Optimizing the extraction of essential oil from cinnamon leaf (Cinnamomum verum) for use as a potential preservative for minced beef

Abstract

Cinnamon leaf essential oil extraction using steam distillation method is a time-consuming and energy-intensive process. Furthermore, a lower yield and a higher rate of product degradation are this method’s main drawbacks. Thus, the goal of this research is to optimize the extraction process parameters of cinnamon leaf essential oil in response to maximizing the yield while retaining quality by using response surface methodology (RSM). The application of extracted essential oil on minced beef to assess its preservative effect was also the other objective of this research. Extraction time (120–210 min), extraction temperature (105–115 ℃), and feed mass (300–600 g) were the chosen independent variables of the optimization experiment using central composite design (CCD). Furthermore, the extracted essential oil’s antibacterial and microbiological preservative activity on minced beef was evaluated. At extraction time of 175.43 min, extraction temperature of 105 °C, and a feed mass of 600 g, the optimum predicted value of cinnamon leaf essential oil yield and cinnamaldehyde concentration (% area) was 2.9% and 34.6%, respectively. Moreover, the second-order polynomial equation fits the experimental data for 20-run experimental data. The chemical composition of cinnamon leaf essential oil extracted at optimal conditions was dominated by eugenol (60.68%) and cinnamaldehyde (33.94%). Additionally, the optimally extracted cinnamon essential oil inhibited the growth of bacteria, particularly gram-positive bacteria. After twenty-one days of storage at 4 °C, total viable count of minced beef seasoned with cinnamon essential oil at concentration of 1.2% (v/v) was lower than 106 CFU/g. To conclude, optimized cinnamon leaf essential oil extraction process provides better yield while retaining its functional properties.

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
https://doi.org/10.1186/s13765-023-00798-y
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Optimizing theextraction ofessential oil
fromcinnamon leaf (Cinnamomum verum)
foruse asapotential preservative forminced
beef
Reta Merid Yitbarek1,2, Habtamu Admassu3,4* , Fekiya Mohammed Idris3,4 and Eskindir Getachew Fentie3,4*
Abstract
Cinnamon leaf essential oil extraction using steam distillation method is a time-consuming and energy-intensive
process. Furthermore, a lower yield and a higher rate of product degradation are this method’s main drawbacks. Thus,
the goal of this research is to optimize the extraction process parameters of cinnamon leaf essential oil in response
to maximizing the yield while retaining quality by using response surface methodology (RSM). The application
of extracted essential oil on minced beef to assess its preservative effect was also the other objective of this research.
Extraction time (120–210 min), extraction temperature (105–115 ℃), and feed mass (300–600 g) were the cho-
sen independent variables of the optimization experiment using central composite design (CCD). Furthermore,
the extracted essential oil’s antibacterial and microbiological preservative activity on minced beef was evaluated. At
extraction time of 175.43 min, extraction temperature of 105 °C, and a feed mass of 600 g, the optimum predicted
value of cinnamon leaf essential oil yield and cinnamaldehyde concentration (% area) was 2.9% and 34.6%, respec-
tively. Moreover, the second-order polynomial equation fits the experimental data for 20-run experimental data.
The chemical composition of cinnamon leaf essential oil extracted at optimal conditions was dominated by euge-
nol (60.68%) and cinnamaldehyde (33.94%). Additionally, the optimally extracted cinnamon essential oil inhibited
the growth of bacteria, particularly gram-positive bacteria. After twenty-one days of storage at 4 °C, total viable count
of minced beef seasoned with cinnamon essential oil at concentration of 1.2% (v/v) was lower than 106 CFU/g. To
conclude, optimized cinnamon leaf essential oil extraction process provides better yield while retaining its functional
properties.
Keywords Essential oil, Extraction time, Extraction temperature, Feed mass, Antibacterial, Antioxidant, Minced beef
*Correspondence:
Habtamu Admassu
hadtess2009@gmail.com
Eskindir Getachew Fentie
eskindir.getachew@aastu.edu.et; eskench@gmail.com
1 Department of Chemical Engineering, Collage of Biological
and Chemical Engineering, Addis Ababa Science and Technology
University, Addis Ababa, Ethiopia
2 Department of Food Engineering, Collage of Engineering and Natural
Science, Debre-Berhan University, Debre-Berhan, Ethiopia
3 Department of Food Process Engineering, Collage of Biological
and Chemical Engineering, Addis Ababa Science and Technology
University, Addis Ababa, Ethiopia
4 Biotechnology and Bioprocessing Center of Excellence, Addis Ababa
Science and Technology University, Addis Ababa, Ethiopia

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
Introduction
Essential oils are concentrated hydrophobic liquid aro-
matic and volatile compounds generated from the cyto-
plasmic fluid of the plant and found as small droplets
located at the intracellular space [1]. For the plant, the
major functions of these oils are either to protect it from
pests and/or predators, or to facilitate plant-to-plant
communication [2]. For a variety of uses, these essen-
tial oils have been utilized for millennia. ey have been
utilized as food additives, perfumery scents, and active
medicinal compounds. From the nearly 3000 plant spe-
cies that have been studied for the composition of their
essential oils up to this point, hundreds have been used
commercially [2].
Cinnamon bark and leaves are frequently utilized to
make essential oils for a variety of applications [3, 4].
ere are over 250 species in the genus Cinnamomum,
with C. verum being the most widely utilized species for
the production of essential oils. Besides, the use of C.
verum essential oil for medical and food additives is a
long-standing tradition [5, 6]. Up to 124 compounds were
identified from essential oil extracted from C. verum [7].
From these compounds, eugenol, cinnamaldehyde, and
camphor are the principal compound found in the essen-
tial oils extracted from leaves, barks and root-bark cin-
namon plant [5, 8, 9]. ese compounds had shown in
the previous studies for their good anti-oxidant, and anti-
microbial effect [3, 10].
Essential oils from aromatic plants can be extracted by
either conventional (hydro-distillation, steam distillation,
solvent extraction etc.) or advanced (supercritical fluid
extraction, microwave-assisted extraction, microwave-
assisted extraction, microwave steam distillation etc.)
techniques [11, 12]. Each of these extraction methods,
however, has advantages and disadvantages of their own.
e most well-known drawbacks of the traditional essen-
tial oil extraction method include the lengthy extraction
period (3–6h), destruction of some compounds that are
temperature-sensitive, simultaneous extraction of other
components, and large solvent residues [13]. Although
advanced extraction techniques are thought to be a use-
ful tool for overcoming the drawbacks of traditional
methods, they are not widely used for the commercial
manufacture of essential oils due to their high initial
investment cost and the requirement of sophisticated
technologies. us, the parameters that guarantee a good
process yield and excellent quality must be in place for
the production process of essential oils using conven-
tional extraction procedures [3].
Due to its low initial cost and ongoing operating
expenses as well as its environmentally friendly approach,
steam distillation extraction is by far the most often used
extraction technology [14]. is extraction method still
has the disadvantage of having a lesser yield of essen-
tial oil. is lower yield is essentially what accounts for
the high price of essential oils. Additionally, essential
oil produced by steam distillation may degrade princi-
pal compounds particularly for those compounds that
are temperature-sensitive. As a result, research is still
being done to determine how to increase yield without
compromising quality of the extracted essential oil [5,
15]. Raw materials quality (plant age, plant part), pre-
treatments (sun, and shade dried), particle size, mass of
feed, and process parameters (extraction time, tempera-
ture, steam flow rate, cooling water flow rate, etc.) are
the main factors that need to be studied, monitored, and
optimized during the steam distillation process in order
to get a better yield and good quality essential oils [16].
Prior studies mainly concentrated on the volatile
compound profiling [4, 17], biological activity [4, 6, 18],
impact of cultivation on chemical composition [10], com-
parison of various extraction techniques [19], and medic-
inal and therapeutic usage of cinnamon essential oil [20].
Besides, process parameter optimization for the hydro
and steam distillation extraction of Cinnamon essential
oil [3, 21] were carried out for limited independent vari-
ables. To the best of our knowledge, no studies have been
conducted to determine how the extraction temperature,
time, and mass of the feed affect the yield and quality of
cinnamon leaf essential oil. Additionally, the use of this
essential oil as a food preservative against microbial
spoilage is an unanswered question that requires further
research. us, the main aim of this study was to opti-
mize the key steam distillation process parameters that
affect the extraction of essential oil from C. verum leaf
material. Furthermore, elucidating the effect of cinna-
mon essential oil concentration effects on the microbio-
logical shelf stability of minced beef was another goal of
this research.
Materials andmethods
Raw material collection, pretreatment andstorage
Fresh cinnamon leaf (Cinnamomum verum Cin.5/82) was
collected from Bebeka Bench Sheko, South West Ethiopia
Peoples’ Region, Ethiopia (6°53ʹ01.4ʺN, 35°25ʹ41.9ʺE).
e botanical identification of the sample was previously
performed at the Wondo-Genet Agricultural Research
Center, under registration number Cin. 5/82. Fresh cin-
namon leaves were first washed with tap water to remove
any traces of dust and debris. en, the leaves were shade
dried for 21 days at room temperature (24 ± 2°C) until
the moisture content is reduced from 44.42 ± 0.03 to
10.32 ± 0.02%. Finally, it was packed in polyethylene plas-
tic and stored at room temperature until further analysis
were carried out.

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
Steam distillation
Shade dried cinnamon leaves were first placed in a 5L
distillation biomass flask. Externally generated steam was
then ejected to the bottom of two serially arranged bio-
mass flasks, the top of which held the ready-to-extract
sample (Fig.1). As the steam passes through the biomass
flasks, the essential oil and water vapor rise to the con-
denser, where they change phases from gas to liquid and
are dropped back into the separatory funnel, which is
located directly beneath the condenser. After the extrac-
tion time was completed, water was drained from the
separatory funnel to separate the visible layer of water
from the extracted oil. e oil was then dried further by
filtering it through anhydrous sodium sulfate. Finally, the
obtained essential oils were stored in amber glass bottle
at the refrigerator (4°C) until further analysis.
e extracted essential oil yield was then calculated
using Eq.1
Process optimization
Response surface methodology (RSM) was used to study
the effect of extraction time (A), temperature (B), and
feed mass (C) on extraction yield (Y1) and cinnamalde-
hyde concentration (Y2). RSM design with the uncoded
and coded levels are tabulated in Table1. Central com-
posite design (CCD) was used to design the experiment
which gives a total of twenty randomized treatments,
including eight fractional factorial points, six axial points,
and six central points.
Volatile compound analysis
e volatile aromatic component profile of extracted cin-
namon essential oil was carried out utilizing gas chro-
matography tandem with mass spectrometry (Agilent,
7890B/G7038A GC/MS System) methods adapted from
[22]. Briefly, 1µL of essential oil sample was ejected to
GC capillary column (HP-5MS UI, 30 m, 0.250 mm,
0.25μm) with the help of auto sampler and separation
was facilitated using helium as carrier gas at flow rate
of 1.0 mL/min. e oven temperature of GC was pro-
grammed as follows: 120 ℃for 3 min isothermal, 120–
260 ℃with a rate of 10 oC /min, 8min isothermal. e
quadrupole mass spectrometer was operated at a split
ratio of (1:20), and the acquisition scan ranged was set
from 50 to 550m/z within electron impact mass spectra
of 70eV ionization energy. e final identification of vol-
atile compounds was performed by comparing retention
indices and mass spectra with the GCMS library (NIST)
and literature.
Total phenolic content
e total phenolic content for each extract was deter-
mined spectrophotometrically using Folin–Ciocal-
teu (FC) procedure as described in [23]. About 100µL
of extracted Cinnamon essential oil was mixed with
100µLof methanol, and 0.1 mL of mixture was trans-
ferred into a volumetric flask and diluted with 0.5mL of
deionized water. Test tubes were filled with 0.2 mL of the
extract and 0.5 mL of Folin-reagent. Ciocalteu’s (diluted
(1)
Yeild
(%)=
Mass of extracted oil (g)
Intial plant biomass (g) *100
Fig. 1 Extraction of cinnamon essential oil via steam distillation
with an experimental setup comprising (1) Condenser, (2) Separatory
funnel, (3) Separatory funnel valve, (4) Steam generator, (5) Sample
holder biomass flask, and (6) Steam injection biomass flask
Table 1 The five levels of the independent variables used in central composite design
Symbol Coded level
−α −1 0 + 1 +α
Time (min) A 89.319 120 165 210 240.88
Temperature (oC) B 103.296 105 110 115 111.704
Feed mass (g) C 401.928 300 450 600 100.928

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
with water 1:10). After being held in the dark for 5min,
the solution received 1 mL of sodium carbonate (7.5%
w/v). e tubes were once more maintained in the dark
for an hour with parafilm on top. Using a spectropho-
tometer UV-vis (Jasco V-530), absorption at 765nm was
measured and compared to a calibration curve for gallic
acid. e gallic acid standard reference curve was con-
structed for the following concentrations, in turn: 0, 20,
40, 60, and 80µg mL− 1. e outcomes were given in mg
gallic acid/g of dried material.
DPPH radical scavenging
Using the stable radical DPPH, the antioxidant activ-
ity was determined in terms of its capacity to donate
hydrogen or scavenge free radicals. e Blois, (1958)
method was employed to conduct the experiments [24].
e rationale behind this colorimetric assay is that as the
radical concentration in a solution decreases, so does
the absorbance at 517nm. Two mL aqueous methanolic
stock solution of cinnamon essential oil extract and 2
mL of a 1 mM DPPH solution was added to a test tube.
e tube was then maintained in the dark for an hour
with parafilm on top. Finally, spectrophotometer UV-
vis (Jasco V-530) was used to measure the absorbance at
517nm wave length and compare the results to a calibra-
tion curve for ascorbic acid. e assay was performed in
triplicate. e inhibition percentage of the DPPH radical
was calculated using the Eq.2.
where I = DPPH inhibition (%), A0 = absorbance of con-
trol sample (t = 0h) and A = absorbance of a tested sam-
ple at the end of the reaction (t = 1h).
Antibacterial activity test
e Agar well diffusion assay was used to assess the anti-
bacterial activity of the cinnamon leaf essential oils [25].
A total of five most common foodborne bacterial path-
ogens (Staphylococcus aureus (ATCC 25,923), Listeria
monocytogenes (ATCC 19115), Escherichia coli (ATCC
25922), Pseudomonas aeruginosa (ATCC 27,853), Acine-
tobactor baumannii (ATCC 19606)) were first obtained
from Ethiopian Public Health Institute (EPHI), Addis
Ababa, Ethiopia. Briefly, each bacterium was first sub-
cultured in nutrient agar at 37 ℃ for 24h. Hundred µL
of standardized inoculums of the test microorganisms
were spread over sterile Muller-Hinton Agar for bacte-
ria. Agar was then cut into 8mm diameter wells with a
sterile cork-borer, and 100 µL of the essential oils with
different concentrations were then placed into different
well. e plates were first incubated at room temperature
(2)
I
(%)=
A
0
-A
A0
*100
for one hour to ensure appropriate oil diffusion into the
agar, and then at 37 ℃for 24h for three days. Each assay
was performed in triplicates. As Ashraf etal. had done
[26], the inhibition zone was eventually determined in
millimeters.
Storage stability test
e methods used by Burt [27] were utilized to analyze
the microbiological shelf stability of minced beef sam-
ples. Ten samples of each treatment were prepared by
mixing 50g of minced beef with various cinnamon essen-
tial oil concentrations. e treated samples were then
packed in sterile polyethylene bags and stored at refrig-
erated (4°C) condition. During the time series analysis,
a ten-gram sample from each treatment was hygienically
taken and added to stomacher bags containing 90 mL of
0.1% saline water for thorough homogenization for about
one minute at 25°C. Serial dilutions of each sample were
then created in 0.1% saline water, and a duplicate 1 ml
sample of each dilution was spread into Plate Count Agar
(PCA). e total viable bacteria were then enumerated
after incubation at 37 ℃ for 48h. Microbial colonies were
enumerated as total viable count for the plats that had
colonies of between 30 and 300.
Statistical analysis
Design Expert Software (V. 13) was used to statistically
assess the experimental data. e best-fitting polynomial
model was chosen by comparing a number of statistical
metrics (lack-of-fit, coefficient of variation, and predicted
and adjusted correlation coefficients). Furthermore, the
analysis of variance of significant differences were iden-
tified by computing the F-value for the probabilities of
0.01, 0.1, and 0.5.
Result anddiscussion
Optimization ofcinnamon leaf essential oil extraction
Steam distillation method is widely used for extract-
ing essential oils from various plant sources due to their
economic feasibility issues. However, it has limitations
in terms of product quality degradation, particular for
temperature sensitive active ingredients. Besides, extrac-
tion of essential oil from the plant source using steam or
hydro-distillation methods is time and energy consum-
ing process. Basically, if steam distillation is used as an
extraction technique, finding quality steam at a lower
temperature is very challenging unless another pres-
sure-reducing system is employed. As a result, further
lowering of the temperature during conventional steam
distillation is nearly impossible. However, by reducing
exposure time, and contact surface between the steam
and the plant material, acceptable essential oil quality can
be maintained. ese issues could be partially addressed

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
by manipulating the extraction process parameters. us,
in this study, RSM optimization technique was applied
for tweaking independent extraction parameters with an
objective to achieve a higher yield without major com-
promisation on the quality of the extracted essential oil.
However, essential oils extraction using stream distilla-
tion is one of the complex processes which applies both
heat and mass transfer unit-operations. us, to opti-
mize and predict such process using white-box model is
very complicated task. Rather, with all due its limitations,
selecting and optimizing the influential process param-
eters for maximizing the yield and quality of essential oil
using RSM is the cost-effective alternative means [28].
It is a mathematical and statistical technique for study-
ing and optimizing multivariable systems by determining
the relationship between the set of independent variables
and the response. Extraction time, temperature, and feed
mass were the independent variables optimized in this
study for maximizing yield and quality. ese parameters
had shown a significant effect on the aforementioned
response for both steam and hydro-distillation extrac-
tion process. Particularly, increasing extraction time and
temperature had shown an increase in the yield of essen-
tial oil [29]. Basically, cinnamaldehyde and eugenol were
major compounds found in all cinnamon leaf extract.
Nevertheless, in this study, the relative concentration
of eugenol was almost similar for all treatment samples.
us, cinnamaldehyde was used to compare the quality
of the extracted cinnamon leaf essential oil. e effect of
independent variables on the response (experimental and
predicted) values are tabulated in Table2.
Inuence ofindependent parameters ontheessential oil yield
Taking other variables into account, each independent
variable had a significant (P < 0.001) effect on the extrac-
tion yield of cinnamon leaf essential oil. Extraction time,
one of the independent variables, had a significant effect
on cinnamon leaf essential oil yield at both the linear and
quadratic levels (Table3). is could be explained by an
increase in mass transfer, which will allow the system to
approach equilibrium as the extraction time increases.
Similarly, both the linear and quadratic levels of extrac-
tion temperature showed a significant effect on essential
oil yield. is could be because the extraction tempera-
ture facilitates rapturing the plant structure to release the
essential oil, which improves the rate of diffusion during
the extraction process [30]. Feed mass had also a sig-
nificant effect on the extraction yield both at linear and
quadratic level (Table3).
Predicted response for the cinnamon essential oil yield
was expressed by second-order polynomial regression
equation in terms of coded values (Eq.3):
Table 2 Experimental and predicted values of extraction yield and Cinnamaldehyde concentration
Run Time (min) Temperature
(℃)Feed Mass (g) Yield (%) Cinnamaldehyde (%)
Experimental Predicted Experimental Predicted
1 120 115 300 2.41 2.46 26.00 26.03
2 165 110 197.731 2.24 2.19 25.87 25.52
3 165 110 450 2.98 3.07 25.93 25.77
4 210 115 300 2.49 2.55 26.5 26.71
5 240.681 110 450 3.14 3.03 25.97 25.69
6 165 101.591 450 2.49 2.46 35.41 34.44
7 89.3193 110 450 2.61 2.59 28.31 27.52
8 165 110 702.269 2.98 2.90 33.31 32.59
9 120 105 300 2.14 2.13 26.85 27.64
10 120 115 600 2.80 2.84 26.40 26.98
11 165 110 450 3.10 3.07 25.86 25.77
12 120 105 600 2.51 2.54 36.33 36.88
13 165 110 450 3.10 3.07 25.23 25.77
14 210 115 600 2.89 2.99 25.89 25.86
15 165 118.409 450 2.84 2.74 26.32 26.22
16 165 110 450 2.99 3.07 26.21 25.77
17 210 105 600 2.94 2.98 33.31 34.04
18 165 110 450 3.11 3.07 25.21 25.77
19 210 105 300 2.45 2.51 26.41 26.59
20 165 110 450 3.11 3.07 25.99 25.77

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
Where: A = Extraction time temperature, B = Extrac-
tion temperature, and C = Feed mass .
Statistical analysis of variance (ANOVA) revealed
that, the experimental data could be best represented by
quadratic polynomial model with coefficient of determi-
nation (R2) values of 0.9809 for cinnamon essential oil
yield (Table3). is R2 values, which was closed to unity,
indicate that our quadratic polynomial model adequately
describes the system. e significance level for quadratic
polynomial model coefficients tabulated in Table3 was
calculated using analysis of variance (ANOVA).
To better visualize the interaction effect of independ-
ent variables on the yield of cinnamon leaf essential oil,
3D plots were employed, with one independent variable
held constant while the other two variables were var-
ied (Fig.2). When the input mass was modest (300 g),
increasing the extraction time resulted in an increase in
extraction yield. is positive effect could be due to the
creation of an environment that results in increased con-
tact time between the solvent and the solute. e same
is true for biomass increase, even at shorter extraction
time (120 min). e combination of time and biomass
feed generates a high essential oil yield (Fig.2). Nonethe-
less, a slight decrease in essential oil yield was observed
for the combination of longer extraction time and higher
feed mass. is could be due to the increased mass trans-
fer resistance caused by the higher feed mass. Similarly,
at low temperatures (105°C), increasing the extraction
(3)
Yield
=−
89.311
+
0.0516A
+
1.5355B
+
0.0097C
−0.0003AB +0.0002AC −0.0001BC
−0.0001A
2
−0.0066B
2
−0.0001C
2
time resulted in a higher extraction yield. Higher extrac-
tion temperature at a lower extraction time resulted in an
increase in essential oil production (120min). is could
be attributed to higher extraction temperatures causing
greater disruption of plant cells. e combined incre-
ment of time and temperature had shown high essential
oil yield. However, longer extraction time and higher
temperature showed a trivial decreases in the cinnamon
essential oil yield (Fig.2B). A loss of some essential oil
in the condenser due to solute-containing steam flow
could explain the relatively lower essential oil at longer
time and higher temperature extraction conditions. e
increase in feed mass and temperature had also resulted
in an increase in the yield of cinnamon essential oil
(Fig.2C). However, increasing feed mass and tempera-
ture resulted in a drop in essential oil yield. is could
be owing to higher feed mass mass transfer resistance or
higher steam temperature, which could cause a loss of
volatiles. During the study of optimizing oil and pectin
extraction from orange peels, Fakayode and Abobi, [31]
also discovered a similar result.
e perturbation plots were used to compare the
effects of extraction time, temperature and feed mass at
a particular point in the design space (Fig.3A). e plot
is specifically used to demonstrate the yield of cinnamon
essential oil yield by varying only one independent vari-
able over its range while holding all the other variables
constant. By default, Design-Expert sets the reference
at the midpoint (i.e. 165min, 110 ℃ and 450g) of all the
independent variables. As the extraction time, tempera-
ture and feed mass were increased the extraction yield
also increased, and vice versa. e feed mass, in particu-
lar, showed a greater increase and reduction in essential
oil yield as it went away from the center (Fig.3A). is
could be directly related to the solute concentration in
the extraction system.
Inuence ofindependent parameters onthequality
ofessential oil
e two primary compounds found in cinnamon leaf
essential oil are eugenol and cinnamaldehyde [8]. ese
two crucial components are responsible for the essen-
tial oil’s distinct aroma and flavor, as well as its unique
therapeutic characteristics. Cinnamaldehyde was used in
this study to optimize the quality of cinnamon essential
oil since the concentration of eugenol was much higher
and constant across all samples. e extraction tempera-
ture and feed mass had a significant effect on the relative
concentration of cinnamaldehyde at both the linear and
quadratic levels (P < 0.001). Although extraction time had
a significant effect on essential oil quality at the linear
Table 3 The regression coefficients values of the responses with
their p-values
Variables Yield (g/g) Cinnamaldehyde (%)
Regression
coecient p-values Regression
Coecient p-values
Intercept 3.07 0.0001 25.77 0.0001
A-Time 0.1319 0.0002 − 0.5422 0.0180
B-Temperature 0.0834 0.0053 − 2.45 0.0001
C-Feed Mass 0.2119 0.0001 2.10 0.0001
AB − 0.0712 0.0429 0.4312 0.1163
AC 0.0163 0.6085 − 0.4488 0.1039
BC − 0.0088 0.7816 − 2.07 0.0001
A² − 0.0918 0.0025 0.2957 0.1446
B² − 0.1661 0.0001 1.61 0.0001
C² − 0.1855 0.0001 1.16 0.0001
R20.9609 0.9791

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Yitbareketal. Applied Biological Chemistry (2023) 66:47
Fig. 2 Response surface plots demonstrating the interaction between A Feed mass (g) and Time (min), B Temperature (℃) and Time (min), C Feed
mass (g) and Temperature (℃) on Yield (%) of cinnamon essential oil; D Feed mass (g) and Time (min), E Temperature (℃) and Time (min), F Feed
mass (g) and Temperature (℃) on cinnamaldehyde (% area)

Page 8 of 13
Yitbareketal. Applied Biological Chemistry (2023) 66:47
level (P < 0.05), it lacked a significant (P > 0.05) effect at
the quadratic level (Table3).
e relative cinnamaldehyde concentration of cinna-
mon essential oil yield can be predicted by the following
second-order polynomial regression equation in terms of
coded values (Eq.4):
Where: A = Extraction time temperature, B = Extrac-
tion temperature, and C = Feed mass .
A 3D surface plot was used to demonstrate the interac-
tion effect of independent variables on the relative con-
centration of cinnamaldehyde (Fig.2). e independent
variable of extraction time had a weak interaction effect
with either extraction temperature or feed mass (Fig.2D,
E). As a result, the influence of time on the quality of cin-
namon essential oil (cinnamaldehyde) was not significant,
at least within the time period designated for this inves-
tigation. is could be owing to the volatile nature of the
cinnamaldehyde component, which causes it to volatilize
at an early stage of the process [16]. While the extrac-
tion period is held constant, increasing the feed mass
increases the relative concentration of cinnamaldehyde
(Fig.2D). Increasing the feed mass resulted in a higher
relative cinnamaldehyde concentration regardless of
(4)
Cinnamaldehyde
=
763.229 −0.2412A −13.753B +
0.2826C
+0.0019AB −0.0066AC −0.0028BC
+0.0015A
2
+0.0645B
2
+0.0052C
2
extraction time or temperature (Fig.2D, F). is could be
due to the increased availability of solute concentration
caused by the increase in feed mass. Furthermore, when
the extraction period was extended, the bigger biomass
showed less quality degradation than the smaller feed
quantity. A significant (P < 0.05) interaction effect was
also seen when extraction temperature was paired with
either extraction time or feed mass (Table3). e rela-
tive concentration of cinnamaldehyde was particularly
reduced at higher temperature processing with longer
extraction time (Fig.2E). ermal-induced decomposi-
tion caused by greater extraction temperatures could
account for the lower relative concentration of cinna-
maldehyde. Besides, the detrimental effect of extraction
temperature on the quality of cinnamon essential oil was
exacerbated by the reduced feed mass compared to its
counterpart (Fig.2F). is could be due to a reduction in
direct contact between the high-temperature steam and
the solute found in bigger biomass.
To better visualize the effect of each independent vari-
able on the cinnamon essential oil quality as it moved
away from the mid-point while all other parameters
stayed constant at specific reference value, a perturba-
tion plot was used (Fig.3B). e relative concentration of
cinnamaldehyde increases as the temperature decreases
from the center point (reference specified by Design-
expert software), and the opposite is fully true. Mean-
while, when the feed mass grew from the reference point,
so did the relative concentration of cinnamaldehyde, and
vice versa.
Fig. 3 Perturbation plot illustrating the change effect of the independent variables from maximum to minimum values on A Yield (%),
and B cinnamaldehyde (% area)

Page 9 of 13
Yitbareketal. Applied Biological Chemistry (2023) 66:47
Optimization ofindependent variables
Different optimum solutions for yield and quality of cin-
namon leaf essential oil were predicted by using design
expert software V.13. Maximization goals were selected
for the optimization of both cinnamon leaf essential oil
yield and cinnamaldehyde relative concentration. For the
optimal values for various independent variable com-
binations, fifty-four distinct numerical solutions were
produced. From the 54 solutions listed, a process param-
eter combination of 175.43min, 105 ℃, and 600g time,
temperature, and feed mass were chosen as the optimum
process parameter combination for the greatest desir-
ability of 0.71, respectively. At these optimized extrac-
tion conditions, the response values were 2.9% extraction
yield and 34.6% relative cinnamaldehyde concentration.
Validation ofthedeveloped RSM models
e adequacy of the model for predicting response val-
ues was tested using optimized cinnamon leaf essential
oil extraction. is was accomplished by carrying out the
extraction experiment at optimal conditions. At optimum
processing conditions, the predicted extraction yield was
2.9% and the cinnamaldehyde relative concentration was
34.6%. Meanwhile, during the validation experiment at
optimized extraction conditions, the extraction yield was
0.289 and the cinnamaldehyde relative concentration
was 33.94%. is finding shows that the experimental
response values were very near to that of the projected
values (Table4).
Chemical composition andantioxidant activity
ofcinnamon essential oil
For additional functionality study, cinnamon essential oil
that was extracted utilizing the steam distillation process
under optimum conditionswas employed. e essential
oil content was evaluated using GC–MS before further
study. is GC–MS study found twenty-four compounds
in total. e first 10 of these compounds account for
more than 98% of the total relative area coverage. When
compared to the other compounds, eugenol was by far
the most prevalent (60.68%). It has been established that
eugenol is first produced in the cinnamon leaves and
that the barks make up a very little portion on the bio-
synthesisprocess. is result is actually in a good agree-
ment with the previous results in which, eugenol is the
primary component of essential oils from cinnamon leaf
oils. Cinnamaldehyde (33.94%) was the second significant
component found in cinnamon leaf essential oil extract
(Table 5). Similar dominance of these two compounds
were also observed for essential oil extracted from five
cinnamon leaves [10]. Other than this two the other com-
pounds shown a lower relative area coverage (< 2%) for
the GC–MS analysis. e percentage area coverage for
the other compounds, excluding these two, was lower
(2%) for the GC–MS analysis. It is nevertheless important
to be aware that the composition of essential oils might
change depending on the timing of harvest, the country
of origin, the stage of a plant’s development, and market
storage conditions [32].
Total phenol, total flavonoid, and DPPH free radical
scavenging potential of cinnamon essential oil extracted
at optimum condition were analyzed to assess the anti-
oxidant activity. is antioxidant activity’s mode of action
is to reduce oxidative stress in the body by scaveng-
ing the generated free radicals of ROS and RNS (reac-
tive nitrogen species). Particularly phenolic compounds
have a reputation for acting as antioxidants due to both
their propensity to donate electrons or hydrogen as well
as the fact that they are stable radical intermediates. In
this study, cinnamon essential oil showed a good antioxi-
dant activity for the aforementioned antioxidant analysis
(Table5). is good antioxidant activity by major comes
from the principal compounds of cinnamon essential oil.
Particularly, cinnamaldehyde demonstrated a good free
radical–scavenging activities in the previous experiments
[30]. Similar, good antioxidant activity was seen for
invitro cinnamon essential oil antioxidant activity study
[17, 33].
Antibacterial activity andminimum inhibitory
concentration
Essential oils represent a potential source of new anti-
bacterial compounds, especially against some genera of
bacteria that cause food deterioration. e antibacterial
Table 4 Optimum conditions, predicted and experimental values of the response variables
Optimum conditions Coded levels Values
Extraction time (min) 0.43 175.43
Extraction temperature (℃) − 1 105
Feed Mass (g) − 1 600
Response variables Predicted values Experimental values
Yield (%) 2.90 2.915 ± 0.72
Cinnamaldehyde concentration (% area) 34.60 33.94 ± 0.61

Page 10 of 13
Yitbareketal. Applied Biological Chemistry (2023) 66:47
activity of cinnamon essential oil was investigated in this
work using invitro disc diffusion assay. Figure3 illustrates
the antibacterial activity of various concentrations of cin-
namon essential oil against five selected foodborne bac-
terium species [Staphylococcus aureus (ATCC 25923),
Listeria monocytogenes (ATCC 19115), Escherichia coli
(ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),
Acinetobactor baumannii (ATCC 19606)]. ese bacteria
were chosen based on their high prevalence in most Ethi-
opian foods [34] as well as the variations in their mem-
brane structures, which play a significant role in how
they react to the applied essential oil.
Given the variety of chemical component present in
cinnamon essential oils, the method of action to pre-
vent microbial growth could not be attributed to a sin-
gle mechanism and may instead target several activities
in the cell. Cinnamaldehyde’s carbonyl group is predicted
to bind to proteins and prevent the bacterial amino acid
decarboxylases from doing their job. Moreover, because
essential oils and their components are hydrophobic,
they can permeate the lipids of the mitochondria and
bacterial cell membrane, upsetting the structure of the
cell and making it more permeable. Cell death will occur
as a result of this significant bacterial cell leakage or the
loss of vital substances and ions.
e minimal inhibition concentration for the applied
essential oil concentrations, however, varied from bac-
terium to bacteria as did the level of the inhibition
zone. Regardless of the bacterial family, the inhibi-
tory effect grows as the essential oil concentration
is increased (Fig. 4, Additional file1: Fig.S2). In this
study, the lowest minimal inhibition concentration was
observed for S. aureus at 0.2% (v/v). while, the highest
minimum inhibitory concentration was observed for
the E. coli (Fig.4). Besides, all the gram-negative bac-
teria in this study showed the higher essential oil con-
centration as compared to the gram-positive bacteria.
is difference is most likely due to the intricacy of
gram-negative bacteria’s double membrane-containing
cell envelope, as contrast to gram-positive bacteria’s
single membrane glycoprotein or membrane-glycopro-
tein-based structures. Furthermore, the highest inhibi-
tion zone (14mm) was observed for the disc inoculated
with S. aureus at 0.8% (v/v) cinnamon essential oil con-
centration (Fig.4). ese inhibition zones are smaller
than those reported in earlier studies [35] that tested
the antibacterial activity of crude cinnamon essential
oil (20mm), however the concentration of the applied
oil was very much higher as compared to this study as
well as recommended dosage applied to the food.
Eect ofessential oil concentrations onstorage stability
ofminced beef
When total viable microbiological counts (TVC) in any
food product reach 7 log CFU/g, the product is consid-
ered spoiled [36]. Figure4 depicts the TVC dynamics of
minced beef seasoned with cinnamon essential oil and
Table 5 Chemical composition and antioxidant activity optimally extracted cinnamon leaf essential oil
Table spaces left blank are for RI values that have not been discovered in the literature under similar operating conditions
S. N Name of the compound Chemical composition
Molecular Formula Rt (min.) Retention index (RI) % Area
RI Literature RI
1 α-Pinene C10H16 5.553 896 917 0.07
2 α-Phellandrene C10H16 7.266 986 996 0.02
3 O-Cymene C10H14 7.796 921 0.45
4 Limonene C10H16 7.918 945 995 1.03
5 Linalool C9H8O 9.862 1039 1090 1.29
6 Cinnamaldehyde C9H8O 14.629 1260 1266 33.94
7 Eugenol C10H12O216.967 966 1373 60.68
8 α-Copaene C15H24 17.439 913 1372 0.16
9 Caryophyllene C15H24 18.573 892 1415 0.38
10 Benzyl benzoate C14H12O226.82 1479 0.21
Antioxidant activity
Total phenols 70.8 ± 0.52 mg GAE/g
Total flavonoids 36.50 ± 0.36 mg QE/g
DPPH 44.65 ± 0.69 IC50 µg/mL

Page 11 of 13
Yitbareketal. Applied Biological Chemistry (2023) 66:47
control samples stored at 4 ℃for 21 days. e raw beef
utilized in this investigation had an initial TVC of 3.82
log CFU/g. e use of cinnamon leaf essential oil sig-
nificantly decreased the growth rate of TVC in minced
beef samples (P < 0.05). More particular, when a higher
concentration of cinnamon essential oils was added to
minced beef, the rate of microbial growth decline was
faster (Fig.5).
However, increasing the essential oil concentration to
more than 1% may have a detrimental influence on the
sensory quality of the food [37]. Nonetheless, even at 0.5%
concentration of cinnamon essential oil treated samples
were within the acceptable range for the twenty-one-
day refrigerated storage in this investigation. Previous
research on the evaluation of effectiveness of cinnamon
essential oil on food deterioration microorganisms yielded
similar results [38, 39]. e control sample, on the other
Fig. 4 Zone of microbial inhibition for different concentrations of cinnamon essential oil against A S.auerus, B L. monocytogenes, C E.coli, D P.
aeruginosa, E A.baumannii bacterial species
Fig. 5 Effect of cinnamon essential oil concentrations on the microbial shelf stability of minced beef

Page 12 of 13
Yitbareketal. Applied Biological Chemistry (2023) 66:47
hand, showed a considerable growth rate after the fourth
day of storage and exceeded the upper limit of 7 log
CFU/g after fifteen days of refrigerated storage (Fig.5).
However, increasing the essential oil concentration of
above 1% might have a negative impact on the sensorial
attribute of the food products [37]. Nevertheless, in this
study even at 0.5% concentrate of cinnamon essential oil
treated samples was with the acceptable range for the
twenty-one days refrigerated storage. Previous studies on
the evaluation of cinnamon essential effectiveness on the
food spoilage microorganisms had also reported similar
findings. Whereas, the control sample had shown a sig-
nificant growth rate after fourth day storage and cross the
maximum limit of 7 log CFU/g after fifteen days of refrig-
erated storage (Fig.5). Several factors, including cell wall
rupture brought on by bioactive substances, cytoplasmic
membrane disruption, cellular component stress brought
on by leakage, altered fatty acid and phospholipid con-
stituents, affecting RNA and DNA formation, and wreck-
ing protein translocation, are cited as the explanations for
this inhibition of microbial growth.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13765- 023- 00798-y.
Additional le1: Fig. S1.Fresh and shade-dried cinnamon leaves. Fig.
S2.Inhibition zones of the Cinnamon essential oil against the common
foodborne microorganisms A Acinetobacter baumannii (ATCC 19606), B
Escherichia coli (ATCC 25922), C Staphylococcus aureus (ATCC 25923), D
Listeria monocytogenes (ATCC 19115), E Pseudomonas aeruginosa (ATCC
27853).
Acknowledgements
The authors would like to acknowledge Addis Ababa Science and Technology
University.
Author contributions
RMY: conceptualization, Methodology, investigation, writing—original draft.
HA: conceptualization, writing—review and editing, supervision. FMI: con-
ceptualization, supervision. EGF: data curation, formal analysis, visualization,
software, writing—original draft.
Funding
Not applicable.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare that they have no competinginterests
Received: 30 December 2022 Accepted: 27 June 2023
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