Extraction of oil from Jatropha curcas L.

seed kernels by
combination of ultrasonication and aqueous enzymatic oil extraction
Shweta Shah, Aparna Sharma, M.N. Gupta *
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
Received in revised form 6 May 2003
Available online 8 April 2004

Abstract

Use of ultrasonication as a pretreatment before aqueous oil extraction and aqueous enzymatic oil extraction was found to be
useful in the case of extraction of oil from the seeds of Jatropha curcas L. The use of ultrasonication for 10 min at pH 9.0 followed by
aqueous oil extraction gave a yield of 67%. However, the maximum yield of 74% was obtained by ultrasonication for 5 min followed
by aqueous enzymatic oil extraction using an alkaline protease at pH 9.0. Use of ultrasonication also resulted in reducing the process
time from 18 to 6 h.

Keywords: Aqueous enzymatic oil extraction; Jatropha oil; Ultrasonication

1. Introduction
Aqueous enzymatic oil extraction (AEOE) has
emerged as a promising technique for extraction of oil
from plant materials (Rosenthal et al., 2001; Sharma
et al., 2002). Its main advantages are that it is environ-
ment friendly and does not produce volatile organic
compounds as atmospheric pollutants (Rosenthal et al.,
1996). One disadvantage associated with AEOE is the
long process time which are necessary for enzymes to
liberate oil bodies. Another factor (sometime neglected)
is the use of enzymes which are not commercially
available. This prevents the use of the process by other
workers. The present work describes the extraction of oil
by use of a commercially available protease preparation
from Jatropha curcas L. seeds. The effect of ultrasoni-
cation as a pretreatment to cut down process time was
also studied.
J. curcas L. is a drought resistant tropical tree and the
oil from its seeds has been found useful for medicinal
and veterinary purposes, as insecticide, for soap pro-
duction and as a fuel substitute (Gubitz et al., 1999).
Various methods for recovering this oil from the seeds
have been investigated. As suitable presses are not
available (Openshaw, 2000), extraction with organic
solvents and water have been the main approaches.
2. Methods
Jatropha seeds were obtained from Dr. Zope, College
of Forestry, Akola, India. Protizymee was purchased
from Jaysons Agritech Pvt. Ltd., Mysore, India. It is
reported to contain mostly acid (pH optimum range 3-
4), neutral (pH optimum range 5-7) and alkaline pro-
teases (pH optimum range 7-10) from Aspergillus flavus
with a specific activity of 2.1 U/mg protein with casein as
substrate. Pectinex Ultra SP-L and Promozyme was
bought from Novo Nordisk Ferment Ltd., Switzerland.
Pectinex Ultra SP-L is an enzyme preparation (Asper-
gillus niger) containing three enzyme activities: xylanase
(404 U/ml), pectinase (338 U/ml) and cellulase (134 U/
ml). Promozyme contains pullulanase activity (434 U/
ml). Crude cellulase preparation from A. niger (0.5-1.0
U/mg solid) were obtained from Central Drug House,
Mumbai, India. Molecular sieves (3 A) were obtained
from E. Merck, Mumbai, India. All other chemicals and
solvents used were of analytical grade.
122 S. Shah et al. / Bioresource Technology 96 (2005) 121-123

2.1. Aqueous and aqueous enzymatic oil extraction of oil
from Jatropha seed kernels
Jatropha seeds were cracked, the shells carefully re-
moved and the kernels thus obtained were used for oil
extraction. The suspension was prepared with powdered
(obtained by using a homogenizer) Jatropha seed kernels
(5 g) in 30 ml distilled water. The suspension was then
incubated at desired temperature with constant shaking
at 100 rpm for specified time period (Aqueous oil
extraction, AOE). The upper oil phase was collected
after centrifugation at 10,000 xg for 20 min and
weighed. Enzyme assisted aqueous oil extraction was
performed similar to AOE (taken as control) except that
the different enzyme preparations i.e. Protizyme e , Cel-
lulase, Pectinex Ultra SP-L, Promozyme as well as
mixture of all these enzymes were added after the pH of
the suspension was adjusted. The amounts of oil
recovered were calculated as percentages of total oil
present in J. curcas seed kernels. The latter was deter-
mined by soxhlet extraction using hexane as a solvent as
per the standard AOAC procedure (Horowitz, 1984).
The solvent extraction of Jatropha seed kernels gave a
yield of 44 g oil/100 g Jatropha seed kernels. Jatropha oil
is reportedly present in the range of 40-60 g oil/100 g
Jatropha seed kernels (Makkar et al., 1997). A value of
44 g oil/100 g Jatropha seed kernels was taken as 100%
recovery of oil while calculating the oil recovery by AOE
and AEOE.
Each extraction was run in duplicate and the yields
were found to agree in duplicates within 3%.
zymes. The best result (47%, w/w oil yield) was obtained
by using Protizyme e (250 mg containing 525 U) at 50
°C. Use of additional enzymes along with Protizyme e
did not improve the oil yield. This is in agreement with
the earlier observation that hemicellulases and cellulases
were less effective in oil extraction from J. curcas L. than
the alkaline protease and their combination with the
proteases did not increase the extraction yield (Winkler
et al., 1997). The above treatment with Protizyme e was
carried out at pH 4.0, 7.0 and 9.0. Protizyme e is a
mixture of three proteases with pH optima of 3-4, 5-7
and 7-10. Fig. 1 shows the effect of using Protizyme e at
three different pH values. In agreement with the result of
Winkler et al. (1997), the alkaline protease component
gave the best results and 64% (w/w) yield could be ob-
tained. Thus, it was established that only alkaline pro-
teases should be used during AEOE from J. curcas L.
seed kernels. Again our yield was lower than 86% re-
ported by Winkler et al. (1997), which may also be due
to the inherent nature of seed material used by us.
64
• Control
60
• Enzyme treated
47
•; 45- 43
38
29
5
£• 30-

15 -
r 17

3. Results and discussion

Use of aqueous oil extraction at various temperatures

was found to yield oil in the range of 17-21% (w/w) only
(Table 1). This is somewhat lower than 38% yield re-
ported by others (Gubitz et al., 1999). In AEOE, en-
zymes are used to facilitate release of oil from oil bodies
enmeshed in protein and cellulosic/hemicellulosic net-
works (Rosenthal et al., 1996). Table 1 also shows the
result of using various commercial preparations of en-
pH
Fig. 1. Effect of varying pH on oil yield by AEOE. The ground
Jatropha seed kernels (5 g) were dispersed in 30 ml distilled water and
stirred to make a suspension. The pH of the suspension was adjusted
to the desired value with 0.1 N NaOH or 0.1 N HCl. To this 250 mg
(525 U) of Protizyme e was added and incubated overnight at 50 °C
with constant shaking at 100 rpm. The oil was recovered as described
in Section 2.

Table 1
Effect of varying temperature on aqueous enzymatic oil extraction
Oil yield (%, w/w)
37 °C 40 °C 50 °C
Control 21 21 17
Pectinex Ultra SP-L 32 33 20
Protizyme e 32 29 47
Cellulase 21 31 19
Pectinex Ultra SP-L + Promozyme + Cellulase + Protizyme e 39 43 49
The suspension of ground Jatropha seed kernels (5 g in 30 ml distilled water, pH 7.0) was incubated overnight at different temperatures with constant
shaking at 100 rpm. Pectinex Ultra SP-L (250 ll), Promozyme (250 ll), Cellulase (250 mg) and Protizyme e (250 mg) and also mixture of all these
enzymes were added to the suspension. A control (AOE) was run at different temperatures in which no enzyme was added.
 S. Shah et al. / Bioresource Technology 96 (2005) 121-123
Ultrasonication is emerging as a powerful tool to
accelerate many chemical and physical processes. It has
also been used to increase the oil yield during AOE in
some cases (Szentmihalyi et al., 2002). The effects of
ultrasonication time on oil yield by both AOE and
AEOE are shown in Table 2. An interesting observation
was that ultrasonication combined with AOE at alkaline
pH gave a yield of 67% which was comparable to 64%
yield obtained by AEOE (as reported above). Consid-
ering that cost of enzyme makes AEOE as a less
acceptable approach economically, ultrasonication as a
pretreatment may be a good technique to be tried along
with AOE. Another useful observation was that AOE,
with or without ultrasonication was more efficient at pH
9.0. Thus, this may explain better result with alkaline
proteases. The maximum yield (74%, w/w) however was
obtained by carrying out AEOE at pH 9.0 with 5 min
ultrasonication as a pretreatment.
All the above experiments (Fig. 1, Tables 1 and 2)
were carried out with a process time of about 18 h
(incubation with enzyme). The data shown in Fig. 2
indicate that using ultrasonication as a pretreatment
allows one to cut down the process time to about 6 h
without reducing the over all yield of 74%. It was
thought prudent to check the presence of moisture in the
extracted oil. For this purpose, a part of the extracted oil
was treated with molecular sieves overnight. It was
found that IR spectra of the untreated oil and oil treated
with molecular sieves (3 A) were identical. Both showed
absence of broad water peak in the region 3700-3500
cm"1.
While a similar approach need to be tried with other
plant materials, the results described here indicate that
ultrasonication pretreatment may be a useful step to
obtain oil by both AOE and AEOE.
Table 2
Effect of time of ultrasonication on the oil yield
Ultrasonication (min) pH
7.0 9.0
Control (% w/w)
5 16 61
10 17 67
15 25 63
AEOE (amount of oil obtained, %, w/w)
5 50 74
10 48 71
15 25 69
The ground Jatropha seed kernels (5 g) were dispersed in 30 ml distilled
water and stirred to make a suspension. The pH of the suspension was
adjusted to 7.0 and 9.0 with 0.1 N NaOH before exposing to ultra-
sonication for different time intervals. Thereafter, Protizyme e (525 U)
was added and incubated overnight at 50 °C with constant shaking at
100 rpm. A control was also run in each case in which there was no
enzyme.
123
<5U
A
60
—A— Control
40 —A— Enzyme Treated
20
A
A
0-
2 4 6 8
Incubation time (h)
Fig. 2. Effect of ultrasonication time on oil yield by AEOE. The
ground Jatropha seed kernels (5 g) were dispersed in 30 ml distilled
water and stirred to make a suspension. The pH of the suspension was
adjusted to 9.0. The suspension was then exposed to sonication for 5
min before adding Protizyme (250 mg) and incubated for different time
intervals at 50 °C with constant shaking at 100 rpm. The oil was
recovered as described in Section 2.
Acknowledgements
This work was partially supported by project funds
from Technology Mission on Oilseeds, Pulses and Maize
(TMOP&M), Council for Scientific and Industrial Re-
search (CSIR) and Department of Science and Technol-
ogy (DST), Department of Biotechnology (DBT), all
Government of India organizations. The authors also
thank Dr. S.K. Khare, IIT, Delhi for his help in obtaining
the J. curcas L. seeds and his interest in this work.
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