Heliyon 7 (2021) e06737Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyonResearch articleCharacterization and antimicrobial activity of microencapsulated citral with
dextrin by spray drying
Ives Yoplac a,b,*, Luis Vargas c, Paz Robert d, Alyssa Hidalgo e
a Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Chachapoyas 01001, Peru
b Escuela de Posgrado, Programa Doctoral en Ciencia de Alimentos, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru
c Facultad de Industrias Alimentarias, Universidad Nacional Agraria La Molina, Av. La Molina s/n, Lima 12, Peru
d Dpto. Ciencia de los Alimentos y Tecnología Química, Facultad de Ciencias Químicas y Farmac
euticas, Universidad de Chile, Casilla 133, Santiago, Chile
e Department of Food, Environmental and Nutritional Sciences (DEFENS), University of Milan, via Celoria 2, 20133, Milan, ItalyA R T I C L E I N F O
Keywords:
Encapsulation
Gram-negative
Gram-positive
Morphology
Spray drying* Corresponding author.
E-mail address: ives.yoplac@untrm.edu.pe (I. Yo
https://doi.org/10.1016/j.heliyon.2021.e06737
Received 3 December 2020; Received in revised fo
2405-8440/© 2021 Published by Elsevier Ltd. ThisA B S T R A C T
Aim of this work was to evaluate the antimicrobial activity and physical characteristics of citral micro-
encapsulated with dextrin (Dx) by spray drying. The encapsulation was optimized using response surface
methodology (RSM), maximizing yield and efficiency, considering as independent variables the citral:Dx ratio
(1:5 and 1:20) and the inlet air temperature (120 and 200 
C). Yield and efficiency under optimal conditions were
71.9% and 99.9%, respectively. Antimicrobial activity against Escherichia coli, Salmonella enterica, Staphylococcus
aureus and Bacillus cereus of the citral microparticles obtained under optimal conditions and of free citral was
evaluated using the disk diffusion methodology. Both compounds showed a broad spectrum inhibitory effect,
being Escherichia coli and Bacillus cereus the most sensitive microorganisms. The inhibition ratio varied between
55 and 75%, and the antibacterial activity was maintained after microencapsulation. The minimum inhibitory
concentrations of free citral were above 0.8 mg/mL. The optimal citral microparticles showed acceptable phys-
icochemical characteristics and broad-spectrum antimicrobial activity. Polymer and emulsifier used in microen-
capsulation protected the functional activity of citral, thus suggesting that these microparticles could be used in
the design of antimicrobial food systems to extend the shelf life of perishable foods.1. Introduction
Citral (3,7-dimethyl-2,6-octadienal) is a natural acyclic monoterpene
aldehyde, composed by two geometric isomers: geranial (trans-citral,
citral A) and neral (cis-citral, citral B), with antifungal, bactericide,
insecticide, deodorant and expectorant activities (Maswal and Dar,
2014). Citral acts predominantly on microorganisms cell membranes by
disrupting structures, causing loss of integrity and altering functions
(Miron et al., 2014). Essential oils containing citral (e.g. lemongrass,
lemon myrtle, lemon verbena, Litsea cubeba, lemon tea tree and melissa)
have shown antimicrobial activity (Hu et al., 2019; Naik et al., 2010;
Nirmal et al., 2018; Sultanbawa et al., 2009). Its isomers showed anti-
microbial activity on Gram-positive, Gram-negative and post-harvest
pathogens (Fancello et al., 2016; Lu et al., 2017; Saddiq and Khayyat,
2010); antimicrobial activity of encapsulated essential oils of lemongrass
and lemon myrtle has been studied on Gram-positive (Enterococcus fae-
calis, Listeria monocytogenes and Staphylococcus aureus) andplac).
rm 8 March 2021; Accepted 1 Ap
is an open access article under tGram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella
enteritidis and Salmonella typhimurium) (Cui et al., 2016; Huynh et al.,
2008; Leimann et al., 2009; Liakos et al., 2016).
Due to its antimicrobial activity, in the food industry the interest on
citral is increasing. However, its use is limited because of chemical
instability, high hydrophobicity, volatility and susceptibility to oxidative
degradation, leading to function alterations. Therefore, microencapsu-
lation technology may help to overcome these limitations (Bakry et al.,
2016; Gharsallaoui et al., 2007; Maswal and Dar, 2014) by protecting the
active compounds from environment (light, oxygen, water, heat), and by
controlling the release (Sutaphanit and Chitprasert, 2014). Encapsulation
of citral and/or essential oils containing citral has been performed using
different methods: by solvent/antisolvent method (Liakos et al., 2016),
liposome-entrapped (Cui et al., 2016) and simple coacervation (Leimann
et al., 2009); by oil-in-water nanoemulsion (Nirmal et al., 2018) and
spray drying (Huynh et al., 2008; Sosa et al., 2014). Spray drying (SD),
the most common and cheap encapsulation method (Balasubramaniril 2021
he CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
I. Yoplac et al. Heliyon 7 (2021) e06737et al., 2013), is a method of immobilization rather than a true method of
encapsulation. Of course, process and formulation optimizations are
required to achieve microparticles with minimal citral on the surface,
maximal encapsulation efficiency and maximal retention within the mi-
croparticles (Huynh et al., 2008).
For SD microencapsulation, the encapsulating agent plays an impor-
tant role on the retention of the volatiles during the process and the shelf-
life of the encapsulated powder (Jafari et al., 2008). Biopolymers used for
citral encapsulation by SD are modified starch with either sucrose or
trehalose (Sosa et al., 2014) and modified starch þ maltodextrin and
whey protein concentrate þ maltodextrin (Huynh et al., 2008). Carbo-
hydrates have been considered good encapsulating agents because they
exhibit low viscosities at high solids concentration as well as good sol-
ubility, leading to high amounts of encapsulated oils (de
Barros-Fernandes et al., 2014). Dextrin is a modified starch obtained by
partial hydrolysis of starch, commonly used in food applications, and a
well-known encapsulating agent for vegetable and marine oils (Bakry
et al., 2016). However, to the best of our knowledge, citral microen-
capsulation with dextrin by SD has not been evaluated. The aims of this
research were to optimize the spray drying encapsulation process of citral
with dextrin by applying response surface methodology (RSM), to
determine the antimicrobial activity of the optimized microencapsulated
citral against Escherichia coli, Salmonella enterica, Staphylococcus aureus
and Bacillus cereus and to evaluate the physical characteristics of the
microparticles obtained under optimal conditions.
2. Materials and methods
2.1. Materials
Soy lecithin (Epikuron 145V) was supplied by Blumos Ltda. (San-
tiago, Chile), dextrin (Amisol® 4810) was donated by Ingredion (Perú),
citral (cis and transmixture96%) and citral analytical standard (99.9%)
were from Sigma-Aldrich (Germany), n-heptane (99%) was from Merck
(Germany). Culture media used for the antimicrobial trials were nutrient
broth (NB), plate count agar (PCA), nutrient agar (NA) and peptone
bacteriological (Merck, Germany). Escherichia coli (E. coli) (reference
number: ATCC® 25922™), Salmonella enterica serovar Typhimurium
(S. enterica) (reference number: ATCC® 14026™), Staphylococcus aureus
(S. aureus) (reference number: ATCC® 25923™) and Bacillus cereus
(B. cereus) (reference number: ATCC® 14579 PK/5) were from Uni-
versidad Nacional Agraria la Molina (UNALM), Total Quality Laboratory
(Lima, Perú).Table 1. Experimental design conditions for the production of citral (Ct) and dextrin (D
drying yield and encapsulation efficiency.
Standard order Independent variables
X1: Temperaturea (
C) X2: Ct:D
1 120 (-1) 5 (-1)
2 200 (þ1) 5 (-1)
3 120 (-1) 20 (þ1
4 200 (þ1) 20 (þ1
5 120 (-1) 12.5 (0
6 200 (þ1) 12.5 (0
7 160 (0) 5 (-1)
8 160 (0) 20 (þ1
9 160 (0) 12.5 (0
10 160 (0) 12.5 (0
11 160 (0) 12.5 (0
12 160 (0) 12.5 (0
a Inlet air temperature.
2
2.2. Preparation of citral encapsulates
Encapsulation of citral (Ct) was performed by SD using dextrin (Dx) as
encapsulating agent and soy lecithin (SL) as emulsifier. A face-center
design (FCD) was applied with 12 runs (4 factorial points, 4 axial
points and 4 central points). The independent variables were Ct:Dx
(1:5–1:20 ratio) and inlet air temperature (IAT,120 200 

– C; Table 1). The
yield of SD process (Y) and encapsulation efficiency of citral (EE) were
the dependent variables. All experiments were performed randomly to
avoid systematic bias.
Infeed solution (100 g) was prepared as follows: lecithin (1 g) was
dispersed in distilled water (30 g) at 50 
C using magnetic stirrer (350
rpm for 15 min). After cooling to 30 
C, citral (1 g) was added and mixed
at 1500 rpm for 3 min using a Polytron homogenizer (PT-2100, Kine-
matica A.G, Switzerland). Dextrin (5, 12.5 or 20 g) was dispersed in
distilled water (63, 55.5 or 48 g) at 50 
C and stirred for 3 h to obtain 1:5,
1:12.5 or 1:20 Ct:Dx ratios, respectively (Table 1). Citral oil-in-water
emulsion was added to the Dx solution and mixed at 1500 rpm for 3
min. The resultant mixture was fed into the spray dryer (B-290, Büchi,
Switzerland). The spray dryer was operated at infeed temperature of 42
 2.5 
C and inlet air temperature of 120, 160 or 200 1 
C. The air flow,
feeding rate and atomization pressure were 600 L/h, 5 mL/min and 0.14
MPa, respectively. Citral microparticles powders were stored at -20 
C in
the dark until analysis.
2.3. Analysis
The encapsulation yield (Y) was determined according to the equa-
tion Yield ð%Þ ¼ WM ðgÞ
W ðgÞ 100, whereWM is the microparticle weight after
SST
SD, and WSST represents the total soluble solids in infeed solution
(Sutaphanit and Chitprasert, 2014).
The EE was computed as EE ð%Þ ¼ 100 CS ð%Þ, where CS is the
citral surface calculated as CS ð%Þ ¼ surface citral
theoretical total citral x 100. Total citral
was determined by gas chromatography as follows: microparticles (0.2 g)
were dispersed in hexane (2 mL), stirred for 1 min and centrifuged at
112,000 g for 1 min. The supernatant was transferred to an amber vial (2
mL) and injected into a 7890A gas chromatograph (Agilent Technologies,
USA) fitted with a DB-5MS fused-silica capillary column (30 m  0.25
mm i.d. x 0.25 μm film thickness, J&W Scientific, USA) and a flame
ionization detector following the method proposed by Ruktanonchai
et al. (2011). The chromatogram showed two peaks corresponding to the
citral isomers, geranial and neral. Citral was quantified using ax) emulsion microparticles by spray drying and results (mean standard error) of
Response variables
x ratio Y1: Yield (%) Y2: Efficiency (%)
42.65  0.65 99.63  0.03
65.53  1.56 99.87  0.00
) 49.66  1.23 97.97  0.02
) 55.96  1.34 97.07  0.01
) 35.41  1.77 99.37  0.00
) 50.43  0.75 99.25  0.14
70.07  0.79 99.75  0.01
) 59.30  0.86 97.70  0.25
) 58.30  1.18 99.22  0.04
) 59.16  1.20 99.19  0.03
) 61.47  1.19 99.14  0.01
) 57.64  1.16 99.12  0.05
I. Yoplac et al. Heliyon 7 (2021) e06737calibration curve (1–2000 μg/mL citral, R2 ¼ 0.999). Results are
expressed as mg citral/g powder.2.4. Characterization of the citral microparticles obtained under optimal
conditions
Moisture content was analysed according to method n
 925.10
(AOAC, 1997). Water activity (aw) was determined by the dew point
method using a HigroLab (Rotronic, USA) at 20  0.3 
C and hygro-
scopicity (Hg) was evaluated according to the procedure described by Cai
and Corke (2000).
The outer structure of the citral microparticles was examined using
scanning electron microscopy (SEM). The citral microparticles were
coated with gold/palladium, using a PS 10E vacuum evaporator and
analysed using a LEO 1420VP SEM (LEO Electron Microscopy Ltd.,
Cambridge, UK) operated at 20 kV. The scanned images were collected
digitally using the EDS 7424 software (Oxford Instruments, Oxford, UK).
Particle size and size distribution were determined by light scattering
using a laser diffraction particle size analyser (Mastersizer X, Malvern
Instruments, Worcestershire, UK). Citral microparticles were dispersed in
water and the results were expressed as volume average (D4,3).2.5. Microbiological assays
2.5.1. Preparation of microorganism suspensions
Bacterial strains used to evaluate inhibitory activity were E. coli,
S. enterica, S. aureus and B. cereus, and were prepared following the
methodology proposed by Wang et al. (2009), with minimal modifica-
tions. Each strain stored in tubes in inclined position at 4 
C was trans-
ferred to Erlenmeyer flask containing NB (100 mL) and incubated at 37

C for 18 h with constant agitation. For the determination of the colony
forming units (CFU)/mL, serial dilutions up to 106 were prepared in
0.1% peptone water. From the last three dilutions, 0.1 mL was trans-
ferred to plates with PCA by surface seeding and incubated at 37 
C for 18
h in an oven (Binder, BD 53, Germany).
The 101 dilution, which had an average cell concentration of 107
CFU/mL, was used for the assessment of antimicrobial activity.
2.5.2. In-vitro antibacterial activity assay of citral microparticles
Antibacterial inhibitory activity was determined by disk diffusion
method according to Wang et al. (2009), with some modifications. Filter
paper discs (Whatman 2, diameter 6 mm) containing 5 μL microparticle
solution (300 mg microparticles in 1 mL diluted heptane plus 1 mL
distilled water) and 5 μL of pure citral mixture solution as a control, with
equivalent concentrations (41 mg of citral in 1 mL of heptane diluted in 1
mL of distilled water), were applied on the plate surface with NA pre-
viously seeded with 0.1 mL test bacterial strains. The plates were incu-
bated at 37  1 
C for 18 h. The experiment was performed in
quadruplicate. The diameter of the transparent inhibition zone against
test microorganism was measured using a manual Vernier (Kern, Ger-
many), and the inhibition ratio was calculated, comparing the inhibition
diameter of the citral microparticles respect to citral (control), as
Inhibition ratio ð%Þ ¼ M
C  100; where M is the inhibition zone diameter
of test citral microparticles and c is the inhibition zone diameter of the
pure citral mixture (control), in mm (Wang et al., 2009).
To verify that the effect of the antimicrobial agents came from citral
and not from heptane (the solvent), an aliquot of pure heptane was used
as a control.
2.5.3. In-vitro antibacterial activity assay of pure citral
Serial dilutions of citral in heptane (0, 0.08, 0.2, 0.4, 0.8, 2.0, 4.0, 8.0,
40.0 and 80.0 mg/mL) were prepared according to Guarda et al. (2011)
and Leimann et al. (2009). The disk diffusion method was used following
the procedure described above. Sterilized filter paper discs were placed3
on the surface of plates with NA previously seeded with the assay bac-
terial strains and 5 μL of serial citral dilutions were added.2.6. Statistical analysis
The RSM experimental design matrix, data analysis, model develop-
ment and optimization were studied using the Design Expert software v.
12 (Stat-Ease Inc., Minneapolis, USA). The average and standard de-
viations of the responses were calculated with Microsoft Excel. One-way
analysis of variance (ANOVA) was performed on the data of the anti-
bacterial trials. When significant differences were found, least signifi-
cance test (LSD; p 0.05) was applied. ANOVA and LSD were performed
using the software Statgraphics 15.2 (StatPoint Inc., USA, 2015).
3. Results and discussion
3.1. Optimisation by response surface methodology
Table 1 shows the results of the face centered design applied to
evaluate the effect of the inlet air temperature and the formulation (Ct:Dx
ratio) on the encapsulation yield and encapsulation efficiency. The Y
ranged between 35.4% and 70.1% and the EE ranged from 97.1% to
99.9%, values comparable to those (Y: 60–98% and EE: higher than 85%)
reported by Sutaphanit and Chitprasert (2014) for basil EO microparti-
cles in gelatin.
The ANOVA (Table 2) evidenced that only the linear term of Ct:Dx
ratio for Y and the quadratic term of the IAT for EE were not significant.
The estimates of the regression coefficients of the second-order poly-
nomial models for the two dependent variables are reported in Table 2.
The yield corresponds to the ratio between the solids content in the feed
solution before spray drying and the solids content after spray drying.
The ANOVA showed that the IAT linear and quadratic terms were the
most significant, followed by the quadratic Ct:Dx ratio. The yield model
explained 90% of the variability (adjusted R2) and was represented by
the following equation:
Yield ¼ -192.42 þ 3.10 IAT - 1.66 Ct:Dx - 0.014 IAT * Ct:Dx - 0.009 IAT2 þ
0.143 Ct:Dx2
The RSM plot (Figure 1) showed that Y was higher for microparticles
with lower Ct:Dx ratio and intermediate IAT. The lower Ct:Dx ratio and
the higher yield of powdered microparticles could be attributed to better
pulverization, smaller size and weight of the droplet inside the dryer
when the solution is more diluted, favoring a better heat transfer and
preventing dust from rapidly precipitating into the drying chamber
(Robert et al., 2017; Vergara et al., 2014). In addition, the best yields
were obtained at intermediate IATs, probably because low temperatures
produce microparticles with higher humidity, helping the powder to
adhere to the drying chamber and thus increasing losses (Urzúa et al.,
2017). On the other hand, high temperatures could volatilize the water
more quickly and result in a sample adhering to the wall of the drying
chamber, reducing the yield (Cao et al., 2018). Similar results were ob-
tained while encapsulating jambul extract in maltodextrin by
spray-drying (Santhalakshmy et al., 2015). Higher yields were instead
obtained by Bhandari et al. (1992) during the microencapsulation at 400

C (IAT) of citral in maltodextrin and gum arabic, without adverse effects
on the EO chemical properties, probably due to the type of encapsulating
agent.
The EE of citral represents the retention percentage of the Ct-SL
emulsion. The ANOVA indicated that the linear and quadratic terms of
Ct:Dx ratio were the most significant on EE, followed by IAT and Ct:Dx
ration interaction. The model explained 99% of the variability (adjusted
R2). The quadratic regression was represented by the following equation:
EE ¼ þ98.50þ 0.0038 IAT þ0.2566 Ct:Dx - 0.0010 IAT * Ct:Dx þ 0.000015
IAT2 - 0.0099 Ct:Dx2
I. Yoplac et al. Heliyon 7 (2021) e06737
Table 2. Estimates of the regression coefficients of the second-order polynomial models and analysis of variance (mean square, MS and significance) for drying yield and
encapsulation efficiency.
Source df Yield Efficiency
Coefficient MS Coefficient MS
Intercept 58.31 99.21
Model 5 192.49*** 1.680***
A-Temperature 1 7.37 325.61*** -0.13 0.102*
B-Ct:Dx ratio 1 -2.22 29.61 -1.09 7.070***
AB 1 -4.14 68.72* -0.29 0.330**
A2 1 -13.73 502.61*** 0.02 0.002
B2 1 8.04 172.22** -0.56 0.831***
Residual 6 9.17 0.012
Lack of Fit 3 15.56 0.021
Pure Error 3 2.79 0.002
R2 0.95 0.99
Adj-R2 0.90 0.99
Pred-R2 0.57 0.94
C.V. % 5.46 0.11
Ct, citral; Dt, dextrin; df, degrees of freedom; Adj-R2, R2 adjusted by df; Pred-R2, R2 in prediction; MS, Mean square; *, p  0.05; **, p  0.01; ***, p  0.001.
Figure 1. Response surface plots for the production of citral (Ct) and dextrin (Dx) emulsion microparticles by spray drying: drying yield and encapsulation efficiency.The RSM plot (Figure 1) showed that the EE of citral mainly increased
with the decrease of the Ct:Dx ratio: the lower the Dx content, the higher
the retention of the citral emulsion in the microparticle, probably as a
consequence of smaller droplet size of the emulsion during spraying in
the more diluted solution, favoring the increase in EE. A possible
explanation for this phenomenon could be the atomization shear effect
on the larger emulsion droplets, coupled with the high speed gradient
and turbulence state at the atomizer "mouth", that could allow large
emulsion droplets to break up into smaller droplets, causing greater loss
of core material from larger emulsion droplets during spray drying
(García et al., 2013; Huynh et al., 2008). Similar results were reported for
garlic EO microparticles in maltodextrin (Balasubramani et al., 2013)
where the highest EE was obtained at 1:6 (EO:maltodextrin) ratios. The
significant IAT-Ct:Dx ratio interaction (Table 2) is evident in Figure 1 for
the different behaviour of EE as a function of IAT at high and low Ct:Dx
ratios. It is interesting to observe that at low Ct:Dx ratio levels, a slight EE
increase occurs when IAT augments; the high temperatures could lead to
a rapid formation of the crust on the drop, which allowed a greater
emulsion retention and water diffusion into hot air.
The lack of fit for yield and EE was not significant indicating that the
mathematical models fit the experimental data within the experimental
domain.4
A numerical optimization was performed to find out the best IAT and
Ct:Dx ratio for maximizing Y and EE of citral microparticles. A desir-
ability value of 0.996 was obtained for the optimal IAT of 187 
C and
Ct:Dx ratio of 1:5, leading to a Y of 70.1% and an EE of 99.9%. It is
important to note that the optimal conditions obtained for Ct-Dx mi-
croparticles according to the statistical design are specific for this system
and may not apply when other biopolymers are used as encapsulating
agents (Vergara et al., 2014). In this context, the biopolymers properties
play a fundamental role in the encapsulation parameters (mainly the IAT)
and the stability of the active compounds (Urzúa et al., 2017). The
optimal IAT found in the present study is within the temperature range
(160 and 200 
C) suggested by other authors studying the encapsulation
process of different matrices such as garlic EO in maltodextrin (Balasu-
bramani et al., 2013), gac red arils EO (Momordica conchinchinensis) in
whey protein and gum arabic (Kha et al., 2014) and coffee EO in gum
arabic (Frascareli et al., 2012).
3.2. Characterization of the microparticles obtained under optimal
conditions
High yield (71.9%) and EE (99.9%), similar to the values predicted by
the model (70.1% and 99.9%, respectively), were found for citral
I. Yoplac et al. Heliyon 7 (2021) e06737microparticles prepared under optimal conditions of Ct:Dx ratio (1:5) and
inlet air temperature (187 
C). Under these optimal conditions and
considering the EE %, a concentration of 137.56 mg citral/g micropar-
ticles was obtained.
The EE was high and superior to studies where dextrin and milk
protein isolate or inulin were used as coating material (Ahn et al., 2008;
García et al., 2013). The EE level obtained in this study could be attrib-
uted to the stability of the feed emulsion (Ct-SL-Dx) in the spray drying
process, which play an important role in the hydrophobic molecules
retention, as well as the emulsifier effect of SL. Furthermore, the high
retention in the dextrin systems was attributed to the higher hydropho-
bicity of the polymer (García et al., 2013).
Previous studies mention that the EE of citral in spray drying is mainly
influenced by the properties of the encapsulating agent (viscosity, solu-
bility), citral:encapsulating agent ratio and temperature (Maswal and
Dar, 2014; Sosa et al., 2014). Temperature, a relevant factor in citral
degradation (Sosa et al., 2014), can be tolerated up to 400 
Cwithout any
negative effect on its chemical properties (Bhandari et al., 1992). In the
present study, despite the high drying temperatures, the EE was high
probably because of the short drying times and/or the rapid crust for-
mation on the drop surface, which allows water diffusion while retaining
EO (Gharsallaoui et al., 2007; Vergara et al., 2014).
The citral microparticles obtained under optimal conditions pre-
sented 5.3  1.3% humidity, 0.2  0.02 water activity, 29.7  0.01 g/
100 g hygroscopicity, 7.08 μm average particle size (D4,3) and particle
size distribution values between 1.04 and 15.05 μm. Moisture, water
activity and particle size were within the range reported for micropar-
ticles obtained by spray drying (Cai and Corke, 2000; Gharsallaoui et al.,
2007). Hygroscopicity was lower than the values obtained by Frascareli
et al. (2012) and Kha et al. (2014) due to the physical-chemical char-
acteristics of the ingredients used, mainly the encapsulating agent (Bakry
et al., 2016).
Figure 2 shows the SEM photographs of the microparticles obtained
under optimal conditions. All had spherical shape with irregular and
rough external surfaces and depressions, indentations or concavities, and
showed a tendency to agglomeration. The depressions could be attrib-
uted to the lack of plasticizing properties of the encapsulating agent and
to the contraction of the microparticles during the drying process,
consequence of the high inlet air temperature. The rapid evaporation in
the initial drying stage and the high pressure of the particles produce
contraction (Frascareli et al., 2012). A similar morphology was observed
in lemon myrtle EO microencapsulated with maltodextrin and modified
starch (Huynh et al., 2008), linseed and rosemary EO with maltodextrin
(de Barros-Fernandes et al., 2014), cardamom oleoresin withFigure 2. Scanning electron microscopy photographs of citral microparticles obtaine
right, agglomerated microparticles.
5
maltodextrin and modified starch (Krishnan et al., 2005), Haematococcus
pluvialis oleoresins with capsul (Bustamante et al., 2016) and coffee EO
with gum arabic (Frascareli et al., 2012), all produced by spray drying
using polysaccharides as wall material (modified starch and maltodex-
trin). Differently, microparticles of sunflower EO in milk protein isolate
and dextrin (Ahn et al., 2008) and lime EO in blend of whey protein and
maltodextrin (Campelo et al., 2018) were spherical, with a homoge-
neous, smooth surface, free of cracks and pores, probably due to the fact
that the dextrin or maltodextrin was combined with a protein-based
polymer, in addition to the effect of the inlet temperature.
3.3. Antibacterial activity of pure citral mixture
The effect of citral on bacterial growth is shown in Table 3. Diameter
means of inhibition zone were significantly different. The lowest inhib-
itory concentration (0.80 mg/mL) was obtained for B. cereus and the
highest (4 mg/mL) for S. enterica.
The bacterial strains studied exhibited a high sensitivity to low citral
concentrations, stronger than those reported by other authors. For
example, pure D-limonene inhibited the growth of E. coli at minimum
concentrations of 25 mg/mL (Donsì et al., 2011) while pure eugenol
inhibited E. coli and S. aureus at a minimum concentration of 10 mg/mL
(Piletti et al., 2017). The results in Table 3 are comparable to the values
reported for antimicrobial agents such as thymol, carvacrol and citrus EO
against S. aureus, S. enterica, L. innocua and E coli (Fancello et al., 2016;
Guarda et al., 2011). Saddiq and Khayyat (2010) concluded that citral
showed greater antibacterial activity compared to industrial antibiotics
such as nalidixic acid, ampicillin and nitrofurantoin.
The inhibitory effect of citral against microorganisms could be
explained by its structural characteristics, as it is an α, β-unsaturated
aldehyde with the carbonyl group adjacent to the α and β carbons. Due to
their position, the α and β carbons are conjugated to the carbonyl group,
allowing the β-carbon to become positively polarized and easily reactant
with nucleophiles (nucleophilic attack) (Wuryatmo et al., 2003). Ac-
cording toWitz (1989), the chemical action of α, β-unsaturated aldehydes
and some of their toxicological effects is based on their ability to act as
direct alkylating agents. These alkylating agents are capable of covalently
bind cellular nucleophilic groups, modifying cellular processes and being
potentially toxic. The antimicrobial effect can also be attributed to the
ability of citral to alter and penetrate the lipid and protein structure of
bacterial cell wall, as recently proposed by Lu et al. (2018). This leads to
protein denaturation and cell membrane destruction, followed by cyto-
plasmic filtration, lysis, and cell death (Saddiq and Khayyat, 2010).d under optimal conditions (3000x). On the left, dispersed microparticles; on the
I. Yoplac et al. Heliyon 7 (2021) e06737
Table 3. Antibacterial effect, expressed as diameter of the inhibition zone (mm; mean  standard error, n ¼ 4), of pure citral mixtures at different dilutions in heptane.
Citral concentration (mg/mL) E. coli1 S. enterica2 S. aureus3 B. cereus4
0.00 -5 - - -
0.08 - - - -
0.20 - - - -
0.40 - - - -
0.80 - - - 7.78d  0.31
2.00 8.68c  0.25 - 6.73c  0.21 8.73c  0.31
4.00 10.00b  0.41 6.65d  0.13 7.08c  0.27 9.88b  0.42
8.00 14.33a  0.61 7.73c  0.59 7.25c  0.17 10.28b  0.18
40.00 14.70a  0.72 12.93b  0.46 10.03b  0.62 13.88a  0.14
80.00 15.25a  0.90 14.05a  0.48 15.03a  0.32 14.30a  0.71
Reference numbers: 1ATCC® 25922™; 2ATCC® 14026™; 3ATCC® 25923™; 4ATCC® 14579 PK/5. 5 Dashes indicate that there was no inhibitory effect.3.4. Antibacterial activity of citral microparticles
The studies carried out with solutions of pure heptane (Table 3) did
not show activity against the bacterial strains under study, demonstrating
that the solvent has no inhibitory effect. The inhibitory effect of citralFigure 3. Growth inhibition of different bacteria species by citral microparticles
expressed as: (A) diameter of inhibition zone at a concentration of 300 mg/mL
and as: (B) inhibition percentage of the citral microparticle compared to an
equivalent amount of free citral. Bars indicate standard error (n ¼ 4). Different
letters on each bar indicate statistically significant differences according to the
LSD test (p < 0.05).
6
microparticles (300 mg/mL) on the growth of E. coli and B. cereus,
S. aureus and S. enterica is presented in Figure 3A. The diameter of the
inhibition zone was significantly different among bacterial strains: Gram-
negative E. coli and B. cereus were more sensitive to citral microparticles
(9.98 and 9.05 mm, respectively) than Gram-positive S. aureus and
S. enterica (7.38 and 6.88 mm, respectively).
Citral microparticle systems would promote enhanced interaction
with microbial cell membranes primarily by the following routes:
increased contact surface area favours passive diffusion through the outer
cell membrane and interaction with the cytoplasmic membrane (Donsì
et al., 2012; Lu et al., 2018); sustained release over time of citral from the
microparticles, driven by the release of citral between the wall material
and the aqueous phase, prolonging its bioactivity (Majeed et al., 2016);
and electrostatic interaction of the positively charged citral microparti-
cles (through the polymer) with the negatively charged microbial cell
walls increase the concentration of these oils at the action site (Chang
et al., 2015). The use of micro-scale delivery systems, such as structures
based on microparticles, microemulsions and liposomes, can increase
passive mechanisms of cellular absorption, reducing resistance to mass
transfer and increasing antimicrobial activity (Bakry et al., 2016; Donsì
and Ferrari, 2016).
These results agree with previous studies of microparticles based on
EO of lemongrass, cloves, thyme, palmarosa, oregano, thymol and
carvacrol, which showed bactericidal properties against E. coli, L. mon-
ocytogenes, L. innocua, B. cereus, B. subtilis, Haemophilus influenzae, Neis-
seria gonorreae, Streptococcus pneumoniae and Vibrio cholerae
(Anaya-Castro et al., 2017; Guarda et al., 2011; Salvia-Trujillo et al.,
2015).
On the other hand, studies comparing the antimicrobial activity of
microparticles of carvacrol, limonene and cinnamaldehyde EO, encap-
sulated with soy lecithin, soy protein and sucrose palmitate, against
Gram-positive and Gram-negative bacteria, did not find significant dif-
ferences (Donsì and Ferrari, 2016). Differently, some studies reported a
higher sensitivity of Gram-positive bacteria than Gram-negatives when
using microparticles of lemongrass EO (Leimann et al., 2009), Citrus
limon EO (Fancello et al., 2016) and citral nanoemulsions (Lu et al.,
2018). The apparently contrasting results among studies are a conse-
quence of the antimicrobial activity dependance on the microparticle
system, type and concentration of the microencapsulated active in-
gredients (Donsì et al., 2012; Salvia-Trujillo et al., 2015). In the present
study, citral with a purity higher than 96%, microencapsulated in
dextrin, was used.
The antibacterial inhibition percentage is the ratio of the diameter
zone of transparent inhibition of the citral microparticles and the inhi-
bition diameter of similar concentrations of free citral. When the inhib-
itory effect (inhibition ratio) of the citral microparticle was evaluated in
comparison with equivalent amounts of free citral, no significant differ-
ences were obtained, and the values varied between 55.6% for S. enterica
and 74.5% for S. aureus (Figure 3B).
I. Yoplac et al. Heliyon 7 (2021) e06737Results showed that, even after the microencapsulation process and
high temperature spray drying, citral maintained its bioactivity. The in-
hibition ratio of the citral microparticle was less than 100%, which could
be due to the lower free mass of the microencapsulated citral compared
to the equivalent amounts of free citral used in the inhibition test; during
the microencapsulation process, dextrin and SL were added to citral, so
the activity of microencapsulated citral was not the same as free citral.
These results were lower than those reported by Leimann et al. (2009) for
lemongrass EOmicroparticles formulated with polyvinyl alcohol, sodium
sulfate and glutaraldehyde and than those reported byWang et al. (2009)
for curcumin microparticles formulated with gelatin and starch. In both
studies, no emulsifiers (e.g. soy lecithin) were used. The presence of SL in
the current research may be the cause of the low inhibition ratio which
hinders the rapid release of citral (Donsì et al., 2011, 2012).
When carvacrol EO, D-limonene and trans-cinnamaldehyde were
microencapsulated with soy lecithin and soy protein, SL was responsible
for the slow release of EO in the aqueous phase, causing antimicrobial
activity for 24 h; in contrast, the availability in the aqueous phase of EO
microencapsulated with sugar esters, Tween 20 and glycerol monooleate
EO was immediate and the bactericidal activity lasted 2 h (Donsì et al.,
2012). In in vitro studies, where a rapid release of the active agent is
required to avoid rapid microbial growth, the use of emulsifiers in
microparticle formulation would reduce the bioactivity of the antimi-
crobials; however, if a bacteriostatic action is desired for a prolonged
period of time to ensure a certain shelf life for a food product, the best
option would probably be the use of SL, to favor a slow release and ensure
a lower concentration of the antimicrobial compound in the aqueous
phase, therefore prolonging its action (Donsì et al., 2011, 2012; Donsì
and Ferrari, 2016). The optimal microparticles obtained in the present
study could be used for the elaboration of active biofilms for the pack-
aging of highly perishable foods.
4. Conclusion
Citral, an acyclic aldehyde with effective antimicrobial activity
extracted from natural sources, was successfully microencapsulated by
spray drying in a dextrin and soy lecithin matrix. The obtained micro-
particles showed high encapsulation efficiency and high emulsion
retention (citral, SL and Dx) during the drying process, favoring citral
molecules retention. Using RSM, the optimal citral microencapsulation
conditions were determined with high yield and encapsulation
efficiency.
The study of the inhibitory activity of optimal citral microparticles
against tested strains of the foodborne pathogens E. coli, S. enterica,
S. aureus, and B. cereus, suggests a broad spectrum of effects. Bactericidal
activity was not significantly affected by the high temperature spray
drying process, and its inhibition ratio was greater than 55%. In addition,
the results of the in vitro assay of minimal inhibitory concentrations of
pure citral (>0.8 mg/mL) indicated that it was effective against the
bacteria strains studied, regardless of their Gram-positive or Gram-
negative classification.
The citral microparticles described in this study could represent a
natural antimicrobial potential for use in the design of food systems and/
or active food packaging, in order to extend shelf life and ensure food
safety.
Declarations
Author contribution statement
Ives Yoplac: Conceived and designed the experiments; Performed the
experiments; Analyzed and interpreted the data; Contributed reagents,
materials, analysis tools or data; Wrote the paper.
Luis Vargas, Paz Robert: Analyzed and interpreted the data;
Contributed reagents, materials, analysis tools or data; Wrote the paper.
Alyssa Hidalgo: Analyzed and interpreted the data; Wrote the paper.7
Funding statement
This work was supported by Fondo Nacional de Desarrollo Científico,
Tecnolo
gico y de Innovacio
n Tecnolo
gica (PE) [179-2015-FONDECYT].
Data availability statement
Data associated with this study has been deposited at 'Repository of
the National Agrarian University La Molina, Peru' under the accession
number http://repositorio.lamolina.edu.pe/handle/UNALM/4157.
Declaration of interests statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
The authors thank Cristina Vergara, Estefanía Gonz
alez, In
es Cea and
Guibeth Morello for their contribution, technical support and
suggestions.
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