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Step-by-step extraction method of citrus functional ingredients using green

solvent

Conference Paper · October 2013

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 Step-by-step extraction method of citrus functional ingredients using
green solvent

Yoshito Sasaki 1), Hideo Iwai 2), Teruaki Matsubara 2), Chiho Uemori 1), Rintaro Hoshino 3),
Masahiro Tanaka 4), Munehiro Hoshino 4), Mitsuru Sasaki 2), and
Motonobu Goto 3)*
1)
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Fukuoka, Japan.
2)
Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan
3)
Department of Chemical Engineering, Nagoya University, Nagoya, Japan
4)
Maruboshi Vinegar･Ascii, Food Technology and biology of technical center (M. A .F. T), Fukuoka,
Japan
* Corresponding author, e-mail : mgoto@nuce.nagoya-u.ac.jp

Abstract
Sour citrus fruits of the various kinds are cultivated in Japan. Citrus junos (yuzu), Japanese typical
sour citrus, have been treated as a raw material for juice processing. Most of the juice processing residue
was disposed without being used despite contain valuable compounds. The parts of yuzu peel account for
approximately 50% of whole fruit. It contains valuable hydrocarbon and carbohydrate such as essential
oil and water soluble dietary fiber consisted of pectin. At first, supercritical carbon dioxide (SC-CO2) was
used as a solvent for extract the essential oil of yuzu peel. The effects of pressure and temperature on the
yield of total oil extract were studied under the following conditions; pressures at 10-30 MPa,
temperatures at 40-80oC. The extract was divided into two layers. The yield of extract tended to increase
with a pressure and temperature increase. The upper layer is an essential oil and the lower layer is
moisture of peel. The highest recovery rate of essential oil was reached 95% at the temperature of 80 oC
and pressure of 30MPa. In second step, sub-critical water was applied to extract pectin from residue of
SC-CO2 extraction. Experimental conditions were carried out under following conditions; pressures at 4-
30 MPa, temperatures at 110-160C. The yield of extracted pectin was greater than 75% at a temperature
range of 120-140C and pressure range of 4-30 MPa. The molecular weight of extracted pectin was
tended to become small molecule along with increasing temperature. Furthermore, it was suggested that
the elimination of methyl groups in the pectin molecule was enhanced the increase of the extraction
temperature.

Keywords Citrus junos, essential oil, pectin

 Step-by-step extraction method of citrus functional
ingredients using green solvent
Yoshito Sasaki 1), Hideo Iwai 2), Teruaki Matsubara 2), Chiho Uemori 1), Rintaro Hoshino 3), Masahiro
Tanaka 4), Munehiro Hoshino 4), Mitsuru Sasaki 2), and Motonobu Goto 3)*
1)
Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka city,
Fukuoka 820-8502, Japan.
2)
Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto
860-8555, Japan
3)
Department of Chemical Engineering, Nagoya University, Furo-cho,Chikusa-ku, Nagoya 464-8603, Japan
4)
Maruboshi Vinegar･Ascii, Food Technology and biology of technical center (M. A .F. T), 2400 Tabara,
Kawasaki-machi, Tagawa-gun, Fukuoka 827-0003, Japan
* Corresponding author, e-mail : mgoto@nuce.nagoya-u.ac.jp , Fax : +81-52-789-3392

Abstract
Sour citrus fruits of the various kinds are cultivated in Japan. Citrus junos (yuzu),
Japanese typical sour citrus, have been treated as a raw material for juice processing. Most of
the juice processing residue was disposed without being used despite contain valuable
compounds. The parts of yuzu peel account for approximately 50% of whole fruit. It contains
valuable hydrocarbon and carbohydrate such as essential oil and water soluble dietary fiber
consisted of pectin. At first, supercritical carbon dioxide (SC-CO2) was used as a solvent for
extract the essential oil of yuzu peel. The effects of pressure and temperature on the yield of
total oil extract were studied under the following conditions; pressures at 10-30 MPa,
temperatures at 40-80oC. The extract was divided into two layers. The yield of extract tended
to increase with a pressure and temperature increase. The upper layer is an essential oil and
the lower layer is moisture of peel. The highest recovery rate of essential oil was reached 95%
at the temperature of 80oC and pressure of 30MPa. In second step, sub-critical water was
applied to extract pectin from residue of SC-CO2 extraction. Experimental conditions were
carried out under following conditions; pressures at 4-30 MPa, temperatures at 110-160C.
The yield of extracted pectin was greater than 75% at a temperature range of 120-140C and
pressure range of 4-30 MPa. The molecular weight of extracted pectin was tended to become
small molecule along with increasing temperature. Furthermore, it was suggested that the
elimination of methyl groups in the pectin molecule was enhanced the increase of the
extraction temperature.

Introduction
Citrus junos, called yuzu in Japan, is an evergreen tree harvested mainly in the southern
part of Japan. Its fruit is processed into juice and, because of its special flavor, is often
preferred to vinegar as an ingredient in sauces and salad dressings. Currently, most of the
juice processing residues are disposed of without being recycled. The residue of citrus fruits is
mainly composed of peel, juice sack and seed. Notably, the peel contains valuable compounds,
such as terpenoids, pectin, and flavonoids. The traditional citrus by-products of cattle feed
pellets and essential oil are still the most important utilization of juice processing residue. And
also, pectin has been conventionally extracted and separated using mild acid or a chelating
agent from citrus fruit waste such as lemon, thereby utilizing industrial wastes.
Carbon dioxide is the most preferred because of its low critical temperature (31.1oC)
suitable especially for thermally labile components. Besides, it is readily available and non-
toxic. Unlike the conventional organic solvent extraction, the extraction process associated
with the use of SC-CO2 does not require complicated process to remove the solvent. Its
presence in foods or beverages is generally regarded as safe and harmless for human
consumption. With these properties, SC-CO2 has been applied to extraction of natural
compounds such as essential oil. [1]
Sub-critical water has unique properties: the hydrogen bond between water molecules
weakens by raising the temperature, and weakening of this hydrogen bond results in a
dramatic change in the dielectric constant. Ionic dissociation of water molecule is progressed
with the rise in temperature, and then catalyzes the hydrolysis reaction. Due to these
properties, it will be expected that pectin extraction can be controlled by pressure and
temperature.
In this work, SC-CO2 extraction of essential oil from scraped wet peel of yuzu was carried
out. As starting material the extraction residue, subcritical water extraction of pectin was
investigated under various hdrothermal conditions. In addition, the properties of extracted
pectin also investigated.

Experimental
SC-CO2 extraction of essential oil
A semi-continuous flow SC-CO2 extraction apparatus (AKICO Co., Ltd., Tokyo) shown in
schematic diagram in Figure 1, was used in the experiments.

Gas meter
PG-1 PG-2 PG-3 PG-4

BPR1 BPR2 BPR3

Flow meter
Thermostat heater

Heater Heater Heater

Extractor
Thermostat heater

Separator

Cooling bath
Separator
CO
Cylinder
2 Cylinder

Heater

Pump
Pomp

Chiller unit

Figure 1 Scheematic diagram of SC-CO2 semi continuous flow extractor

Approximately 80 g of scraped citrus peel was placed 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 was flowed into the
extractor covered by a thermostat heater that was maintained at the operating temperature.
The supplied pressure of liquid 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 and a dry gas meter. Extracted oil was collected from the pressurized
separator and weighed right after collection, then stored at -20oC prior to component analysis.
Extracts were collected every 30 min for the first 60 min, and thereafter, collected every 60
min for a total extraction time of 300 min. Extraction experiments were carried out at
temperatures of 40, 60 and 80oC, pressures of 10, 20 and 30 MPa, with CO2 flow rate of
0.19-0.21 g/s.

Subcritical water extraction of pectin

A schematic diagram of the semi-continuous flow extractor is shown in Figure 2. The
maximum working conditions of the apparatus are 450C and 45 MPa. A 0.5-g portion of raw
material was charged in the extractor (SUS 316; 8.7 mm i.d.×118 mm length, 7.0 mL), and
the extractor was capped with gasket filters (average pore size 20 m) to prevent sample
particles from flowing out.
 Back pressure
T2 P regulator

Extraction cell
HPLC pump

Filter
Degassed water
Oven T1 Cooling bath
Cooling water 7mL / fraction
Figure 2 Schematic diagram of semi continuous water flow extractor

Distilled water was degassed and continuously delivered through a heating coil placed
in an oven into the extractor by a high-pressure pump (PU-980; Jasco Co., Ltd.) at water flow
rates of 0.5, 1.0, and 2.0 mL/min. The pressure in the extractor was controlled by a back-
pressure regulator (HBP-450; Akico Co., Ltd.) at 4, 10, 20, and 30 MPa. The extraction
temperature was monitored by the thermocouples at the inlet and outlet of the extractor. Prior
to extraction, degassed water (60 mL) was delivered to the extractor at 80C to remove the air
and water-soluble substance. Initially, one fraction was collected. Then, the reaction
temperature was immediately raised from 80C to the desired temperatures of 110, 120, 130,
140, 150, and 160C. The extracted solutions were fractionated, and 10 fractions (7 mL) were
collected at each temperature and pressure condition.

Results
SC-CO2 extraction of essential oil
In order to determine the optimum condition in the range of experimental conditions
investigated in this study, the yield of total extract from yuzu peel was plotted against
temperature and pressure. Figure 3 shows the yield of total extracted oil of yuzu peel at
various temperatures and pressures obtained in 300 min. The yield of essential oil increased as
the pressure of SC-CO2 increased (along with increasing density). The maximum yield of
93% was obtained at the condition of 80oC and 30MPa.

93%
100

90

80

70
Yield [%]

60

50

40
30
30

20
40 20
]
Pa
[M

60
re

Tem
p e ra
su

ture
es

[ oC] 10
Pr

80

Figure 3 Effect of temperature and pressure on the yield of yuzu essential oil

The amount of recovered essential oil was four times as much as that of cold pressed oil.
In addition, functional components included in the water layer was also investigated and
shown in Figure 4. As a result, hesperidin of nearly 70% in the raw material was extracted.
Pectin was not extracted almost due to having high polarity. Therefore, it was expected that
the pectin of most is remaining into extraction residue.

80 1.0
Hesperidin recovery rate [%] 80oC, 30MPa 80oC, 30MPa
70

Pectin recovery rate [%]

0.8
60
50 0.6
40
0.4
30
20 0.2
10
0 0
1 2 3 4 5 6 1 2 3 4 5 6
Fraction Fraction
Figure 4 Extraction behaviors of hesperidin and pectin in the aqueous layer

Subcritical extraction of pectin

Figure 5 shows the effect of pressure on pectin yield at various temperatures. At 120°C,
pectin yield improved with decreasing pressure. However, the extraction pressure did not
influence the yield of pectin between 130 and 140C. Furthermore, the yield of pectin was
poorer at temperatures greater than 140C, indicating that pectin molecules are beginning to
decompose. As a result, the highest yield was obtained at temperatures under 140°C,
independent of pressure. It is thought that the important factor is not pressure, but temperature,
for extraction and separation of polar substance with hydrogen bond from natural products
using subcritical water.

80

80.00

75
76.86

73.71
Yield [%]

70
70.57

67.43
65

64.29

60
61.14
5
58.00
10
Pres

15
sure

20
150
25 140
[MP

130
30
a]

120 o
]
ture [ C
Tempera

Figure 5 Effect of temperature and pressure on the yield of pectin

Properties of extracted pectin

Figure 6 shows the gel filtration chromatogram of pectin extracted under the various
conditions. Under hydrothermal conditions, the molecular size of pectin decreased with
increasing extraction temperature. This result indicated that different molecular weights of
pectin can be recovered by altering the extraction temperature.
 Mw [kDa] (Standard pullulans)
106 105 104 4×103
12000
120oC 15
10000 140oC 35 12
160oC 57
8000 HCl

RI response
6000
635
4000

2000

0
6 7 8 9 10 11
Retention time [min]
Figure 6 Molecular weight distribution chromatograms of the pectin extracted under the
various conditions

Figure 7 shows the effect of extraction conditions on the Degree of Esterification (DE)
of the pectin molecule. It was suggested clearly that the extraction temperature affects the
degree of esterification in pectin molecule. It is possible to recover pectin with different
gelling properties by adjusting the extraction temperature

Pressure 4MPa
45
Degree of metyl esterification [%]

40
35
30
25
20
15
10
5
0
HCl 120oC 140oC 160oC
Extraction conditions
Figure 7 Effect of extraction conditions on the Degree of Esterification (DE) of the pectin
molecule

Conclusion
Supercritical CO2 and subcritical water were applied individually to extract valuable
compounds from citrus waste effectively. The maximum yield of SC-CO2 extraction was
obtained at a temperature of 80oC and pressure 30 MPa. Sub-critical water extraction enabled
effective separation of pectin without the use of chemicals, such as acid, at a yield of greater
than 75%. Pectin molecule with different properties were recovered by altering the extraction
temperature. Even though sub-critical water extraction enabled effective separation of pectin
without the use of chemicals, pectin is very cheap product. It is necessary to examine the
conditions of extraction as pectin oligosaccharide with highly function.

References :
[1] B.C. Roy, M. Hoshino, H. Ueno, M. Sasaki, M. Goto, Supercritical carbon dioxide extraction of the volatiles
from the peel of Japanese citrus fruits. J. Essent. Oil Res., 2007, 19, 78-84
[2] R.J. Braddock, Handbook of Citrus By-products and Processing Technology, Willy Inter Science, New York,
1999, 120-121.

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