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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 Various methods for recovering this oil from the seeds
have been investigated. As suitable presses are not
Aqueous enzymatic oil extraction (AEOE) has available (Openshaw, 2000), extraction with organic
emerged as a promising technique for extraction of oil solvents and water have been the main approaches.
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., 2. Methods
1996). One disadvantage associated with AEOE is the
long process time which are necessary for enzymes to Jatropha seeds were obtained from Dr. Zope, College
liberate oil bodies. Another factor (sometime neglected) of Forestry, Akola, India. Protizymee was purchased
is the use of enzymes which are not commercially from Jaysons Agritech Pvt. Ltd., Mysore, India. It is
available. This prevents the use of the process by other reported to contain mostly acid (pH optimum range 3-
workers. The present work describes the extraction of oil 4), neutral (pH optimum range 5-7) and alkaline pro-
by use of a commercially available protease preparation teases (pH optimum range 7-10) from Aspergillus flavus
from Jatropha curcas L. seeds. The effect of ultrasoni- with a specific activity of 2.1 U/mg protein with casein as
cation as a pretreatment to cut down process time was substrate. Pectinex Ultra SP-L and Promozyme was
also studied. bought from Novo Nordisk Ferment Ltd., Switzerland.
Pectinex Ultra SP-L is an enzyme preparation (Asper-
J. curcas L. is a drought resistant tropical tree and the gillus niger) containing three enzyme activities: xylanase
oil from its seeds has been found useful for medicinal (404 U/ml), pectinase (338 U/ml) and cellulase (134 U/
and veterinary purposes, as insecticide, for soap pro- ml). Promozyme contains pullulanase activity (434 U/
duction and as a fuel substitute (Gubitz et al., 1999). 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.

45. 1.122 S. 43 38 Each extraction was run in duplicate and the yields 29 were found to agree in duplicates within 3%.000 xg for 20 min and and 7-10. curcas L. To this 250 mg enmeshed in protein and cellulosic/hemicellulosic net. Again our yield was lower than 86% re- recovered were calculated as percentages of total oil ported by Winkler et al. AOE). Jatropha seed kernels (5 g) were dispersed in 30 ml distilled water and zymes are used to facilitate release of oil from oil bodies stirred to make a suspension. Thus. gave the best results and 64% (w/w) yield could be ob- lulase. 1984).0 and 9. The above treatment with Protizyme e was at 100 rpm for specified time period (Aqueous oil carried out at pH 4. it was established that only alkaline pro- mixture of all these enzymes were added after the pH of teases should be used during AEOE from J. This is somewhat lower than 38% yield re. 1997). Jatropha oil is reportedly present in the range of 40-60 g oil/100 g 64 Jatropha seed kernels (Makkar et al.. Protizyme e is a extraction. en. the shells carefully re. The upper oil phase was collected mixture of three proteases with pH optima of 3-4. Promozyme as well as tained. Aqueous and aqueous enzymatic oil extraction of oil zymes. Shah et al. in Section 2.1 N NaOH or 0. pH 7. 5-7 after centrifugation at 10. Use of additional enzymes along with Protizyme e Jatropha seeds were cracked. A control (AOE) was run at different temperatures in which no enzyme was added. The amounts of oil seed kernels. 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. Cel. Pectinex Ultra SP-L (250 ll). In agreement with the result of performed similar to AOE (taken as control) except that Winkler et al. Enzyme assisted aqueous oil extraction was three different pH values. 7.1 N HCl. Fig. . 5 £• 30- 15 - r 17 3. (1997). 1999). Pectinex Ultra SP-L. did not improve the oil yield. A value of • Control 60 44 g oil/100 g Jatropha seed kernels was taken as 100% • Enzyme treated recovery of oil while calculating the oil recovery by AOE 47 and AEOE.0. / Bioresource Technology 96 (2005) 121-123 2.0. Cellulase (250 mg) and Protizyme e (250 mg) and also mixture of all these enzymes were added to the suspension. The solvent extraction of Jatropha seed kernels gave a yield of 44 g oil/100 g Jatropha seed kernels. The best result (47%. •. to the inherent nature of seed material used by us.e. The oil was recovered as described result of using various commercial preparations of en. which may also be due present in J.0) was incubated overnight at different temperatures with constant shaking at 100 rpm. Table 1 also shows the with constant shaking at 100 rpm. The ground ported by others (Gubitz et al. the suspension was adjusted. Protizyme e . curcas seed kernels. The latter was deter. (1997). mined by soxhlet extraction using hexane as a solvent as per the standard AOAC procedure (Horowitz.. the alkaline protease component the different enzyme preparations i... The suspension was prepared with powdered were less effective in oil extraction from J. 1 shows the effect of using Protizyme e at weighed. Table 1 Effect of varying temperature on aqueous enzymatic oil extraction Oil yield (%. This is in agreement with moved and the kernels thus obtained were used for oil the earlier observation that hemicellulases and cellulases extraction. (525 U) of Protizyme e was added and incubated overnight at 50 °C works (Rosenthal et al. 1996). In AEOE. 1997). The pH of the suspension was adjusted to the desired value with 0. w/w oil yield) was obtained from Jatropha seed kernels by using Protizyme e (250 mg containing 525 U) at 50 °C. Results and discussion Use of aqueous oil extraction at various temperatures pH was found to yield oil in the range of 17-21% (w/w) only (Table 1). than (obtained by using a homogenizer) Jatropha seed kernels the alkaline protease and their combination with the (5 g) in 30 ml distilled water. Effect of varying pH on oil yield by AEOE. curcas L.1. The suspension was then proteases did not increase the extraction yield (Winkler incubated at desired temperature with constant shaking et al. Fig. Promozyme (250 ll).

Rose hip (Rosa canina L. 1999. was added and incubated overnight at 50 °C with constant shaking at Winkler. 67. 100 rpm. H. 2. Biochem.. J. V. Effect of time of ultrasonication on the oil yield Makkar.. Exploitation of the tropical oil seed plant Jatropha curcas L. 449^156. ultrasonication as a pretreatment may be a good technique to be tried along 0- with AOE..0 9..1 N NaOH before exposing to ultra. A. It was thought prudent to check the presence of moisture in the This work was partially supported by project funds extracted oil. G. The maximum yield (74%. A.. adjusted to 9. Then.0 with 0. water and stirred to make a suspension. 45. Gupta. 2002.K. Horowitz. Rosenthal. Protizyme e (525 U) different extraction methods. Chem. The suspension was then exposed to sonication for 5 All the above experiments (Fig. K. Appl. Aqueous enzymatic 10 17 67 processes for edible oil extraction. N.. M.M. R. Foidl.K. Technol. 28.0 Openshaw. Enzyme Microb. Khare. Council for Scientific and Industrial Re- found that IR spectra of the untreated oil and oil treated search (CSIR) and Department of Science and Technol- with molecular sieves (3 A) were identical. Gubitz. Niranjan.. AEOE (amount of oil obtained. K. Gubitz.0. 2002). Khare. A.. 1996.0. Thereafter. . Thus. w/w) Combined effect of operational variables and enzyme activity in 5 50 74 aqueous enzymatic extraction of oil and protein from soybean. 15 25 69 Sharma.0 and 9. S. Consid- 20 ering that cost of enzyme makes AEOE as a less A A acceptable approach economically. 7. 499-509. W. this may explain better result with alkaline proteases. Shah et al. P. IIT.. Washington... 73-82. 19. S. %. enzyme. Am. B.. It was (TMOP&M). Bioresour. An interesting observation —A— Control was that ultrasonication combined with AOE at alkaline 40 —A— Enzyme Treated pH gave a yield of 67% which was comparable to 64% yield obtained by AEOE (as reported above).. 79. Another useful observation was that AOE. M. Trabi. Both showed ogy (DST). Trinca.L. A review of Jatropha curcas: an oil plant of Control (% w/w) unfulfilled promise. For this purpose. Steiner. M. 1.. a part of the extracted oil from Technology Mission on Oilseeds. 82. Becker.. Soc..S. 2 4 6 8 with or without ultrasonication was more efficient at pH Incubation time (h) 9. K.. DC. S. curcas L. While a similar approach need to be tried with other plant materials. Biomass Bioenergy 19. Delhi for his help in obtaining the J. 195-201. D. Wink. M. all absence of broad water peak in the region 3700-3500 Government of India organizations. The pH of the suspension was Szentmihalyi. The oil was recovered as described in Section 2.. Illes.0 with 5 min ground Jatropha seed kernels (5 g) were dispersed in 30 ml distilled water and stirred to make a suspension. Tables 1 and 2) min before adding Protizyme (250 mg) and incubated for different time were carried out with a process time of about 18 h intervals at 50 °C with constant shaking at 100 rpm. Food Chem..P. 2000. Bioresour. M.. L. 1984.. Gilmour. Methods of analysis of the Association of Official Table 2 Analytical Chemists. Pyle. 10 48 71 Enzyme Microb. Technol. The obtained by carrying out AEOE at pH 9. Pyle. 2 indicate that using ultrasonication as a pretreatment allows one to cut down the process time to about 6 h Acknowledgements without reducing the over all yield of 74%. Technol. Lakatos. Staubman.) oil obtained from waste hip seeds by sonication for different time intervals. K. S. the results described here indicate that ultrasonication pretreatment may be a useful step to References obtain oil by both AOE and AEOE. E. 2001. Technol. Department of Biotechnology (DBT). thank Dr. (incubation with enzyme). 1-15. / Bioresource Technology 96 (2005) 121-123 123 Ultrasonication is emerging as a powerful tool to <5U accelerate many chemical and physical processes. The effects of 60 ultrasonication time on oil yield by both AOE and AEOE are shown in Table 2. Pulses and Maize was treated with molecular sieves overnight. 65. Vinkler. A control was also run in each case in which there was no Enzyme supported oil extraction from Jatropha curcas seeds.. Niranjan. G..... Enzyme assisted aqueous The ground Jatropha seed kernels (5 g) were dispersed in 30 ml distilled extraction of peanut oil. The authors also cm"1. 5 16 61 Rosenthal. 1997. 215-218. D. Sporen.. 3152-3157. adjusted to 7. It has A also been used to increase the oil yield during AOE in some cases (Szentmihalyi et al. The pH of the suspension was ultrasonication as a pretreatment.. seeds and his interest in this work.. Studies on Ultrasonication (min) pH nutritive potential and toxic constituents of different provenances of Jatropha curcas L. 15 25 63 402-420. Effect of ultrasonication time on oil yield by AEOE. 2002..M. Agric. Oil. The data shown in Fig. w/w) however was Fig. Mittelbach. J.N. K. Biotechnol. F. W.L. 1997..