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Austin Chemical Engineering
Research Article

Step-Wise Pressure Controlling Approach forSupercritical Carbon Dioxide Extraction of Essential Oilfrom Thick Peel of Bitter Orange (Citrus Aurantium)
Hoshino Y1,2, Tanaka M2, Takamizu A3, Suetsugu
T3, Hoshino M2,4, Wahyudiono1, Kanda H1 andGoto M1*
1
Department of Materials Process Engineering, Nagoya
University, Japan
2
Maruboshi Vinegar Ascii, Food Technology and Biologyof Technical Center (MAFT), Japan
3
Maruboshi Vinegar Co.Ltd, Japan
4
ASCII Co.Ltd, Japan

Abstract*Corresponding author: Goto M, Department ofMaterials Process Engineering, Nagoya University, Japan

Supercritical carbon dioxide (SC-CO2) extraction has been applied for therecovery of essential oil from plant material. In conventional SC-CO2 extraction,
CO2 is continuously flowed at a constant pressure from dried sample. In thiswork, step-wise pressure controlling approach was applied to essential oilfrom fresh bitter orange peel without drying pretreatment. The extracts containwater as well as essential oils. Extraction was carried out at temperatures of
40 to 80oC; and pressures of 10 to 30 MPa. The recovery of essential oil was
considerably high at 95.4 %, more than 3.4 times the recovery obtained byconventional semi-continuous method.

Received: July 14, 2020; Accepted: August 20, 2020;
Published: August 27, 2020

Keywords: Citrus aurantium; Essential oil; Supercritical carbon dioxide;
Pressure control.

Introduction
Citrus aurantium, bitter orange or called daidai in Japan, isa popular variety of sour citrus. Bitter orange has been mainly
cultivated in Wakayama prefecture in Japan and is being harvested
from December to January. Bitter orange fruit is covered with thickand hard peel which has pleasant flavor. Its juice has been used infood products such as sauces and chilled sweets because of its uniqueand desirable flavor. Most of citrus essential oils have been collected
as byproducts in juice production process. Bitter orange juice has
been produced in small scale, because only a few areas cultivate bitterorange trees. For this reason, the amount of bitter orange essential
oil cannot be recovered in commercial scale. There are reports of its
extraction using steam distillation, hydrodistillation and cold press
method in laboratory scale [1-6]. The aromatic components containedin the essential oils of citruses are monoterpenes, sesquiterpenes,
and oxygenated compounds. It is known that the monoterpenes,
such as limonene, take a little part of the aroma. On the other hand,sesquiterpene and some of the oxygenated compounds contribute to
the strong fragrances and peculiar characteristic flavor of the citrus.
The characteristic flavor components of cold-pressed bitter orange
essential oil have been investigated using GC-olfactometry andsensory analysis [4].
Essential oils from some citrus peel are focused as a medicinal
applications [3,6,7]. The essential oil obtained from Citrus aurantium,
have been used to study medicinal activity such as central nervous
system action [6], anxiolytic and sedative effect [7], and cholinesterase
inhibitory activity [3].
In the extraction of natural compounds such as essential oil,the use of supercritical fluid has been proposed. Supercritical fluid
has liquid-like density and gas-like viscosity, thus it is considered to
be a promising solvent that combines high solvent power and hightransport capacity. Of the many possible fluids of choice, carbon

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dioxide is the most preferred because of its low critical temperature(31oC) suitable especially for thermally labile components such asaroma compounds. Besides, it is readily available and non-toxic. Unlike
the conventional organic solvent extraction, the extraction process
associated with the use of supercritical CO2 does not leave harmful
solvent residues in the extracts. Its presence in foods or beverages isgenerally regarded as safe and harmless for human consumption. CO2
is nonflammable, non-toxic, environmentally-friendly, and cheap. In
addition, SC-CO2 has low critical temperature, low viscosity and it
is easy to separate from the extract. Some researchers reported thesupercritical extraction of essential oil from various citrus peels suchas orange [8, 9], and grapefruit [10], bergamot [11], unshu [12], yuzu[13], and yuzu and kabosu [14,15]. In these reports, citrus peel was
pretreated either by drying, crushing or a combination of these steps
prior to a commonly used semi-continuous flow type of extraction.
In this work, a more efficient extraction method of the citrus
essential oil from the unusually thick peel of bitter orange using
step-wise pressure controlling approach was investigated. Scraped
fresh bitter orange peel without pre-drying treatment was used as asample material. The results were compared with the typical semicontinuous extraction method, and optimum extraction condition
was investigated.

Materials and Methods
Materials and chemicals
Sample of bitter orange fruit was harvested in December 2009 at
Fukuoka prefecture, Japan. The outer bitter orange peel was scraped
automatically using a skinning machine (KR-ROBO-03, Mitsuwa
Co. Ltd., Niigata, Japan) which separate the flavedo part containing
albedo of approximately 2.4 mm thick. Its peel was scraped, placed
inside an air-tight wrapping plastic bag, and then stored in a freezer
at -20oC until extraction experiments were performed. No pre-drying
treatment was carried out on the sample. The feed material contained

Citation: Hoshino Y, Tanaka M, Takamizu A, Suetsugu T, Hoshino M, Wahyudiono, et al. Step-Wise Pressure
Controlling Approach for Supercritical Carbon Dioxide Extraction of Essential Oil from Thick Peel of Bitter Orange(Citrus Aurantium). Austin Chem Eng. 2020; 7(3): 1079.

Goto Mwater of approximately 80wt%. Prior to extraction experiments, thesample was gently thawed to room temperature. Carbon dioxide(99.9%) was obtained from Uchimura Sanso Co., Japan.
Steam distillation
Approximately 40g of scraped citrus peel was minced in 500 mLof distilled water, and placed into a 1L round flask. The flask was thenconnected to a distilling receiver for essential oil testing with Liebig
condenser. Distillation was carried out at a temperature of 100oC for
24 h. The average amount of oil content in the wet peel obtained bysteam distillation was 1.42%
Semi-continuous SC-CO2 extraction
A semi-continuous flow SC-CO2 extraction apparatus (AKICO
Co., Ltd., Tokyo) shown in schematic diagram in Figure 1, was usedin the experiments. Approximately 80 g of scraped citrus peel wasplaced in a 500 mL extractor having a height of 20 cm and inside
diameter of 7 cm. Liquid CO2 from a cylinder with siphon attachment
was passed through a chiller kept at 0oC, and compressed CO2 wasflowed into the extractor covered by a thermostat heater that wasmaintained at the operating temperature. The supplied pressure ofliquid CO2 was controlled by Back-Pressure Regulator (BPR) 1, while
the pressure in the extractor was controlled by BPR 2. The exit fluid
from the extractor was expanded to a pressurized separator at 2 MPa
and 0oC by BPR 3. CO2 flow rate was measured by a flow meter anda dry gas meter. Extracted oil was collected from the pressurized
separator every 60 min for a total extraction time of 300min, andweighed right after the collection. Extraction experiments werecarried out at temperatures of 40, 60 and 80oC, pressures of 10, 20
and 30 MPa, with CO2 flow rate of 0.95-1.05g/s.

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selective detector and a HP-5MS column (30 x 0.25 mm (5%-Phenyl)methylpolysiloxane column, film thickness x 0.25µm).

Results and Discussion
Components of essential oil
From the GC-MS chromatogram of essential oil extractedwith supercritical CO2, major components identified are shown incomparison with that obtained by steam distillation. About 76 peaks
were observed by GC-MS for essential oil obtained by supercritical
CO2. The composition of essential oils between supercritical fluid
extraction and steam distillation is slightly different, oils obtainedby supercritical fluid extraction has higher content for limonene andlinalool whereas lower content for α-pinene, myrcene, γ-terpinen,and α-farnesene. The components reported in literatures for the same
citrus fruit are roughly similar; Maekawa et al. [1] (cold pressing:limonene 86.23%, β-pinene 5.50%, myrcene 1.60%, caprylaldehyde
1.93%, linalool 0.52%), Deterre et al. [5] (cold-pressing: limonene
90.47%, α-pinene 1.21%, b-pinene 0.21%, myrcene 4.37%, linalool
0.66%), Tundis et al. [3] (hydrodistillation: limonene 65.8%, α-pinene
1.8%, β-pinene 0.3%, myrcene 2.9%, ocimene 0.3%, γ-terpinene
0.1%, linalool 1.8%, β-caryophyllene 0.3%), and Pultrini et al. [6](hydrodistillation: limonene 97.83%, myrcene 1.43%, octanal 0.45%).
Considering the difference in harvesting season, geographicallocation, variety, and maturity, there is no significant difference inthe composition and content.
Menichini et al. [16] compared the essential oil of Citrus
medica L. cv. Diamante among hydrodistillation, cold-pressing, andsupercritical carbon dioxide extraction. Although the oil composition

Step-wise pressure controlling SC-CO2 extraction
In the case of step-wise pressure controlling extraction approach,approximately 80 g of scraped citrus peel was placed in the extractor.
Liquid CO2 was then loaded into the extractor until the desired
extraction conditions were attained. SC-CO2 extraction was carriedout under the same temperature and pressure conditions of semicontinuous experiments. The pressure was controlled in step wise
consisted of a series of pressurization period, static high pressure
period and depressurization period. In the static period, CO2 flow
was stopped. The static period of step-wise pressure controlling
extraction was set to 10, 15, 30, 60 and 90 min. After the holding
time, which allowed CO2 to penetrate into the sample matrix has
elapsed, the system was depressurized to ambient, while collecting
essential oil at the separator. These series of step-wise pressurizationstatic-depressurization steps were conducted six times under various
conditions.

Figure 1: Schematic diagram of SC-CO2 extraction apparatus.

Recovery of extracted oil
The recovery (%) was defined as the weight of extracted oils perweight of oil obtained by steam distillation as shown below.
Yield (%) = (weight of extracted oil)/(weight of steam distilledoil)×100
GC-MS analysis of essential oil
The essential oils obtained by supercritical CO2 extraction andsteam distillation were analyzed by GC-MS. The GC-MS is Agilent
7890N series gas chromatograph equipped with an Agilent 5975 mass

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Figure 2: Yield of bitter orange essential oil through the semi-continuous
extraction method.

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Goto M

Figure 3: Effect of holding time on the yield of bitter orange essential oil at
60oC, 30 MPa.

Austin Publishing Groupof 30 MPa are shown in Figure 3. The recovery increased withincreasing holding time despite similar amount of CO2 consumption.
The difference in the recovery of bitter orange essential oils werehardly observed between 60 and 90 min. By treatment under
static pressurized condition, thick bitter orange peel underwent
swelling under supercritical CO2 conditions. It was thought that
SC-CO2 penetrated and diffused through the cell matrix followedby dissolution of the target essential oil into CO2, and was highly
dependent on density changes during depressurization step. Longer
holding time may lead larger amount of essential oil dissolved inCO2. During depressurization step, CO2 containing the essential oil
penetrated in sample matrix expanded by pressure reduction andflow out to the fluid phase by convective flow. The mass transfer by
the convective flow is much faster than diffusion process. In semicontinuous extraction at constant pressure, mass transfer is limited todiffusion, whereas it is both convective flow and diffusion in pressure
controlling extraction process.
Effect of pressure and temperature on step-wise pressure
controlling extraction

Figure 4: Effect of pressure on the yield of essential oil.

was similar between hydrodistillation and cold-pressing, components
and composition of oil obtained by supercritical fluid extraction was
significantly different. Most of essential oil components were notcontained but 85% was citropten which was 2.5% in cold-pressing oil.
One of the reason of this difference may be collection of extracted oil
after the supercritical fluid extraction. Volatile components could not
be easily collected in the collection vessel from gaseous carbon dioxide
flow. In our work, the collection part was carefully designed and also
water was extracted with essential oil which make the collection ofvolatile components easier.
Preliminary studies on semi-continuous extraction
Figure 2 shows the yield of total oil extract of bitter orange peelby semi-continuous extraction method at various temperature andpressure conditions for 300 min. The yield increased with increasing
extraction temperature. The yield was the highest at a temperatureof 80oC and pressure of 20 MPa. However, its yield was only 28%of those obtained by steam distillation. This relatively low yieldcompared conventional steam distillation method could be due to thedifficulty of supercritical CO2 to penetrate and diffuse through thesample matrix because of its thickness of the peel. The supplied CO2
simply passed through the samples and could hardly isolate the target
materials. Thus, in the succeeding experiments step-wise pressure
controlling extraction approach was considered, instead.

The holding time of the static time was fixed at 90 min to
investigate the effect of pressure and temperature on the recoveryof essential oil. Figure 4 shows the effect of pressure on the recoveryof bitter orange essential oil. Under this isothermal condition, theamount of extracted essential oil increased with increasing pressure.
This result indicates that the CO2 density plays an important role inoil extraction. At higher pressure, more CO2 penetrates into matrix,
resulting in more essential oil dissolved in CO2.
Figure 5 shows the effect of temperature on the yield of essentialoil. Contrary to the effect of pressure as shown in Figure 4, the yield
increased with temperature despite the decrease in CO2 density
at isobaric condition. Two factors relating to the properties of thetarget compounds and SC-CO2 could contribute to this behavior. The
first factor is the vapor pressure effect of essential oil with the risein temperature, and the second relates to the mass transfer rate ofessential oil from the sample matrix to the extracting SC-CO2.
Optimum condition for batch-wise extraction
The holding time was fixed at 90 min to investigate the optimum
extraction condition under various temperatures and pressures. Figure
6 shows the optimum condition to extract the bitter orange essential
oil through the step-wise pressure controlling extraction approach.
The increase in SC-CO2 density as the pressure increased obviously
had a positive effect on the recovery of essential oil. However, theEffect of holding time on step-wise pressure controlling
extraction
The effect of holding time of the static period on step-wisepressure controlling extraction was investigated, and the results forthe recovery of essential oils at a temperature of 60oC and pressure
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Figure 5: Effect of temperature on the yield of essential oil.

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Goto M

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3.4 times the recovery obtained by semi-continuous method. In
addition, CO2 consumption was reduced to 38%. Future directions
should consider a combination of semi-continuous and step-wise
operation for a more efficient and effective extraction.

Acknowledgments
This work was supported by Kyusyu Bureau of Economy, Tradeand Industry.
References
Figure 6: Optimum extraction condition obtained by batch-wise extraction.

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Figure 7: Comparison of the yield and CO2 consumption between semicontinuous and batch-wise extraction approaches for 5 hours.

recovery of essential oil tends to increase at higher temperatures of 60
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Conclusions
The step-wise pressure controlling approach was found suitablefor SC-CO2 extraction of essential oil from unusually thick fresh
citrus peel like that of bitter orange as compared to that of the typicalsemi-continuous method. Even from wet citrus peel, essential oil
was able to be extracted. Since volatile compounds such as essential
oil would volatilize and disappear during drying process, extraction
from wet sample is valuable. Additionally, volatile components can beeasily recovered with water. Optimum condition for extraction was
obtained at a temperature of 80oC and pressure of 20 MPa, similar
to that of a typical semi-continuous operation. At this condition, therecovery of essential oil was considerably high at 95.4%, more than
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