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Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.11 (2): 1055-1059. 2013 www.world-food.net
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Extraction of agarwood (Aquilaria crassna) oil by using supercritical carbon dioxide

extraction and enzyme pretreatment on hydrodistillation

Nuttawan Yoswathana
Department of Chemical Engineering, Faculty of Engineering, Mahidol University, Nakornprathom Province, 73170, Thailand.
e-mail: nuttawanyoswathana@hotmail.com

Received 2 January 2013, accepted 28 April 2013.

Abstract
Agarwood (Aquilaria crassna) is a fragrant wood containing economically important essential oil. It has been traded for use as incense, perfume and
traditional medicine. In this study, the effect of three different methods for extraction of agarwood oil and the chemical compositions of extracted
agarwood oil were studied. Conventional hydrodistillation method had two major disadvantages namely; long extraction time of about 5 days and high
energy consumption with low yield (0.075% w/w). Pretreatment of agarwood with technical enzymes before hydrodistillation affected the extractability
of essential oil positively (0.20% w/w). Extraction of agarwood oil using supercritical carbon dioxide at 600 bar resulted higher yield (0.47% w/w)
compared to hydrodistilation at distinct shorter extraction time of 3 h and process temperature (85°C). Analyzing of the extracts using Gas
Chromatograph/Mass Spectrometer (GC/MS) had shown that the composition of essential oils differed from each other. The major compounds in
essential oil from hydrodistillation were selina-3,7(11)-diene (17.21%), δ-selinene (12.36%), 1,3,5-trimethyl-6-methyldiene-tricyclo [3.2.1.0(2,7)
[oct6-3-en-8exo-ol (10.26%) and 4a,5-Dimethyl- 3-(1-methyldiene)-4,4a,5,6,7,8-hexa hydro-2(3H)-napthalene (9.6%). Pretreatment of agarwood
with technical enzymes resulted aristol-9-en-8-one (28.77%), β-guaiene (14.94), guaia-3,9-diene (14.32%) and 7,7-dichlorobicycl0 [3.2.0] hept-2-en-
6-one (7.58%) as the principal substances. For supercritical carbon dioxide (ScCO2) extraction (600 bar, 85°C, 3h) of agarwood was three substances;
furoscrobiculin B (27.98%), furosardorin A (19.07%), and bicyclo[4.4.0]des-5-ene, 1,5-dimethyl-3-hydroxy-8-(1-methylene-2-hydroxyethyl-1)
(18.68%) as the main substances in agarwood oil.

Key words: Agarwood oil, supercritical carbon dioxide (ScCO2), hydrodistillation.

Introduction
Aquilaria crassna (Agarwood) is one of the highly valuable
fragrant woods that containing economically important essential
oil. It is member of Thymceleceae family and Magnliopsida class.
Agarwood is tropical tree that distributes in rain forest southeast
(Cambodia, Laos, Thailand and Vietnam) 1. In Thailand, local name
of agarwood is also called Krisana, is widely cultivated in
Chanthaburi and Trat provinces. The components of essential oil
have revealed various kinds of compound that consist of the
major sesquiterpenes compounds (γ-selinene, selina-3, 11-dien-9-
one, selina-3, 11-dien-14-al) and derivate choromones 2. In ancient,
agrwood oil has been consumed as perfume in the world especially
in Middle East because of its protective effect against parasite. In
addition, it has been used for religious purpose in Islam and
Buddhism as well as traditional medicine in Southeast Asia. In
2001, the organization of conserve the agrwood (CITES; The
Convention on International Trade in Endangered Species of Wild
Fauna and Flora) are founded by many countries in Asian in order
to govern the conservation and utilization of tropical species and
determine resource of agarwood 1. The conventional methods for
extraction of essential oil from agarwood performed either by
hydrodistillation or steam distillation. The procedure for production
of essential oil from agarwood start with immersion of the
agarwood in water about 5 days followed by distillation at boiling
temperature (100°C) for 10-15 days. The yield of essential oil after
this method is about 0.2% 3. Therefore, the conventional methods
have the major drawbacks of the operating process such as long
extraction time (total about 15-20 days) and high energy
consumption. On the other hand, agarwood oil is expensive product
with sell price approximately 3,000-4,000 bath for 1 tola of oil (1
tola = 12.5 cc.) 4. In the past several years, supercritical carbon
dioxide (ScCO2) extraction has been used as alternative methods
for extraction. Due to carbon dioxide is an inert, non-flammable,
safe, and inexpensive. In addition, it is a non-polar fluid (dipole
moment = 0) that can dissolve any kind of hydrophobic
compounds or slightly polar compounds 5. Moreover, it can
changed to critical fluid under the mild critical temperature (Tc =
31.1°C) and critical pressure (Pc = 73.8 bar). The physical properties
in this critical region enhance positive effect to extraction involving
low density, low viscosity, high diffusion rate compare to either
gas or liquid behaviors 6. In 1981, supercritical carbon dioxide was
applied for decaffeination of coffee in U.S. 7. Supercritical carbon
dioxide become more attention for extraction of other substances
such as antioxidants, herb and essential oil from natural product
in order to replace the conventional organic solvent extraction

Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013 1055
(ethanol, chloroform, methanol and petroleum ether) and
conventional thermal extraction (steam distillation and
hydrodistillation). The conventional extraction methods generally
used high boiling temperature that can lead to degradation of
heat-sensitive compound. As well as remaining of solvent residue
in product that has negative effect to the quality of extracted
products 6, 8. Many authors reported the successfully extraction
of essential oils from different raw materials (eucalyptus leave,
mint leave, thyme leave, and chamomile flower) using supercritical
carbon dioxide 9-12. The solvating power of ScCO2 extraction
depends on pressure and temperature. Density gives an estimate
of the joint effects of both variables on the solvating power.
Besides the operational conditions, the equilibrium solubility of
pure compounds is influenced by molecular weight, polarity and
presence of functional groups. When considering extraction from
a solid substrate, kinetics a yields also depends on the interaction
with the solid matrix 13. The cell wall is composed of cellulose,
hemicellulose, lignin and pectin. Hydrolases such as cellulases,
hemicellulases and pectinases break down the cell wall. Besides,
research have attracted increasing interest to apply enzyme for
cleavage the strong linkages of plant polysaccharides (cellulose,
hemicellilose). It is a strong woody structure and act as a barrier
for essential oil extraction. Therefore, using of the cellulase, xylase
and other digestive enzyme 14, 15 could be easier to extract and
might enhances the yield of agarwood oil. The aims of this work
were to compare the extraction yield by supercritical carbon dioxide
(ScCO2), pretreatment of technical enzymes and hydrodistillation
and to analyze chemical constituents of agarwood oil by GC-MS.
Materials and Methods
Plant material and chemicals: Aquilaria crassna (agarwood)
used in this study was cultivated by artificial method to stimulate
the resin production. This agarwood was purchased from Bo-rai,
Trat province. The plant material was dried in the oven, cut into
small pieces and grounded to size of 3.0 mm. Carbon Dioxide,
purity 99.9%, was purchased from Thai Industrial Gases Public
Company Limited, Thailand. Ethanol, analytical grade 99.9% was
purchases from Merck & Co., Inc. Germany. Cellulase and xylase
(Dr. Luca, Germany), alcalase and rohalase (AB-Enzyme, Germany)
were used as technical enzyme.
Hydrodistillation: Of the dried heartwood agarwood 200 g was
soaked in 1 L water at room temperature for 5 days. Then,
hydrodistillation was carried out at boiling temperature of water
for 30 hours. The obtained agarwood oil in Clevenger-type
apparatus was taken and stored at ambient temperature until
analyze the chemical constituents.
Pretreatment with technical enzymes: 200 g of dried heartwood
of agarwood was mixed with 1800 ml of 1% H2SO4 and immediately
autoclave at 121°C for 15 min. After that the sample was cooled
down until 55°C. Finally, the sample was neutralized to pH 4.0-4.5
with 5N NaOH and mixed with 12 ml of enzyme mixture (5 ml
cellulase, 5 ml xylase, 1 ml alcalase and 1 ml rohalase). The prepared
sample was then incubated for 3 days at 55°C in a water bath
followed by using hydrodistillation. The extracted agarwood oil
was stored at ambient temperature until analyze the chemical
constituents.
1056
Supercritical carbon dioxide (ScCO2) extraction: Supercritical
Carbon Dioxide (ScCO2) Extraction was carried out in pilot-plant
scale equipment with 1 litre volume of extractor (SIB-Foodtech,
Germany) as shown in Fig. 1. Two hundred grams dried heartwood
of agarwood was added in a 1 meter long cloth extraction bag and
the extraction bag was inserted into the extractor vessel. The ScCO2
extraction was performed at different extraction pressure (200, 400
and 600 bar) and at different extraction temperature (30, 50, 70 and
85°C). For all ScCO2 extraction experiments the extraction time (1
and 3 h) and CO2 flow rate (approximately 0.5 kg/h) was constant.
The extracted agarwood oil during ScCO2 extraction was lead to a
receiver containing 100 ml ethanol. The ethanol containing
extracted agarwood solution was vacuum evaporated in a
rotavapor (R-114 Buchi, Switzerland). The extracted agarwood oil
was measured and selected the condition of maximum extracted
yield by removing the wax form extracted agarwood oil. Freezing
the solution at -21°C in freezer unit until wax was solidified then
filtered. The filtered solution was vacuum evaporated in a
rotavapor. Finally, the amount of total extract was determined by
gravimetric method. The extracted was kept at ambient temperature
until analyze the chemical constituents with GC-MS.
T3 V3 Pressure of extracted
S
E
Pressure of
separator
T2 T1 V1
CO2 pump
V2
hot water
Separator Extractor 99.9 %
CO2 tank
Cooling machine
Figure 1. Schematic diagram of supercritical carbon dioxide (ScCO2)
extraction equipment.
Analysis of agarwood oils by using gas chromatography-mass
spectrometry (GC-MS): Chemical constituents of extracted
agarwood oils were analyzed by a Gas chromatography (Triplus)
equipped with polaris mass spectrometer. The column used was
Zebron capillary, ZB-5 ms (30 M × 0.25-mm i.d., film thickness 0.25
µm). Condition: split mode and injection of 1 µm. The operating
temperature was 60°C for 5 mins, ramp of 3°C per min up to 240°C
and 240°C for 1 min. Helium was used as the carrier gas.
Statistical analysis: All experiments were performed 3 to 4 times
and average values were reported. Statistical analyses were carried
out using Microsoft Excel 2007.
Results and Discussion
Hydrodistillation: The effect of distillation time on the extracted
agarwood oil (A. crassna) during hydrodistillation is shown in
Fig. 2. According to the essential oil yield, after 1 day
hydrodistillation about 0.1% v/w essential oil could be extracted,
increased the yield up to 0.17% v/w could be obtained after 5
days distillation. Also, the yield of essential oil increased with
Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013
 0.20 0.6
0.18
0.5
0.16
0.14 0.4

Yield (w/w)
y = 0.018x + 0.084
Yield (v/w)

0.12 2
R = 0.9878 0.3
0.10
y = 0.0059x - 0.0152
0.08 0.2 2
R = 0.9666
0.06
0.1
0.04
0.02 0
0 10 20 30 40 50 60 70 80 90
0.00
0 1 2 3 4 5 6 Temperature (degree celsius)
Day
Figure 4. The effect of temperature on the oil yield of A.
Figure 2. Relationship between oil yield of agarwood (A. crassna at extraction pressure of 600 bar.
crassna) and distillation time.

increasing the extraction time and related linearly (R2 = 0.99). It is temperature can increase the vapor pressure of the extractable
also important to conduct additional experiments to investigate compounds. Although, increasing temperature leads to reduce
the effect of longer extraction time than 5 days on the essential oil the density of ScCO2 (for a constant pressure) and reduce the
extraction during hydrodistillation. power of the supercritical solvent 13.

Supercritical carbon dioxide (ScCO2) extraction
Effect of pressure: Pressure effects on the oil yields are
demonstrated in Fig. 3. The ScCO2 extraction yield at low pressure
of 200 bar and 70°C after 1 h extraction (0.17% w/w) was higher
than hydrodistillation for 5 days at 100°C (0.075% w/w). This
indicates that the ScCO2 method is an effective and short time
method for extraction of essential oil from agarwood. Further
increasing the pressure from 200 to 400 bar, the result of essential
oil from 400 bar got 2 times higher yield of 200 bar at same extraction
temperature and time (1 h). At extraction pressure at 600 bar higher
yield compare to at 400 bar was obvious but this yield increasing
was only slightly higher that at 400 bar. The most relevant extraction
process parameter is the extraction pressure that can affect the
extractability of compounds. The higher extraction pressure leads
to increase the density of ScCO2 that increased the larger the
solvent power of ScCO2 and the solubility of essential oil in ScCO2
The essential oil yield: Generally, CO2 is a non-polar fluid (dipole
moment = 0) that can dissolve any kind of hydrophobic
compounds or slightly polar compounds 5. In previous work
reported that essential oil was extracted together with cuticular
wax 8, 13. In this work, extracted oil had cuticular wax that was
undesired product. It was removed by using low temperature 13.
Fig. 5 shows the oil yield before and after dewax in agarwood oil
that the condition of maximum extracted yield was 600 bar, 85°C
and 1 h. The extracted agarwood oil was performed the yield of
0.51% w/w. After dewax, the essential oil yield was about 0.31%
w/w.
0.51
0.6
0.5
Yield (W/W)

0.31
13 0.4
.
0.3

0.2
0.360
0.40 0.1
0.315
0.35
0
0.30 (a) wax (b) dewax
Yield (W/W)

0.25 Figure 5. Comparison of extracted oil yield: (a)

0.17
0.20 Wax and oil, (b) Dewax.
0.15
0.10
0.05 Comparison of essential oil yield from 3 different methods of
0.00 extraction: Fig. 6 shows that the essential oil yield from agarwood
200 400 600
Pressure (bar)
by using 3 different extraction methods. Hydrodistillation gave
very low (0.075% w/w) agarwood oil. This indicated that
Figure 3. The effect of pressure on the oil yield of
A. crassna at extraction temperature of 70°C. hydrodisitillation was not efficiently method for agarwood
essential oil extraction because of low yield and high energy
Effect of temperature: The yield of agarwood oil could be observed consumption. The color of agarwood oil from hydrodistillation
in Fig. 4. The higher extraction temperature could positively affect was yellow color. Adding technical enzymes mixture during soaking
the more amount extractability of essential oil. At 30°C, the yield (5 days, at ambient temperature) before hydrodistillation improved
was about 0.15% w/w. Then the temperature at 85°C leaded to 3 the yield of essential oil. Up to 0.21% w/w essential oil extraction
times higher yields at the same extraction pressure and time (600 could be measured after hydrodistillation of enzymes pretreated
bar, 1 h). This indicated that the extraction on agarwood oil sample. This indicated the advantage of apply technical enzymes
will be improve with increasing the extraction temperature in in the agarwood oil extraction process. Technical enzymes such
linear relation (R2 = 0.97) at pressure of 600 bar. Due to the high as cellulase and hemi-cellulase could degrade the woody plant

Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013 1057
 extraction process of 3 h compared to conventional method (up to
0.6 ≥ 10 days). The low viscosity, high diffusivity and nearly similar
0.47, 3 h
0.5 density of ScCO2 compare to conventional organic solvents
promote the extractability of essential oil using ScCO2 method.
Oil ield (%W/W)

0.4
0.3 0.21, 30 h
0.2
0.075, 30 h
0.1
0 sample and air, no oxidation reaction could be occurring during
Hydrodistillation pretreatment with ScCO2
extraction enzyme
Figure 6. The agarwood oil yield obtained by
hydrodistillation, pretreatment with enzyme and ScCO2
extraction.
cell wall materials (cellulose and hemi-cellulose) and improve the
mass transfer during hydrodistillation. The color of subsequent
hydrodistillated agarwood sample was intensive (brown
yellowish) compare to hydrodisitillation sample without enzyme
pretreatment. The highest essential oil yield could be obtained
using ScCO2 extraction. After ScCO2 extraction at 600 bar, 85°C
and very short extraction time of 3 h up to 0.47% w/w essential oil
was measured. The color of ScCO2 extraction was brown yellowish.
In addition, ScCO2 extraction of agarwood oil do not need soaking
process. This ScCO2 extraction allows very short essential oil
The energy consumption during ScCO2 extraction is far lower
than conventional hydrodistillation. Shorter extraction time and
very lower energy consumption for evaporation of solvent make
ScCO2 process an environmentally friendly and interesting method
for essential oil extraction. Because of no direct contact between
ScCO2 extraction so that essential oil with high quality could be
obtained.
Comparison chemical constituents of agarwood oil from 3
different methods of extraction: Comparison between chemical
compositions of essential oil extracted using hydrodisitllation,
enzymes pretreated and subsequent hydrodistillated agarwood
sample as well as ScCO2 extraction as shown in Tables 1-3 that
reported remarkable different extracted compounds in essential
oil. The GC-MS analysis of essential oil showed about 17-30
compounds for all samples but the concentration of compounds
was totally different for different extraction methods. Whereas
essential oil from hydrodistillation (Table 1) contained higher
amount of selina-3,7(11)-diene (17.21%) and δ-selinene (12.36%),
respectively.
For pretreatment with technical enzymes and subsequent

Table 1. Chemical constituents of agarwood oil by hydrodistillation (soaking with water).

Retention
Identification % area
time
32.01 o-(-)-spathulenol 9.86
35.49 Bicyclo[4.4.0]dec-5-ene,1,5-dimethyl-3-hydroxy-8-[1-methylene-2-hydroxyethyl] 2.48
37.87 ȕ-Guaiene 4.41
38.27 į-Selinene 12.36
38.42 Agarospirol 3.85
38.81 Guaia-3,9-diene 7.83
39.11 Selina-3,7(11)-diene 17.21
39.85 10-epi-T-eudesmol 7.38
40.57 Į-Guaiene 8.86
41.95 1,3,5-Trimethyl-6-methylidene-tricyclo[3.2.1.0(2,7)]oct6-3-en-8-exo-ol 10.26
44.37 4a,5-Dimethyl-3-(1-mehtylidene)-4,4a,5,6,7,8-hexahydro-2(3H)-Napthalenone 9.68
50.03 Hexadecanoic acid (CAS) 3.73
54.37 5,19-cyclo-5a-androst-6-ene-3,17-dione (CAS) 2.05

Table 2. Chemical constituents of agarwood oil by hydrodistillation (soaking with technical enzymes).
Retention
Identification % area
time
22.14 1-Phenyl-2-buten-1-ol 0.69
32.02 Caryophyllene oxide 2.52
33.07 Methyl-6,9-octadecadiynoate 3.66
34.96 Cedrane-8,13-diol 0.90
35.72 9-Isopropyl-1-methyl-2-methylene-5-oxatricyclo[5.4.0.0(3,8)]undecan 1.32
36.91 Furanodiene 2.11
37.17 Eudesma-4,11-diene-3-one 2.85
37.87 į-Selinene 2.50
38.28 Guaia-3,9-diene 14.32
38.83 10-epi-T-eudesma 5.06
39.11 ȕ-Guaiene 14.94
39.65 7,7-dichlorobicyclo[3.2.0]hept-2-en-6-one 7.58
40.58 (3,8,8-Trimethyl-1,2,3,4,5,6,7,8-octanhydro-2-naphthalenyl)methyl acetate 4.36
40.90 7-(1,3-dimethyl-buta-1,3dienyl)-1,6,6-trimethyl-3,8-dioxa-tricyclo[5.1.0.0 2,4] 3.68
41.95 6-(1,3-Dimethyl-buta-1,3-dienyl)-1,5,5-trimethyl-7-oxa-bicyclo[4.1.0]hept-2-ene 2.04
42.44 2H-Cyclopropa[g]benzofuran,4,5,5a,6,6a,6b-hexahydro-4,4,6b-trimethyl-2-(1-methylethenyl) 2.68
44.37 Aristol-9-en-8-one 28.78

1058 Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013
Table 3. Chemical consitiuents of agarwood oil by ScCO2 extraction present the retention time
and area percentage.
Retention
Identification % area
time
38.45 calarene 0.50
38.84 ȕ-Guaiene 1.07
39.12 agarospirol 4.71
40.27 cholic acid 1.56
40.57 (3,8,8-Trimethyl-1,2,3,4,5,6,7,8-octanhydro-2-naphthalenyl)methyl acetate 5.39
41.39 3-Ethyl-3-hydroxyandrostan-17-one 4.29
42.45 6-(1,3-dimetyl-buta-1,3-dienyl)-1,5,5-trimethyl-7-oxa-bicyclo[4.1.0]hept-2-ene 4.29
45.53 Bicyclo[4.4.0]dec-5-ene, 1,5-dimethyl-3-hydroxy-8-(1-methylene-2-hydroxyethyl-1) 18.68
49.42 Furosardorin A 19.04
50.03 Furoscrobiculin B 27.98
50.93 10,12,14-Nonacosatriynoic acid 4.48
55.78 4,5-methylene-9,10-phenanthra[D]benzo[B]furan 6.53
56.18 Stigmast-3-en-3-ol 1.32
60.84 Androst-4-en-9-methylthio-11-ol-3,17-diene 3.93

hydrodistillated agarwood sample as viewed in Table 2 that was 2

aristol-9-en-8-one (28.77%), β-guaiene (14.94), guaia-3,9-diene
(14.32%) and 7,7-dichloro bicycl0[3.2.0] hept-2-en-6-one (7.58%),
respectively.
In the case of ScCO2 extraction as shown in Table 3, three major
substances were observed. The concentration of furoscrobiculin
B (27.98%), furosardorin A (19.07%), and bicyclo[4.4.0] des-5-
ene,1,5-dimethyl-3–hydroxy-8-(1-methylene-2-hydro xyethyl-1)
(18.68%) was higher amount the other substances in essential oil.
dioxide. The ScCO2 can extract high molecular compounds and
non-polar. Several studies have shown the ScCO2 extraction also
could extract other complex compounds such as waxes, oleoresin
and long chain hydrocarbon 7. Using ScCO2, higher molecular
weight compounds can be extracted from agarwood as well as
alcohol terpenes are readily solubilized by carbon dioxide.
Conclusions
Hydrodistillation yielded very low (0.75% w/w) agarwood oil.
Pretreatment of agarwood with technical enzymes before extraction
significantly increased the extraction yield of agarwood oil (up to
0.21% w/w). The most effective method for extraction of agarwood
oil was ScCO2 extraction. Using ScCO2 extraction could be up to 5
times more essential oil (0.47% w/w) and shorter time for extraction
(3 h) than hydroditillation. The pressure and temperature extraction
conditions have effect to extraction yield of agarwood oil. The
higher pressure and temperature lead to higher yield during ScCO2
extraction. Also, the ScCO2 extraction of agarwood has distinct
advantages because of shorter extraction time, higher extraction
yield of agarwood oil and less energy consumption. The
composition of extracted essential oils from 3 different extraction
methods differed from each other. The ScCO2 extraction showed
less oxygenated compounds.
Acknowledgements
The authors wish to thank Thai Traditional and Herbal
Development Center, module 1, Biotechnology Pilot Plant,
Thailand Science Park, Pathumtani, and Faculty of Engineering,
Mahidol University.
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Journal of Food, Agriculture & Environment, Vol.11 (2), April 2013 1059