FARMASETIKA LANJUTAN

(Antimicrobial nanoemulsion formulation based on thyme (Thymus vulgaris) essential

oil for UF labneh preservation)

FITRIA
NIM 227014022

PROGRAM STUDI MAGISTER FARMASI

FAKULTAS FARMASI
UNIVERSITAS SUMATERA UTARA
MEDAN
2023
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1

Available online at www.sciencedirect.com

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

Original Article

Antimicrobial nanoemulsion formulation based on

thyme (Thymus vulgaris) essential oil for UF labneh
preservation

Samah M. El-Sayed*, Hoda S. El-Sayed

Dairy Science Department, National Research Centre, 33 El Bohouth St. (Former El Tahrir St.), Dokki, P.O. 12622,
Giza, Egypt

article info abstract

Article history: The present study was evaluated and fabricated the thyme essential oil (TEO) nano-
Received 11 September 2020 emulsion form to use in preservative of UF labneh. The antimicrobial and chemical
Accepted 20 December 2020 composition of the oil was determined by well diffusion method and Gas chromatography
Available online 29 December 2020 emass spectroscopy (GCeMS). The TEO nanoemulsion was characterized by Dynamic light
scattering (DLS), Transmission electron microscope (SEM), and time killer effect. Inclusion
Keywords: TEO exhibited high inhibitory effects against numerous pathogenic strains, in which the
Thyme essential oil diameter of inhibition ranged from 10 to 36 according to different concentrations of TEO.
Nanoemulsion The GCeMS analysis of TEO has detected 15 compounds mainly thymol (43.63%). TEO
UF labneh nanoemulsion was almost spherical with a diameter of 52 nm and able to eliminate
Antimicrobial properties pathogenic counts within 60 min. The fortified labneh with different concentrations (0.1,
Shelf life 0.2, and 0.3%) TEO nanoemulsion was kept the quality of labneh up to six weeks. The
counts of psychrotrophic, mold, and yeasts were indicated in the eighth week compared
with control. The addition of TEO nanoemulsion was affected slightly on chemical prop-
erties during storage. Therefore, the highest total scores were given to labneh with 0.1%
TEO nanoemulsion. The results suggested that the formulated TEO nanoemulsions might
be a promising agent to prolong the shelf life and enhanced the aroma of labneh.
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

and antioxidant actions [2]. Because of known antimicrobial, it

1. Introduction
Essential oils are aromatic compounds gained from plant
materials and are distinguished by its extreme volatility and
complex structure [1]. Essential oils gained from plants or
spices are rich sources of biological composites as phenolic
acids and terpenoids which possess antibacterial, antifungal,
is widely considered for use in pharmaceutical, agricultural,
food, and cosmetic products [1,3]. Concerning antimicrobial
activity, numerous articles have verified the possibility of
essential oils to prevent the multiplication of several types of
microorganisms in food products, like Listeria sp., Escherichia
coli, Salmonella sp., Bacillus sp. and Aspergillus sp. [4e6]. Their

* Corresponding author.
E-mail address: Samah_mosbah80@yahoo.com (S.M. El-Sayed).
https://doi.org/10.1016/j.jmrt.2020.12.073
2238-7854/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
1030 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1
antimicrobial action is highly dependent on the existing
structure and functional groups.
Thyme (Thymus vulgaris) essential oil is the most inspected
and implemented essential oils for its antimicrobial proper-
ties. According to essential oil compositions, several chemo-
types of thyme have been recognized, as thymol, carvacrol,
borneol, geraniol, linalool, sabinene hydrate and a number of
multiple-component chemotypes [7]. Thyme essential oil is
recognized for its antimicrobial properties, antifungal, and
antioxidant action. Thyme essential oil is a blend of more than
30 different compounds, in which the main components are
carvacrol (55e85%) and thymol (0e5e10%), have the strongest
antimicrobial action attributable to their phenolic composi-
tion. The antimicrobial mechanism of carvacrol and thymol
able to destroy bacterial cells by changing the permeability of
the cell membrane and resulting leakage of cell components
[8e10]. It’s more effective against Gram-negative bacteria but
with reduced activity against Gram-positive Lactobacillus and
Bifidobacterium [11].
Many literature studies prove that thyme is non-toxic to
gut probiotics. Karmakar et al., (2012) [12] demonstrated that
the combination of essential oil and probiotic bacteria can be
managed together to cure the pathogenic infection in the
human gut. This is because the essential oils are effective in
lower concentrations against the pathogens whereas they
have high Minimum inhibitory concentration (MIC) values for
probiotics. Si et al., (2006) [11] found that when mixed different
essential oil which content eugenol, thymol, and other com-
pounds with pig cecal digesta, established high efficacy
against some pathogens strains with little inhibition effect on
the total number of lactobacilli and bifidobacteria. The per-
centage of inhibition was noticed at the concentration of
300 mg/ml thymol and gotten 20%, 26%,37%, and 80% for Bifi-
dobacterium longum, Bfidobacterium breve, Lactobacillus planta-
rum, and Lactobacillus acidophilus, respectively. Ouwehand
et al., (2010) [13] confirmed that Lactobacillus reuteri appeared to
be very resistant to the thyme oil. In contrast, Bifidobacterium
animalis ssp. lactis and B. longum were particularly resistant
against the concentration of 50 and 500 mg/l of thyme
essential oil. Also [14], demonstrated that the combination of
oregano extract with peppermint and thyme essential oils had
a positive effect on the gastrointestinal tract of animals by
increasing the number of probiotics bacteria.
The attention for use of food preservation is continuously
increased due to consumer demands to replace food pre-
servatives with natural materials with a tangible effect [15,16].
From the view of technological point, applying the essential
oils as a food preservative presents some disadvantages due to
the strong flavor and smell, which could alter the sensory
characteristics of the product [17]. Additionally, essential oils
are readily degraded by heating and exposure to light and
oxidation [18], and are difficult to dissolve in the aqueous
medium [19]. Therefore, to overcome these imperfections,
nanoemulsion techniques have been projected, as they during
storage enhanced the chemical stability, increase the distri-
bution of essential oils in aqueous formulations, reduce the
change in sensory processes, and in some cases, improve their
antimicrobial activity [20]. Provides their small particles,
nanostructures were able to encourage inert cellular absorp-
tion devices, which reduces mass transfer as well as increases
antimicrobial activity. Likewise, nanoemulsions can be
designed for good stability and low turbidity, and are suitable
for a wide range of commercial applications as in food as
preservative agents [21]. Accordingly, there is an increasing
challenge in the food industry to develop consumer confi-
dence to use nano-food ingredients [22].
Labneh, the old fermented milk product that is consumed
in the Middle East countries, is gained from yogurt after
removing part of the whey. Likewise, due to the presence of
acidic flavor and milky white color, the labneh is smooth, soft,
and dispersible in a cream-like consistency. Labneh is formed
by strains of lactic acid bacteria, which ferment the lactose
present and produce organic acids, mainly lactic acid [23]. The
labneh shelf life was commonly short even if it stored at the
coldness [24]. The high microbial load of a labneh during
storage and packaging leads to unfamiliar flavors and un-
wanted physical-chemical changes that eventually lead to
product rejection [23]. One of the most acceptable ways to
prolong the life of fresh food products is to use essential oil
[1,25,26]. Most essential oils are classified as GRAS (generally
recognized as safe) and the antimicrobial activity of essential
oils leads to the prolonged of different foods shelf life [1].
Moreover, the survivability of lactic acid bacteria did not weak
by essential oil but may be increased at adjusted the essential
oil concentrations [23,27]. Elama et al., (2019) [28] evaluated
the antimicrobial activity of several essential oils when
applied in concentrated yogurt (Labneh) with the percent
250 ml/kg. The study showed that the addition of essential oil
as clove, rosemary, cinnamon able to prolong the shelf life of
the Labneh. Therefore, the essential oils in basic or nano-
emulsion form were promising agents in dairy products for
their antimicrobial action, antioxidant, bio-preservative and
for enhanced aroma [29,30].
Therefore, this study was designated to evaluate the anti-
microbial activity of TEO and determine the main components
of TEO. Moreover, TEO nanoemulsion was fabricated to pre-
serve the labneh. Firstly, TEO nanoemulsion was character-
ized by TEM, DSL, and time killer against pathogens was
detected. Secondary, the microbiological, chemical, and sen-
sory properties of UF labneh during the storage period for
eight weeks were assessed.
2. Materials and methods
2.1. Materials
Thyme essential oil was gained from the oil extraction unit
from the National Research Centre (NRC), Giza, Egypt. UF-
retentate used in the manufacturing of UF labneh was ob-
tained from Dairy Industry Units, Animal Production Research
Institute, Ministry of Agriculture, Giza, Egypt. All substances,
microbiological media and chemicals used in the current
research were of analytical grade and obtained from Oxiod,
Egypt.
2.1.1. Microbial strains
Microbial strains used in this study were collected from the
Dairy department at National Research Centre, Egypt as (Ba-
cillus cereus B-3711, E. coli O157: H7 9637, Listeria monocytogenes
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1
598, Salmonella typhimirum 14028s, Staphylococcus aureus 8325,
Aspergillus niger 3858a and Aspergillus flavus B-3357). Bacterial
strains were incubated at 35  C in tryptone Soya broth and
fungal strains were incubated at 25  C in potato dextrose broth
until observable turbidity reached 0.5 “McFarland” standard
solution.
2.1.2. Lactic acid strains
Lactic acid strains used in manufacturing UF labneh as (L.
acidophilus CH-2, Lactobacillus bulgaricus Lb-12 DRI-VAC and
Streptococcus thermophilus CH-1) were collected from Dairy
department at National Research Centre, Egypt.
2.2. Methods
2.2.1. Gas chromatographyemass spectroscopy (GCeMS)
analysis for thyme essential oil (TEO)
Samples were analyzed by GCeMS using a Hewlett Packard
model 5890. The gas chromatograph is equipped with 5 series
Mass selective detector 8644 (HP) with flame ionization de-
tector (FID) on a fused silica 132 capillary column DB-5 (25 min
length, 0.32 mm i.d., and 0.5 mm film thickness). Gas chro-
matography conditions include a temperature maintained at
60  C for 2 min and it was then automated at 4  C/min to
270  C. The split injector temperature was 270  C and the MS
conditions were kept at 280  C and 42 ev. According to Gunther
& Joseph (1978) [31]; the qualitative analysis was based on the
percentage area of each peak of thyme oil compounds.
2.2.2. Antimicrobial activity of thyme essential oil (TEO)
Thyme essential oil (TEO) was diluted in dimethyl sulfoxide
(DMSO) to obtain 0.1: 3.0% concentration. Antimicrobial action
was accomplished by the method of agar well diffusion
[25,26]. The nutrient agar medium (Oxoid) was added to petri
dishes and permitted to harden. 0.1 ml of each tested strain
has been spread to the surface of the agar. The plates were left
for 2 h at 37  C for those tested strains saturated in agar and
prepared different wells in the medium by a cork borer
(0.5 cm). 100 ml of a different concentration of the oregano oil
was added to each well. All plates were incubated for 24 h at
37  C, after which the inhibition zone was measured around
each well in millimeters.
2.2.3. Preparation of TEO nanoemulsion
Nanoemulsion of TEO was carried out by mixed 80% w/w of
distillate water with 10% (w/w) of TEO, respectively, and 10%
(w/w) of tween at a total mass of 100 ml for the solution. The
mixture was stirred at 800 rpm with a magnetic type stirrer for
30 min to get a homogeneous solution. The resulting emulsion
was sonicated for 20 min by a 25 kHz ultrasonic Homogenizer
(USH650, max power: 650 W) [32]. The TEO nanoemulsion was
stored at room temperature for used as a natural preserve
agent in manufacture UF labneh.
2.2.4. Particle size measurement for TEO nanoemulsion
Nanoemulsion droplets diameters for TEO nanoemulsion
were measured using dynamic light scattering (NICOMP 380
ZLS, Dynamic light scattering (DLS) instrument, USA).
1031
2.2.5. Transmission electron microscope (TEM) analysis
TEM analysis was carried out to study the structure and
morphology of the prepared nanoemulsion by using JEOL JEM-
1230 transmission electron microscope. The sample was
diluted with deionized water; a small drop of nanoemulsion
was dropped to 200-mesh formvar-coated copper grids at
room temperature, and negatively stained with uranyl acetate
for 3 min. The excess liquid and prepared samples were dried
using the Whatman filter paper at room temperature.
2.2.6. Killing kinetics assay of TEO nanoemulsion
Different concentration of TEO nanoemulsion (0.05, 0.1, 0.5,
1.0 and 3.0%) was used for studying the killing kinetics assay
by indicated the reduction of number log CFU over 90 min [33].
Activated strains of E. coli, S. aureus, and A. flavus were indi-
vidually centrifuged (5000 rpm, 15 min) and washed with sa-
line solution. Test strains were diluted to obtain 1  106 CFU/
ml and challenged with different TEO nanoemulsion con-
centrations using nutrient broth medium. After 0, 5, 15, 30, 45,
60, 75, and 90 min from the time of incubation at 37  C, 0.1 ml
of the interacted sample was taken from each tube, diluted,
and spread on top of nutrient agar plates. The colonies were
enumerated after incubated for 24 h at 37  C.
2.2.7. UF laneh prepared using TEO nanoemulsion
UF labneh was made according to El-Sayed et al. (2017) [25,26].
Ultrafiltration retentate was pasteurized at 72  C for 5 min and
rapidly cooled to 42  C. The pasteurized UF-retentate has
inoculated with Lactic acid strains Lb. bulgaricus 1%, S. ther-
mophilus 1%, and Lb. acidophilus 1%. The inoculated ultrafil-
tration retentate was distributed into four portions, the first
part was severed as control and the other 3 portions were
fortified with 0.1, 0.2, and 0.3% of TEO nanoemulsion treat-
ments (T1, T2, and T3). All treatments packed in polystyrene
cups (50 ml). The cups were incubated at 42  C till coagulation
and after that, cold-stored (5 ± 2  C) for eight weeks.
2.2.7.1. Chemical analysis of UF labneh during cold storage for
eight weeks. 50 g of each UF labneh samples was analyzed for
dry matter, total nitrogen, ash, titratable acidity (TA), total
nitrogen, and fat contents for fresh samples and during stor-
age as declared by AOAC (2012) [34]. Digital pH meter (Hanna,
Germany) with a combined electrode using for measured pH
values of labneh.
2.2.7.2. Assessment of UF labneh bacteriological quality during
cold storage for eight weeks. Ten grams of labneh samples
were homogenized with 90 ml of tri-sodium citrate (2% w/v).
Decimal dilutions were performed in 9 ml saline solution. The
lactic acid bacteria were enumerated by MRS agar (Oxoid) [35]
and incubated at 37  C for 48 h. The mesophilic bacterial
counts were aerobically incubated using nutrient agar (Oxoid)
at 35  C for 48 h [36]. Psychrotrophic bacteria were detected by
nutrient agar and incubated at 5  C for 7 days [37]. Yeasts and
molds counts were detected by acidified potato dextrose agar
(Oxoid) pH 3.5 [36] and the colonies counted after 4 days at
25  C. All microbiological results were recorded log CFU/gm.
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terpinene (6.56%), becaryophyllene (5.65%) and carvacrol

Table 1 e Chemical composition of thyme essential oil
(3.18%). Other constituents, such as terpinolene (1.78%), car-
(TEO).
yophyllene oxide (1.09%), Borneol (1.07%), and bepinene
No. Compounds Area percentage %
(1.02%) were detected in small amounts. The other constitu-
1 Thymol 43.63 ents were present in a total amount of less than 1%. These
2 p-cymene 22.86 findings are in agreement with previous studies, who exam-
3 Bronyl acetate 8.70
ined the chemical composition of the same essential oil and
4 g-terpinene 6.56
5 becaryophyllene 5.65
they reported that thymol, p-cymene, and g-terpinene are
6 Carvacrol 3.18 major compounds in the TEO [40e42]. Furthermore, Yamazaki
7 Terpinolene 1.78 et al. (2004) [43] reported that phenolic compounds such as
8 Caryophyllene oxide 1.09 thymol and carvacrol have strong antimicrobial effects espe-
9 Borneol 0.071 cially against E. coli O157: H7. Moreover, Xu et al. (2008) [44] and
10 bepinene 1.02
Ballester-Costa et al. (2013) [45] demonstrated that thymol and
11 a-phellandrene 0.99
carvacrol compounds have the ability to facilitating the par-
12 geterpinol 0.98
13 a-thujene 0.72 titioning of the bacterial cytoplasmic membrane cells espe-
14 Thymol methyl ether 0.68 cially into the bacterial lipid bilayer, eventually leading to
15 a-terpinene 0.49 bactericidal effect. Therefore it can be used as a good preser-
ving agent in the food industry.

2.2.7.3. Sensory evaluation of UF labneh during cold storage for
eight weeks. 50 g of labneh samples were evaluated as rec-
ommended by the method of Obi et al. (2010) [38] for color &
appearance, body & texture, odor, taste, and overall accept-
ability, by 10 panelists had previous knowledge of the sensory
quality of dairy products from the staff of the dairy depart-
ment, National Research Centre (NRC), Egypt, using the nine-
point hedonic scale at eight weeks of the storage period [(1)
Very bad, (2) Bad, (3) Imperfect, (4) Sufficient, (5) Mediocre, (6)
Satisfactory, (7)Good, (8) Very good, (9) Excellent].
2.2.8. Statistical analysis
The data were analyzed according to Statistical Analysis
System Users Guide SAS (2004) [39], (SAS Institute, Inc., USA).
All data of labneh were carried out in triplicate and mean
values.
3. Results and discussion
3.1. Chemical composition of thyme essential oil (TEO)
The results obtained by GC/MS analysis of the TEO are pre-
sented in (Table 1). A total of 15 components representing
99.4% of the total detected components were identified. The
components detected at the highest quantities in TEO were
thymol (43.63%), p-cymene (22.86%), Bronyl acetate (8.70%), g-
3.2. Antimicrobial activity of thyme essential oil (TEO)
The results of the antimicrobial in this research confirmed
the presented results of chemical composition. The results
in (Table 2) indicated that TEO has a different degree of
inhibition diameter against tested pathogens. The most
virulence effect observed against B. cereus (19e36 mm),
followed by E. coli (16e32 mm) and S. aureus (13e33 mm).
Also, all TEO tested doses exhibited a significant effect
(p < 0.05) on each of the pathogenic microbes, in which
the inhibition effect increased with the TEO concentration.
Moreover, the TEO has virulence effect against fungi,
which mainly cause a problem in dairy products and other
food. The degree of inhibition observed against A. niger
and A. flavus was ranged between 13e31 mm and
10e32 mm, respectively. There was a relationship between
the antimicrobial activity of TEO and their different
compounds as thymol, p-cymene, g-terpinene,
becaryophyllene and carvacrol as confirmed by previous
chemical composition data and other authors [42,46,47].
Likewise, Moro et al. (2013) [48] found that TEO has shown
acceptable antimicrobial activity in vitro besides patho-
gens and spoilage microbes. Burt (2004) [1] stated that
essential oil able to break the outer membrane of patho-
genic bacteria, lead to leakage lipopolysaccharides, and
create pours in the cytoplasmic membrane.

Table 2 e Antimicrobial effect of TEO against pathogenic strains (%).

Pathogenic stains 0.1 0.5 1.0 2.5 5
Diameter of inhibition zone (mm)
S. typhamirum 11D ± 0.50 17C ± 0.60 20C ± 0.88 23C ± 1.06 23D ± 0.90
E. coli 16B ± 0.55 21B ± 0.92 24B ± 1.12 26B ± 0.94 32B ± 1.18
S. aureus 13C ± 0.40 20B ± 0.89 23B ± 1.14 26B ± 0.98 33B ± 1.17
B. cereus 19A ± 0.85 25A ± 0.10 27A ± 1.92 30A ± 1.18 36A ± 1.18
L. monocytogenes 11D ± 0.35 15D ± 0.11 17D ± 0.66 23C ± 2.15 29CD ± 0.89
A. niger 13C ± 0.45 20B ± 0.88 23B ± 1.15 27B ± 1.155 31C ± 1.17
A. flavus 10D ± 0.30 20B ± 0.90 22B ± 1.10 25BC ± 0.10 32B ± 1.12

Data expressed as (mean ± SD) of 3 replicates. Within the same column, means with different letters are significantly different (p  0.05).
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3.3. Particle size analyzer
The mean particle diameter of TEO nanoemulsion was eval-
uated by the DLS method and the particle size distribution is
compiled in (Fig. 1). The mean diameter of prepared nano-
emulsion was about 52 nm, Solans, et al., (2003) [49] pointed
that numerous types of Nano formulations have been cate-
gorized by their stability and small droplets size, ranging from
20 to 200 nm. This, along with an enhanced surface area for
interaction, greater thermodynamic stability, and improved
bioavailability, all of this makes nanoemulsion as a good
active ingredient for food preserve applications. Furthermore,
the value of polydispersity index (PDI) was 0.15, Jaiswal et al.,
(2015) [50] displayed that the greater value of polydispersity
showed the fewer uniformity of droplet size of nanoemulsion,
that PDI represents the homogeneity of droplet size in nano-
emulsion. Moreover, a sample with a very wide size distribu-
tion has a polydispersity index value higher than 0.7 is not
suitable for DLS analysis [51]. In our study, the TEO nano-
emulsion has low PDI value which indicates the overall sta-
bility and good homogeneity.
3.4. Nanoemulsion morphology
TEM analysis was done to confirm the nanoemulsion droplet’s
formulation size and structure. The prepared nanoemulsion
containing 10 v/v% thyme oil using 10 v/v% Tween, and 80 v/v
% water was visualized by TEM (Fig. 2). The TEM image dis-
played that most of the droplets of nanoemulsion were almost
spherical and also it had approximately uniform shape and
size. Moreover, the TEM image reveals that the thyme essen-
tial oil is evenly distributed in the aqueous phase. Corre-
spondingly, the nanoemulsion with 10 v/v% thyme essential
oil showed nanometer particle size at range 20e52 nm which
is an acceptable dispersive capability and in agreement with
the results of the DLS test. These results confirmed by Has-
sanin et al., (2017) [52] indicated by TEM micrograph that
nanoemulsion of the thyme essential oil was spherical in
shape and moderately mono or di-dispersed and was in the
range of 26.6e45.3 nm. Similarly, Sundararajan et al., (2018)
[53] found that the loaded essential oils of the nanoemulsions
had spherical shape and regular distribution.
Fig. 1 e Particle size distribution of TEO nanoemulsion.
1033
3.5. Killing kinetics assay of TEO nanoemulsion
Time kill kinetics were determined by the count of remaining
viable strains at changing time points (5, 15, 30, 45, 60, 75, and
90 min) after exposure to the test TEO nanoemulsion. The
killing assay against A. flavus was indicated that different
concentrations of TEO nanoemulsion showed a different
reduction especially when used 3.0% concentration (Fig. 3, A).
At the concentrations 0.05, 0.1, 0.5, 1.0 and 3.0%, the viable
count of A. flavus was reduced significantly (p  0.05) around
3.0, 4.0, 4.4, 6.0 and 6.0 log cycles at 90 min, respectively. After
60 min, the Viable A. flavus count was destroyed at a con-
centration of 1.0 and 3.0% compared with control. Succes-
sively, at these concentrations, TEO nanoemulsion acts as the
bactericidal (no viable cells detected) for A. flavus. Similarly
(Fig. 3, B), shows the results of kill kinetics assay of active TEO
nanoemulsion against S. aureus, where a reduction in the
count by 2.2, 2.7, 3.2, 4.55 log cycles at 90 by 0.05, 0.1, 0.5 and
1.0% TEO nanoemulsion concentrations. Additionally, the
used concentration 3.0% able to kill S. aureus (no viable cells
detected) at 75 min compared with control. Moreover, the
lowest concentration (0.05%) was able to use as a bacterio-
static agent, which can reduce S. aureus during 90 min by 2.2
log cycles. The same trend of results observed with E. coli, that
the TEO nanoemulsion at different concentrations was
significantly (p  0.05) reduced the viable count of E. coli during
90 min (Fig. 3, C).
The E. coli exhibited a bactericidal response to TEO nano-
emulsion for all tested concentrations at 90 min. The viable E.
coli counts reduced by 0.86, 1.86, 2.35, 3.25 and 3.8 log cycles for
0.05, 0.1, 0.5, 1.0 and 3.0% TEO nanoemulsion concentrations
at 90 min. The results found that TEO nanoemulsion exhibited
strong antimicrobial activities and able to destroy the cells.
The bacteriostatic concentration of TEO nanoemulsion
(ranged between 0.05 and 1.0) was successful in reduction
around 2e4 log cycles within 60 min. Accordingly, the TEO
nanoemulsion was displayed as bactericidal or bacteriostatic
agents depending to the used concentrations and there may
be possibilities for its use as an additive to foodstuffs [54e57].
Numerous researchers have recognized the nanoemulsion of
Fig. 2 e TEM image of TEO nanoemulsion.
1034 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1

Fig. 3 e Time killer kinetics of TEO nanoemulsion (A) A. flavus, (B) S. aureus, (C) E. coli (1) 0, (2) 5 min, (3) 15 min, (4) 30 min, (5)
45 min, (6) 60 min, (7) 75 min and (8) 90 min.
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Table 3 e Chemical composition of fresh labneh with different ratios of TEO nanoemulsion.
Treatments Dry matter Fat Fat/DM Protein Ash
Control 30.54A ± 0.045 9.50B ± 0.100 31.10C ± 0.330 11.92A ± 0.070 2.81A ± 0.100
T1 30.52A ± 0.155 9.50B ± 0.100 31.12C ± 0.330 11.92A ± 0.700 2.80A ± 0.045
T2 30.50A ± 0.100 9.53A ± 0.057 31.25B ± 0.105 11.90A ± 0.053 2.77A ± 0.160
T3 30.48B ± 0.140 9.57A ± 0.057 31.40A ± 0.100 11.88A ± 0.080 2.75B ± 0.055

Data expressed as (mean ± SD) of 3 replicates. Within the same column, means with different letters are significantly different (p  0.05).
Control: labneh without TEO nanoemulsion, T1: labneh with nanoemulsion solution contains 0.1% TEO, T2: labneh with nanoemulsion solution
contains 0.2% TEO, T3: labneh with nanoemulsion solution contains 0.3% TEO.

essential oils to their ability to enter through microbial cell
wall membranes and cytoplasmic membranes into the inside
cells and thus break the cell structures, rendering them more
permeable to the surrounding essential oils [58e61].
3.6. Chemical composition of UF labneh
This increase is related to the fat content of TEO nano-
emulsion and to decrease in dry matter in this treatment (T3).
This result agrees with El-Sayed et al. (2017) [25,26].
3.7. Chemical properties of UF labneh fresh and during
eight weeks of cold storage

Data presented in (Table 3) refers to the chemical composition
of fresh labneh with different ratios of TEO nanoemulsion.
The dry matter, fat, fat/dry matter (DM), protein, and ash
content of labneh treatments ranged from (30.48 to 30.54%),
(9.50 to 9.57%), (31.10 to 31.40%), (11.88 to11.92%), and (2.75 to
2.81%), respectively. Dry matter and ash content were signif-
icantly decreased in T3 which contains 0.3% TEO nano-
emulsion compared with other treatments T1, T2, and control
samples. The addition of TEO nanoemulsion had a significant
(p  0.05) effect on fat and fat/DM while it had no significant
(p  0.05) influence on protein content.
The fat and fat/DM content increased by rising the added
ratio of TEO nanoemulsion and the highest value was
observed in treatment contained 0.3% TEO nanoemulsion.
Table 4 displays the chemical properties of labneh prepared by
the addition of 0.0 (control), 0.1, 0.2, and 0.3% TEO nano-
emulsion during cold storage for eight weeks. The statistical
analysis illustrated that there were significant differences
(p  0.05) observed in the dry matter content between control
and labneh treatments except in fresh time. Dry matter con-
tent of labneh samples was in the range of 30.50, 30.52, 30.52,
and 30.54% for T3, T2, T1 and control samples respectively at
the fresh time of manufacturing, and then the dry matter
content increased to 31.22, 31.31,31.60, and 31.88% in the same
previous pattern at the end of the cold storage period. The
highest values of dry matter were noted in control samples in
fresh and after storage periods compared with other treat-
ments. Moreover during storage, dry matter content increased

Table 4 e Chemical properties of UF labneh fresh and during eight weeks of cold storage.
Parameters Storage period (weeks) Treatments
Control T1 T2 T3
Af Af Af Af
Dry matter (%) Fresh 30.54 ± 0.06 30.52 ± 0.16 30.52 ± 0.10 30.50 ± 0.14
First 30.78Ae ± 0.19 30.66Be ± 0.01 30.59Ce ± 0.21 30.55Ce ± 0.10
Second 30.88Ad ± 0.30 30.80Bd ± 0.16 30.70Cd ± 0.11 30.62Dd ± 0.10
Fourth 30.94Ac ± 0.31 30.91Ac ± 0.06 30.86Bc ± 0.17 30.73Cc ± 0.17
Sixth 31.32Ab ± 0.05 31.27Bb ± 0.58 30.93Cb ± 0.31 30.85Db ± 0.35
Eighth 31.88Aa ± 0.19 31.60Ba ± 0.12 31.31Ca ± 0.05 31.22Da ± 0.25
Acidity (%) Fresh 0.88Af ± 0.02 0.86Af ± 0.02 0.84Ae ± 0.02 0.84Ad ± 0.02
First 0.95Ae ± 0.02 0.90Be ± 0.02 0.88Bd ± 0.02 0.86Bd ± 0.02
Second 1.14Ad ± 0.05 1.05Bd ± 0.01 0.91Cd ± 0.05 0.88Cd ± 0.05
Fourth 1.20Ac ± 0.03 1.15Bc ± 0.01 0.96Cc ± 0.01 0.95Cc ± 0.01
Sixth 1.28Ab ± 0.03 1.22Bb ± 0.01 1.16Cb ± 0.02 1.11Db ± 0.03
Eighth 1.34Aa ± 0.02 1.27Ba ± 0.01 1.22Ca ± 0.02 1.17Da ± 0.02
pH Fresh 5.48Aa ± 0.10 5.50Aa ± 0.10 5.51Aa ± 0.10 5.52Aa ± 0.01
First 5.40Bb ± 0.05 5.46Ab ± 0.01 5.48Ab ± 0.10 5.50Ab ± 0.01
Second 5.29BCc ± 0.01 5.33Bc ± 0.10 5.36Bc ± 0.02 5.43Ac ± 0.10
Fourth 5.20Cd ± 0.015 5.26Bd ± 0.01 5.31Bd ± 0.01 5.38Ad ± 0.04
Sixth 5.11De ± 0.01 5.18Ce ± 0.01 5.25Be ± 0.01 5.30Ae ± 0.01
Eighth 5.05Df ± 0.05 5.12Cf ± 0.04 5.18Bf ± 0.03 5.25Af ± 0.02

Data expressed as mean of three replicates. Means between columns (effect of treatments) showing the same capital letters are not significantly
different (p  0.05). Means between rows (effect of storage) showing the same small letters are not significantly different (p  0.05). Control:
labneh without TEO nanoemulsion, T1: labneh with nanoemulsion solution contains 0.1% TEO, T2: labneh with nanoemulsion solution con-
tains 0.2% TEO, T3: labneh with nanoemulsion solution contains 0.3% TEO.
1036 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1

Fig. 4 e A. Lactic acid counts of UF labneh samples during storage period. B. Mesophilic bacterial counts of UF labneh
samples during storage period.
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1 1037

Table 5 e Microbiological quality of UF labneh during storage.

Microbiological content Storage period (Weeks) Treatments
Control 1 2 3
Psychrotrophic bacterial counts Fresh N.D N.D N.D N.D
First N.D N.D N.D N.D
Second N.D N.D N.D N.D
Fourth 2.43Ca ± 0.313 N.D N.D N.D
Sixth 4.00Ba ± 0.152 1.81Bb ± 0.236 1.66Bb ± 0.152 1.16Bc ± 0.115
Eighth 4.80Aa ± 0.104 2.50Ab ± 0.167 2.10Bb ± 0.124 1.20Bc ± 0.154
Mold and yeast counts Fresh N.D N.D N.D N.D
First N.D N.D N.D N.D
Second N.D N.D N.D N.D
Fourth 2.19Ca ± 0.170 N.D N.D N.D
Sixth 3.74Ba ± 0.233 N.D N.D N.D
Eighth 4.88Aa ± 0.325 1.36Ab ± 0.268 1.10Ab ± 0.228 N.D

Data expressed as mean of three replicates. Means between columns (effect of treatments) showing the same capital letters are not significantly
different (p  0.05). Means between rows (effect of storage) showing the same small letters are not significantly different (p  0.05). Nil: not found
counts. Control: labneh without TEO nanoemulsion, T1: labneh with nanoemulsion solution contains 0.1% TEO, T2: labneh with nanoemulsion
solution contains 0.2% TEO, T3: labneh with nanoemulsion solution contains 0.3% TEO. N.D: Non detectable.

significantly (p  0.05) due to moisture loss which is associated
with natural evaporation [62]. Also, the total acidity and pH
values of the labneh samples during eight weeks of cold
storage are shown in Table 4. There were no significant dif-
ferences (p  0.05) observed in the total acidity and pH values
between treatments in fresh time. The total acidity values
between all treatments of prepared labneh significantly
increased throughout the storage period. The highest value of
total acidity was observed in control samples, whereas the
lowest values were recorded in T3 which prepared by the
addition of 0.3% TEO nanoemulsion. This mainly may be
related to the growing number of lactic acid bacteria in the
control samples. Similarly, Habib et al. (2014) [63] suggesting
that the total viable count and starter culture may be influ-
enced by added the essential oil into the product.
Contrary to that the pH values significantly (p  0.05)
decreased in control samples and the labneh prepared with
TEO nanoemulsion (T1, T2, and T3) during the storage period.
Furthermore, significant differences were noted between the
control sample and the other treatments, the pH of the control
sample had the lowest values ranged from 5.48 to 5.05
compared with the labneh preserved with TEO nanoemulsion.
Moreover, the pH values were increased significantly (p  0.05)
by raising the ratio of TEO nanoemulsion. The results are
disagreement with the opinion of Shan et al. (2011) [64] and El-
Sayed et al. (2020) [65] who reported that the natural herbal,
extracts, and oils involved high levels of phenolic compounds
were caused principally of the low pH in cheese.
3.8. Assessment of UF labneh bacteriological quality
during cold storage for eight weeks
3.8.1. Counts of lactic acid bacteria
Data presented in (Fig. 4A) mentions to the lactic acid counts
of labneh with different ratios of TEO nanoemulsion. Total
lactic acid counts significantly (p  0.05) increased in the first
weeks of storage to range between 10.05 and 9.43 log CFU/g
and remained in the same log for two weeks. In the fourth
week, the viable counts of lactic acid significantly (p  0.05)
decreased in all samples, which the counts ranged between
8.75 and 8.31 log CFU/g. Moreover, at the end of storage, the
viable counts were decreased but in the safe level for lactic
acid and probiotics to donate a healthy effect to the human.
Which the counts were ranged between 7.37 and 7.16 log CFU/
g at eight weeks of storage. Also, this data exposed that the
used TEO nanoemulsion concentration has a slight effect on
the viability of lactic acid bacteria in labneh during storage.
According to Gammariello et al. (2008) [66] informed that TEO
limited the microorganism’s growth responsible for milk
spoilage without affecting the dairy useful bacteria. Assem
et al. (2019) [67] found that eugenol extracted from extracted
from carnation calli did not have any effect on lactic acid
bacteria.
3.8.2. The mesophilic bacterial counts
Increasing TEO nanoemulsion in the UF labneh amount
significantly (p  0.05) affected in mesophilic bacterial counts
as (Fig. 4B). Initial total bacterial count in labneh was about
4.64e4.19 log CFU/g and then continuously reduced to reach
2.26, 2.13 and 2.03 log CFU/g using amount 0.1, 0.2 and 0.3% of
TEO nanoemulsion, respectively at the eight weeks of storage,
in compared with control (5.49 log CFU/g). These findings
indicate that TEO nanoemulsion has proven to be unusually
effective on mesophilic bacterial counts in labneh. According
to Ben Jemaa et al. (2016) [68] by adding TEO or its nano-
emulsion, bacterial growth in various contaminated milk has
been controlled and improved microbiological and chemical
properties. Also, S‚ahan et al. (2004) [69] indicated the total
bacterial counts gradually decreased in labneh during storage
using vegetable oil.
3.8.3. Microbiological quality of UF labneh
Quality of labneh was indicated by psychrotrophic bacterial
and mold and yeast counts during storage (Table 5). It is
1038 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1

evident, that the production of labneh by UF-retentate, is best
to apply for industrial-scale because this procedure declines
the handling of equipment and improve the keeping quality of
labneh, and this clarifies why the shelf life of control labneh
exceed two weeks. Consequently, during the first two weeks
did not detect any psychrotrophic bacterial counts. But, in the
fourth week was detected little numbers of psychrotrophic
bacteria in control (2.43 log CFU/g) and with the storage period,
the psychrotrophic bacteria were proliferated in control. Also,
the psychrotrophic bacterial counts were detected in other
treatments in the sixth week. The viable psychrotrophic bac-
terial counts were reached to 4.80, 2.50, 2.10 and 1.20 log CFU/g
for control, T1, T2, and T3, respectively at the end of the
storage period.
The same trend of results was observed for mold and
yeast counts (Table 5), which in the first two weeks did not
detect mold and yeast counts. In the fourth week were
appeared mold and yeast counts in the control sample (2.19
log CFU/g). This small count was proliferated during storage
to reached 4.88 log CFU/g in the eighth week for the control
sample. Moreover, the counts of mold and yeast were
detected in T1 and T2 and reached 1.36 and 1.10, respec-
tively at the end of the storage period. Furthermore, during
the storage period, T3 was free from mold and yeast counts.
These results due to the preservatives effect of TEO nano-
emulsion. These results are in perfect agreement with the
study of Moro et al. (2013) [48] who established that TEO has
suitable antimicrobial activity against pathogens and
spoilage microbe associated with cheese contamination.
Similarly, Zantar et al. (2013) [70] noted that using 0.05% TEO
from the first week of production can inhibit mold and yeast
counts in flavored goat cheese.

Table 6 e Sensory evaluation of UF labneh samples during eight weeks of cold storage.
Character Treatments
Assessed C T1 T2 T3
Fresh
C&A (9) 7.55Ba ± 0.48 7.57Ba ± 0.33 8.30Aa ± 0.75 8.50Aa ± 0.33
B&T (9) 7.58Ab ± 0.75 7.21Ab ± 0.80 7.08Ac ± 1.50 6.57Bc ± 1.60
O (9) 7.11Ab ± 0.66 6.82Ab ± 0.80 5.23Bb ± 1.50 5.02Bb ± 1.50
T (9) 7.20Aa ± 1.50 6.91Ab ± 0.48 5.73Bb ± 0.80 5.43Bb ± 1.60
OA (9) 7.43Ab ± 0.62 6.88Bb ± 0.45 5.42Cb ± 0.35 5.21Cb ± 0.48
Week
C&A (9) 7.34Ba ± 1.33 7.46Ba ± 1.50 8.04Aa ± 0.39 8.21Aa ± 0.75
B&T (9) 7.93Ab ± 0.48 7.52Bb ± 0.80 7.17Bb ± 0.51 6.89Cb ± 0.75
O (9) 7.34Ab ± 0.80 6.93Bb ± 0.54 5.36Cb ± 0.48 5.18Cb ± 1.75
T (9) 7.41Aa ± 1.60 6.99Bb ± 1.50 5.80Cb ± 1.75 5.54Cb ± 1.60
OA (9) 7.56Aa ± 0.80 7.07Ba ± 0.35 5.63Cb ± 0.39 5.52Cb ± 0.51
2 weeks
C&A (9) 7.21Ba ± 0.40 7.30Aa ± 0.33 7.74Aa ± 0.40 8.15Aa ± 0.48
B&T (9) 8.14Aa ± 0.48 7.89ABb ± 0.75 7.51Bb ± 0.35 7.32Bb ± 0.75
O (9) 7.66Aa ± 0.75 7.23Aa ± 0.48 5.56Bb ± 0.40 5.29Bb ± 1.50
T (9) 7.45Aa ± 0.55 7.26Aa ± 0.40 5.88Bb ± 0.75 5.61Bab ± 0.80
OA (9) 7.76Aa ± 0.75 7.11Ba ± 0.35 5.84Ca ± 0.80 5.67Ca ± 0.80
4 weeks
C&A (9) 6.11Cb ± 0.48 7.04Bb ± 1.33 7.32ABb ± 1.60 7.75Ab ± 1.33
B&T (9) 8.58Aa ± 0.62 8.22Aa ± 1.50 8.06Aba ± 0.48 7.85Ba ± 1.60
O (9) 7.98Aa ± 1.75 7.39Aba ± 1.60 5.73Ba ± 1.50 5.34Ba ± 1.50
T (9) 7.51Aa ± 0.40 7.35Aa ± 0.48 5.94Bb ± 1.50 5.78Ba ± 1.50
OA (9) 7.98Aa ± 0.48 7.23Aba ± 1.33 5.95Ba ± 0.35 5.73Ba ± 0.62
6 weeks
C&A (9) e 6.60Bb ± 0.50 7.13Ab ± 0.80 7.56Ab ± 0.66
B&T (9) e 8.43Aa ± 0.48 8.26Aa ± 0.62 8.15Aa ± 0.75
O (9) e 7.54Aa ± 0.80 5.92Ba ± 0.75 5.63Ba ± 0.80
T (9) e 7.44Aa ± 0.62 6.23Ba ± 0.48 5.94Ba ± 0.62
OA (9) e 7.31Aa ± 0.35 6.13Ba ± 0.33 5.81Ba ± 0.44
8 weeks
C&A (9) e 6.20Bb ± 0.75 6.82Ac ± 0.80 7.08Ac ± 0.62
B&T (9) e 8.65Aa ± 0.75 8.44Aa ± 0.66 8.32Aa ± 0.48
O (9) e 7.72Aa ± 0.48 6.09Ba ± 0.39 5.82Ba ± 0.62
T (9) e 7.58Aa ± 0.62 6.53Ba ± 0.60 6.05Ba ± 0.50
OA (9) e 7.47Aa ± 0.33 6.32Ba ± 0.60 5.95Ba ± 0.50

C&A: Color and Appearance B&T: Body and Texture O: Odor T: Taste OA: Overall Acceptability.
Data expressed as mean of three replicates. Means between columns (effect of treatments) showing the same capital letters are not significantly
different (p  0.05). Means between rows (effect of storage) showing the same small letters are not significantly different (p  0.05). Control:
labneh without TEO nanoemulsion, T1: labneh with nanoemulsion solution contains 0.1% TEO, T2: labneh with nanoemulsion solution con-
tains 0.2% TEO, T3: labneh with nanoemulsion solution contains 0.3% TEO.
 j o u r n a l o f m a t e r i a l s r e s e a r c h a n d t e c h n o l o g y 2 0 2 1 ; 1 0 : 1 0 2 9 e1 0 4 1
3.9. Sensory evaluation of UF labneh during cold storage
for eight weeks
The scores recorded for color & appearance, body & texture,
odor, taste, and overall acceptability displayed that the addi-
tion of TEO nanoemulsion to UF labneh considerably affected
the sensory characteristics (Table 6). There is a significant
difference in the mean color & appearance scores between
labneh treatments and throughout the storage period. The
control samples had a clean natural white color moreover;
other treated samples presented a more bright white color
increased with an increasing percentage of TEO nano-
emulsion. Furthermore, the results confirmed that there was
homogeneity and velvety feeling in color and appearance
scores in labneh treated by TEO nanoemulsion compared to
control samples this is in agreement with their mean scores as
shown in Table 6, which is in accord with the finding of Liu
et al., (2019) [71].
Body and texture differed slightly among all the treatments
and during the storage period and this may be reflected the
influence of total solids and the acidity content on the body
and texture of labneh as effective values, as it was confirmed
by the results of Nsabimana et al. (2005) [72]. The highest
values of body and texture were observed in the control
samples when fresh and after four weeks, followed by the
labneh preserved by 0.1, 0.2, 0.3% TEO nanoemulsion respec-
tively after eight weeks of stored.
Among the tested sensory attributes, odor and taste scores
of labneh treatments decreased with increasing ratio of TEO
nanoemulsion whereas during storage all labneh treatments
improvement significantly specifically after two weeks of
storage, this is in agreement with the finding of Ghalem and
Zouaoui (2013) [73]. T2 and T3 are significantly very low for
odor and taste scores when fresh and after eight weeks of
storage compared to other treatments (control and T1) which
averagely displayed acceptable and good taste Table 6.
In this work, increasing the ratio of TEO nanoemulsion
might have led to lower overall acceptability of labneh
whereas, the overall acceptability of all treatment enhance-
ment during the storage period. The labneh preserved by 0.1%
TEO nanoemulsion and stored for eight weeks displayed
higher overall acceptability comparing with the other treated
samples. Also, after four weeks of stored period yeasts and
molds are seen in the surface of control labneh samples.
4. Conclusion
Modern food science recommended the involvement of the
ingredients that have natural antimicrobial activities to in-
crease the shelf life of some food products. Based on these
results, it can be concluded that T. vulgaris essential oils
fabricated in nanoemulsion form showed excessive inhibitory
action against pathogenic and spoilage strains. Moreover, the
addition of TEO nanoemulsion to UF labneh as a preserved
agent had a significant effect on chemical, microbiological,
and sensory properties. The TEO nanoemulsion at 0.1% has
shown to prolong the shelf life up to 6 weeks at cold storage of
UF labneh, with acceptable taste and aroma.
1039
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.
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