous study by Bizzo et al. [29], using Cachet et al. methodology
[30]. Results were expressed as mg/g of essential oil. Samples were
injected in triplicate.
2.5.2. Antimicrobial activity
The antimicrobial activity of the free essential oil was evaluated by
the well diffusion method described by Harris et al. [31].
The degree of sensitivity or resistance against Listeria monocytogenes
was tested by measuring the inhibition halo (mm). Briefly, 6 mm wells
were drilled in plates containing TSA medium (trypticase soy agar) and a
culture of Listeria monocytogenes at 102 and 104 CFU/mL. The wells were
filled with 100 μL of the samples. After incubating the plates at 37 ◦ C for
24 h, inhibition halos were measured. The analysis was carried out in
duplicate and ethyl alcohol (95 % analytical grade) was considered the
negative control.
2.5.3. Encapsulation efficiency of the beads
The content of marjoram essential oil was determined by extracting
the essential oil from the beads as previously described (item 2.5.1). The
volume of essential oil collected was multiplied by a density factor of

3
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Fig. 1. Partial GC-FID chromatograms from representative samples of free (A) and encapsulated (B) marjoram essential oil recovered from the beads formulated with
1.25 g/100 mL alginate, 1.25 g/100 mL WPI, and 0.175 mol/L CaCl2. (1) sabinene, (2) α-terpinene, (3) γ-terpinene, (4) trans-sabinene-hydrate, and (5) terpinen-4-ol.
GC column: HP-5MS, 30 m.

0.893 g/mL to calculate the mass of recovered essential oil. where: MEO = marjoram essential oil.
Encapsulation efficiency was calculated according to Eq. (4) [22]:
EE = (Total amount of loaded MEO)/(MEO content in the emulsion) × 100 2.5.4. Morphology of the beads
(4) The internal and external morphology of the beads were evaluated

4
K.E.L. Mazza et al.
Fig. 2. Viscosity of emulsions prepared with different alginate and WPI concentrations, as a function of shear rate (○ 0.8 g/100 mL alginate/0.8 g/100 mL WPI (1), ●
0.8 g/100 mL alginate/1.7 g/100 mL WPI (2), □ 1.7 g/100 mL alginate/0.8 g/100 mL WPI (3), ■ 1.7 g/100 mL alginate/1.7 g/100 mL WPI (4), Δ 1.25 g/100 mL
alginate/1.25 g/100 mL WPI (5), ▴ 1.25 g/100 mL alginate/1.25 g/100 mL WPI (6), ◆ 1.25 g/100 mL alginate/1.25 g/100 mL WPI (7)).
by scanning electron microscopy. To reveal the inner bead, the freeze-
dried samples were cut with a craft knife. Samples were freeze-dried
and coated with a thin layer of gold, using a gold wire. Then, they
were observed in a scanning electron microscope (Tescan Vega 3, Tes­
can®, Kohoutovice, Czech Republic), operating at 15 kV.
2.5.5. Size and circularity of the beads
Beads' size (area, in mm2 and diameter, in mm) and shape (circu­
larity) were determined using a digital image processor, ImageJ [32].
Feret's diameter was applied to evaluate the beads sizes. This parameter
measures the longest distance between any two points along the selec­
tion boundary. The surface area is a result of a projection around the
bead, using the average diameter [24]. The circularity of the wet beads
was obtained by Eq. (5) [33]. A digital camera was used to capture
images of the wet beads. Results of each sample were obtained after
analyzing 100 beads.
( / ) The bactericidal effect of marjoram essential oil has already been
Circularity = 4πr area perimeter2 (5)
2.6. Statistical analysis
Statistical comparisons were based on at least triplicate results, and
all data were presented as mean and standard deviation. Data were
tested for normality and homogeneity.
For the rotational central composite design, the analysis of variance
(ANOVA), test for the lack of fit, determination of regression co­
efficients, and the generation of three-dimensional graphs were per­
formed with Statistica 7.0 (StatSoft, Tulsa, US) software.
Correlation analysis between surface area and Feret's diameter was
run with GraphPad Prism v.6.0 (GraphPad software 2012, Inc., La Jolla,
CA, US). Significance was established at p < 0.05. t-test was used to
make comparisons between composition results at p < 0.05 (Statistica
7.0 software).
3. Results and discussion
3.1. Characterization of marjoram essential oil
3.1.1. Chemical profile
Thirty (30) compounds were identified in the free marjoram essential
oil (Table 1). According to GC-FID data (Fig. 1), oxygenated mono­
terpenes constituted the major fraction of marjoram essential oil (51.1
5
International Journal of Biological Macromolecules 233 (2023) 123478
%), followed by hydrocarbon monoterpenes (43.5 %). Marjoram
essential oil also presented low amounts of hydrocarbon sesquiterpenes
(2.9 %), oxygenated sesquiterpenes (0.2 %), and other compounds (2.4
%). The major constituents in free marjoram essential oil were terpinen-
4-ol, γ-terpinene, α-terpinene, sabinene, and trans-sabinene-hydrate,
being classified as an alcohol monoterpene chemotype, as reported by
the literature in marjoram essential oil from distinct origins
[5,6,9,11,34–36]. After encapsulated, marjoram essential oil recovered
from the beads was absent in one of its main compounds, trans-sabinene-
hydrate.
3.1.2. Antimicrobial activity
The free marjoram essential oil was able to inhibit the microor­
ganism Listeria monocytogenes. The inhibition halo at the concentration
of 104 CFU/mL was 10 mm and at 102 CFU/mL, the inhibition halo was
12 mm.
described by other authors against several microorganisms [7,8], and its
inhibitory capacity can be different according to the chemical compo­
sition of the essential oil. Although the major compound, terpinene-4-ol,
is known to have antimicrobial activity, the effect of an essential oil is
not attributed to a single component, but to the mixture of active
components, that may provide synergistic or additive interactions [37].
Therefore, the present qualitative result indicates the possible
application of marjoram essential oil in products susceptible to the
presence of Listeria monocytogenes.
3.2. Characterization of the emulsions
3.2.1. Emulsion viscosity
Emulsions' rheological behavior was evaluated by steady-shear flow
curves. Fig. 2 shows the viscosity of the emulsions produced with
different alginate and WPI concentrations, as a function of shear rate.
The emulsions prepared with lower alginate concentration (0.8 g/
100 mL) showed Newtonian behavior, i.e., had no variation in viscosity
at different shear rates. On the other side, Herschel-Bulkley mathemat­
ical model described better the flow characteristics of the emulsions
prepared with higher alginate concentrations (1.25 and 1.7 g/100 mL),
as shear rate increase promoted viscosity reduction. Thus, these emul­
sions could be characterized as non-Newtonian fluids with shear-
thinning behavior.
As expected, alginate concentration was the most important variable
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Table 2 3.2.2. Emulsion droplet size

Droplet size and polydispersity of the emulsions produced with different alginate Emulsions' mean diameter and polydispersity index (Span) are shown
and WPI concentrations. in Table 2. In this case, only the central and some factorial points were
Tests Alginate (g/100 mL) WPI (g/100 mL) D [2,3] (μm) Span considered, since the concentration of the CaCl2 solution was not
1 0.8 (− 1) 0.8 (− 1) 1.73 1.365
considered a variable, as it is not related to the emulsion formulation.
2 0.8 (− 1) 1.7 (+1) 1.44 1.319 Alginate concentration was the factor that most affected the droplet
3 1.7 (+1) 0.8 (− 1) 1.25 1.247 size (Fig. 3a). The increase in this polymer concentration led to a
4 1.7 (+1) 1.7 (+1) 1.10 1.18 decrease in the droplets' size, that may be attributed to the increase in
5a 1.25 (0) 1.25 (0) 1.32 1.056
the emulsion viscosity, which difficult the droplets' movement, avoiding
5a 1.25 (0) 1.25 (0) 1.29 1.127
5a 1.25 (0) 1.25 (0) 1.29 1.116 coalescence and flocculation. Similar behavior was observed by Chan
[26], that also reported a decrease in the size of droplets with an in­
WPI: Whey protein isolate; D [2,3]: Sauter Diameter; Span: particle size scat­
crease in alginate concentration. In addition, WPI has emulsifying
tering index.
a properties and is able to reduce surface tension, resulting in the for­
Central point.
mation of smaller droplets when used at higher concentrations [39]. The
droplet size is directly related to the emulsions' stability, as small
affecting emulsions viscosity, which could be attributed to its well-
droplets indicate a low occurrence of droplet agglomeration, suggesting
known thickening effect [19]. This is important information concern­
greater kinetic stability [40]. As previously mentioned, droplet size
ing emulsions stability and droplet size since higher emulsion viscosity
strongly influences the encapsulation efficiency and, thus, is an impor­
could reduce destabilizing phenomenon by increasing the resistance to
tant property to be controlled in the production of essential oil beads.
coalescence. Emulsions stability and droplet size, in turn, directly affects
The droplet mean diameter of the analyzed emulsions ranged from
the encapsulation efficiency and, consequently, the applicability of the
1.10 to 1.73 μm (Table 2). These sizes are smaller than those reported by
resulting beads. WPI concentration had a very slight influence on
Benavides et al. [12] (2.28–3.77 μm) for thyme essential oil emulsified
emulsions viscosity. WPI gel formation property, which is Ca2+ induced
with sodium alginate, using the same type of homogenizer at different
and heat dependent, was not our aim [38].
speeds. The smaller sizes are probably due to the use of WPI as an

Fig. 3. Effect of alginate and WPI concentrations on the (a) mean droplet size and (b) polydispersity of emulsions.

Table 3
Encapsulation efficiency (EE), circularity, area, and Feret diameter of the beads, for the 17 trials of the experimental design.
Tests Alginate (g/100 mL) WPI (g/100 mL) CaCl2 (mol/L) EE Circularity Area (mm2) Diameter (mm)
(%)

1 0.8 (− 1) 0.8 (− 1) 0.1 (− 1) 60.9 0.885 2.87 2.03

2 0.8 (− 1) 0.8 (− 1) 0.25 (+1) 66.0 0.876 2.76 2.01
3 0.8 (− 1) 1.7 (+1) 0.1 (− 1) 51.2 0.906 2.18 1.79
4 0.8 (− 1) 1.7 (+1) 0.25 (+1) 55.8 0.887 2.29 1.83
5 1.7 (+1) 0.8 (− 1) 0.1 (− 1) 50.7 0.900 3.99 2.45
6 1.7 (+1) 0.8 (− 1) 0.25 (+1) 50.9 0.907 4.20 2.46
7 1.7 (+1) 1.7 (+1) 0.1 (− 1) 45.7 0.910 3.97 2.39
8 1.7 (+1) 1.7 (+1) 0.25 (+1) 45.7 0.880 3.66 2.31
9 0.5 (− 1.68) 1.25 (0) 0.175 (0) 60.0 0.743 1.89 2.09
10 2.0 (+1.68) 1.25 (0) 0.175 (0) 50.7 0.815 4.00 2.44
11 1.25 (0) 0.5 (− 1.68) 0.175 (0) 60.8 0.905 3.35 2.21
12 1.25 (0) 2.0 (+1.68) 0.175 (0) 51.1 0.902 3.67 2.32
13 1.25 (0) 1.25 (0) 0.05 (− 1.68) 45.6 0.882 3.80 2.35
14 1.25 (0) 1.25 (0) 0.3 (+1.68) 50.8 0.904 4.14 2.46
15a 1.25 (0) 1.25 (0) 0.175 (0) 50.6 0.862 3.89 2.50
15a 1.25 (0) 1.25 (0) 0.175 (0) 51.4 0.893 3.95 2.48
15a 1.25 (0) 1.25 (0) 0.175 (0) 56.5 0.894 3.93 2.44

WPI: Whey protein isolate.

a
Central point.

6
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Table 4 authors also verified that “curing” time above 20 min led to a decrease in
Coded second-order regression coefficients for encapsulation efficiency (EE), the amount of oil in the beads.
circularity, area, and Feret diameter of beads produced by ionic gelation. Lower concentrations of alginate and WPI resulted in higher encap­
Coefficient EE Circularity Area Diameter sulation efficiency (Fig. 3(e)) and were considered more appropriate for
β0 54.416** 0.880** 3.937** 2.482**
producing marjoram essential oil beads. However, other factors such as
β1 − 4.140** 0.012 0.679** 0.1860** the beads' size and shape should also be considered when establishing
β11 0.385 − 0.029** − 0.392** − 0.105** the most suitable polymer concentration.
β2 − 3.398** 0.0007 − 0.086** − 0.032*
0.597 0.015 − 0.192** 0.105**
β22 −
3.3.2. Beads' morphology by SEM
β3 1.365 − 0.001 0.034* 0.009
β33 − 2.143 0.012 − 0.029 − 0.056** The SEM images obtained from freeze-dried beads containing mar­
β12 1.212 − 0.006 0.075** 0.026 joram essential oil, prepared with different alginate concentrations are
β13 − 1.187 0.0006 − 0.012 − 0.011 shown in Fig. 4.
β23 − 0.087 − 0.006 − 0.037 − 0.004 All the analyzed beads exhibited rough external surfaces with small
Fcalculated 11.43 2.58 10.99 2.96
Ftabulated 3.68 3.68 3.68 3.68
pores (Fig. 4 (a), (c), and (e)).
R2 0.93631 0.76818 0.93671 0.80186 Beads' internal morphology was characterized by a porous structure,
which may indicate the presence of microchannels inside them (Fig. 4
β0: mean; β1: Sodium alginate linear; β11: Sodium alginate quadratic; β2: WPI
(b), (d), and (f)). The morphology of the beads obtained in this work
linear; β22: WPI quadratic; β12: Sodium alginate × WPI; β3: CaCl2 linear; β33:
CaCl2 quadratic; β13: Sodium alginate × CaCl2; β23: WPI × CaCl2.
agrees with previous works, which revealed that alginate gels have a
*
Significant at p < 0.06. characteristic porosity, which can allow the diffusion of small molecules
**
Significant at p < 0.05. into or out of them [10,14,16], thus affecting the encapsulation effi­
ciency. Soliman et al. [23] described the interior of the bead as a three-
emulsifier, which reduces the interfacial tension and makes the dimensional porous sponge, trapping the essential oil in the alginate's
disruption of emulsion droplets during homogenization easier [39]. egg box structure.
All the emulsions showed a polydisperse distribution since Span
values were higher than 0.4 (Table 2) [41]. According to Fig. 3b, both 3.3.3. Beads' size and circularity
WPI and alginate concentration significantly influenced this parameter All the studied variables (sodium alginate, WPI, and CaCl2) influ­
(p < 0.05). enced the beads' area and diameter (Table 4). Sodium alginate was the
variable that most affected these parameters (Fig. 3 (b) and (c)). The
increase in alginate concentration led to larger diameters, which is
3.3. Encapsulation of marjoram essential oil related to the increase in emulsion viscosity.
As described by Lee et al. [19], Feret's diameter is the most used
The mean values of encapsulation efficiency, surface area, Ferret parameter to measure alginate beads. However, when dealing with
diameter, and circularity of the beads obtained by ionic gelation are beads of low circularity or with deformations, this tool may be not very
shown in Table 3. efficient. Therefore, the beads' surface area determination was a more
Table 4 shows the regression coefficients for the coded second-order suitable option. Nonetheless, both data can be used as a size parameter.
polynomial equation, the F calculated values, and the determination The size and shape of the beads may vary depending on the viscosity
coefficients (R2). The model obtained for encapsulation efficiency was of the alginate solution, the concentration and the structure of the
suitable, showing no lack of fit and satisfactory determination co­ polymers, the diameter of the dripping needle or nozzle used in the
efficients. Thus, its surface response was presented. emulsion dispersion, and the distance between the tip of the needle and
The effect of the variables on the studied responses are shown in the gelling bath. The last one, responsible for promoting a rearrange­
Fig. 4. ment of the shape of the liquid drop. When increasing the height (>100
cm), the impact force of the liquid drop in the gelation bath can cause
3.3.1. Encapsulation efficiency deformation in the beads, such as forming pear-shaped beads. On the
Encapsulation efficiency is related to the amount of essential oil other hand, shorter distance can form tear- or pear-shaped beads (<10
encapsulated in alginate beads. It is an important property, for example, cm) [42].
to determine the necessary amount or concentration of additives In addition, the emulsion output flow affects the uniformity of the
necessary upon the application in a food product. Encapsulation effi­ beads, since manual extrusion-dripping generates beads with low uni­
ciency varied from 45.6 to 66 % (Table 4), values close to those observed formity while dripping with a pump commonly produces more uniform
by Hosseini et al. [10] in the microencapsulation of essential oil of beads [16,43].
Satureja hortensis by ionic gelation. The beads produced in the present work showed a mean diameter
According to Fig. 3(a) and (e) and Table 4, WPI and especially so­ ranging from 1.79 to 2.5 mm (Table 3), using a 0.70 mm diameter
dium alginate concentrations negatively affected the encapsulation ef­ needle. Similar results were observed by Chan [26] and Chan et al. [44].
ficiency, i.e., the increase in these variables led to a decrease in the The measurement of beads' size is important to have an appropriate fit
encapsulation efficiency. As previously discussed, higher sodium algi­ when applying the additive in a product, aiming for a good essential oil
nate and WPI concentrations led to smaller droplet sizes. Considering delivery while attending to consumers' expectations.
that the alginate beads obtained by ionic gelation are generally very Most of the beads had good circularity, with values close to 1
porous [14], the smaller droplets may have diffused more easily (Table 3). The closer to 1, the more perfect is the circle, and the closer to
throughout the pores, explaining the lower encapsulation efficiency 0, it can be considered an elongated polygon. Although this tool only
values. This porosity was observed in the marjoram essential oil beads measures the two-dimensional image, it was used to estimate the beads'
produced in the present work, as described in the morphological anal­ shape. In this way, results suggested that the beads had a round shape,
ysis (Fig. 4). except for the beads produced in the lower concentrations of alginate.
According to Soliman et al. [23], higher alginate concentration leads It is well known that the alginate concentration influences the
to the formation of a denser three-dimensional network, which can emulsion viscosity, as stated in item 3.3.1, and can alter fluidity during
decrease the encapsulation agents' ability to encompass large amounts of dripping, affecting the shape of the beads. Thus, a lower viscosity so­
oil, consequently, decreasing encapsulation efficiency. In their study on lution can generate beads deformation when the drop viscosity and/or
the encapsulation of thyme, clove, and cinnamon essential oils, the surface tension are not sufficient to overcome gelling solution surface

7
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Fig. 4. Pareto Chart for encapsulation efficiency (a), Feret diameter (b), area (c), circularity (d) and Response Surface of encapsulation efficiency for CaCl2 con­
centration of 0.175 mol/L (e). L – linear terms; Q – quadratic terms.

tension [12,42]. On the other hand, if the surface tension is too high, the
impact force of the emulsion in the interface of the air-gelation bath may
cause deformations in the beads [19]. Therefore, it is imperative for the
process success to determine the best concentrations of the encapsula­
tion agents.
According to Chan et al. [44], the beads produced by external ionic
gelation are usually spherical, regardless of their size. The sphericity of
alginate beads affects their mechanical and chemical stability and may
promote reduced strength in less spherical beads, leading to undesirable
rupture, for example, and could also affect controlled-release rates. The
sphericity also improves the aesthetics of the beads, a desirable char­
acteristic for food and pharmaceutical ingredients [19]. In addition,
more circular beads usually have better flow, facilitating transportation
and storage.

8
K.E.L. Mazza et al. International Journal of Biological Macromolecules 233 (2023) 123478

Fig. 5. SEM of freeze-dried beads prepared with 1.25 g/100 mL WPI, 0.175 mol/L CaCl2 and different alginate concentrations: (a) and (b) 0.5 g/100 mL alginate; (c)
and (d) 1.25 g/100 mL alginate; (e) and (f) 2 g/100 mL alginate. Being (a), (c) and (e) the surface bead, and (b), (d) and (f) the inner bead; (a) magnification 2000×,
(c) and (e) magnification 3000×, and (b), (d) and (f) magnification 5000×. Arrows indicate some pores.

9
K.E.L. Mazza et al.
3.3.4. Chemical composition of the encapsulated essential oil
The marjoram essential oil recovered from the alginate beads
showed a similar chemical profile to the free essential oil (Table 1,
Fig. 5). However the beads had a limited capacity to retain the essential
oil, as also observed by Benavides et al. [12], leading to a 21 % reduction
in the total content of compounds.
Terpinen-4-ol, the major compound, was the most affected by
encapsulation, a significant decrease from 403.11 mg/g, in the free
essential oil to 262.52 mg/g (p < 0.05), in the recovered essential oil,
representing 35 % of the loss. The influence of the encapsulation process
on marjoram essential oil chemical compounds content may influence
some characteristics of the beads. Marjoram essential oil is a complex
substance composed of a broad spectrum of compounds with distinct
biological, technological, and sensory properties. Any alteration caused
by the production process on its chemical composition may affect
product properties, such as antimicrobial and antioxidant activity, and
organoleptic parameters. Thus, investigating the chemical composition
of the final product is important to screening its possible applications.
4. Conclusion
The essential oil of marjoram, rich in terpinen-4-ol, presented anti­
microbial activity against Listeria monocytogenes.
The emulsions prepared with higher WPI concentration presented
smaller droplet sizes probably due to the good emulsifying properties of
WPI, promoting emulsion droplets disruption during homogenization.
Sodium alginate and WPI concentrations influenced the encapsula­
tion efficiency, size, and shape of the beads in distinct ways. Lower
alginate and WPI concentrations resulted in higher encapsulation effi­
ciency and lower circularity. To produce highly appealing beads for the
cosmetic and food industry, both technological properties, such as high
encapsulation efficiency, and physical characteristics, such as good
circularity, are desirable. Therefore, the results suggest that an inter­
mediate concentration of the factors (1.25 g/100 mL sodium alginate,
1.25 g/100 mL WPI, and 0.175 mol/L CaCl2) would be more appropriate
to produce marjoram essential oil beads which meet consumers and
quality parameters.
The present results led us to conclude that this study was a well-
succeed initial attempt to use biocompatible and sustainable in­
gredients to develop a natural additive with flavoring or preservative
potential, by using a relatively simple, low-cost and low-energy
demanding method. However, a limitation of this method is the
scaling-up, since the ionic gelation process is still not trivial in the food
industry.
CRediT authorship contribution statement
Karen Mazza: Conceptualization, Investigation, Data Curation,
Formal analysis, Visualization, Writing - Original Draft. André Costa:
Formal analysis, Writing - Reviewing & Editing. Janine Silva: Re­
sources, Writing - Reviewing & Editing. Daniela Alviano: Resources,
Data Curation. Humberto Bizzo: Supervision, Resources, Writing-
Reviewing and Editing. Renata Tonon: Supervision, Conceptualization,
Project administration, Resources, Writing- Reviewing and Editing,
Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by CAPES [Finance code 001, grant number
88882.425027/2019-01], FAPERJ [grant numbers E-26/200.399/2020,
10
International Journal of Biological Macromolecules 233 (2023) 123478
E-26/202.325/2019, E-26/202.326/2019 and E-26/202.710/2019]
and CNPq [grant numbers 408330/2016-3, 310659/2018-3, and
310248/2018-3].
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