Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning Shweta Shah, Aparna Sharma, M.N. Gupta* Department of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India Received 15 April 2003; accepted 16 October 2003Abstract Three phase partitioning has been evaluated for extraction of oil from Jatropha curcas L. seeds. This process consisted ofsimultaneous addition of t-butanol (1:1, v/v) and 30% (w/v) ammonium sulphate to the slurry prepared from Jatropha seedkernels. Combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9, led to 97%oil yield within 2 h.Keywords: Enzyme assisted three phase partitioning; Jatropha seed kernel oil; Three phase partitioning1. Introduction this process with solvent extraction. Hexane is the sol- vent which is generally used in that process. t-Butanol Recently, we have described a new approach for has a higher boiling point (84 °C) than hexane (69 °C),extraction of oil from plant materials, in general, and hence addition of volatile organic solvents to the at-soybean in particular (Sharma et al., 2002a). In this ap- mosphere during TPP will be much lower even if openproach, called three phase partitioning (TPP), appro- systems are used (Rosenthal et al., 1996). Thus, sim-priate amounts of ammonium sulphate and t-butanol pler extraction designs will be possible with TPP.were added to an aqueous suspension of soybean meal. Jatropha is a genus comprising 70 species grow-This led to the formation of three distinct phases: up- ing in tropical and subtropical countries (Openshaw,per organic phase, lower aqueous phase and interfa- 2000). The seed kernel contains 40-60% (w/w) oilcial precipitate (consisting mostly of proteins). The oil (Makkar et al., 1997). The presence of some antinu-was found in the organic solvent phase and could be tritional factors renders this oil unsuitable for use inrecovered by evaporating the t-butanol. TPP is simple cooking (Makkar et al., 1997; Gubitz et al., 1998).to carry out, has short processing time (about 1 h), and Some efforts are being made for removal of theseis easy to scale up. It may be also useful to compare antinutritional factors (Haas and Mittelbach, 2000). Meanwhile, as such, it has been used for production of biodiesel (Foidl et al., 1996). The present work demonstrates the application of TPP in extraction of oil from seed kernels of Jatropha
276 S. Shah et al. / Industrial Crops and Products 20 (2004) 275-279curcas L. An important further step in the process 3. Results and discussiondevelopment has been the pretreatment of the plantmaterial with enzymes before TPP. The solvent extraction of Jatropha seed kernels gave a yield of 44 g oil/100 g Jatropha seed kernels. Jat- ropha oil is reportedly present in the range of 40-60 g2. Materials and methods oil/100 g Jatropha seed kernels (Makkar et al., 1997). A value of 44 g oil/100 g Jatropha seed kernels was Jatropha seeds were obtained from Dr. Zope, Col- taken as 100% recovery of oil when calculating thelege of Forestry, Akola, India. ProtizymeTM was oil recovery by TPP.purchased from Jay sons Agritech Pvt. Ltd., Mysore, The effect of varying ammonium sulphate duringIndia. It is reported to contain mostly acid (pH op- TPP is shown in Fig. 1. About 83% (w/w) oil could betimum range 3-4), neutral (pH optimum range 5-7) obtained by using 30% (w/v) ammonium sulphate. Inand alkaline proteases (pH optimum range 7-10) many other cases as well, where TPP has been used forfrom Aspergillus flavus with a specific activity of separation of proteins/enzymes generally ammonium2.1U/mg protein with casein as substrate. All other sulphate 30% (w/v) has given best results (Dennisonchemicals and solvents used were of analytical and Lovrein, 1997; Sharma and Gupta, 2001a). Ingrade. the case of extraction of soybean oil by TPP also, 30% (w/v) ammonium sulphate was found to be op- 2.1. Three phase partitioning for extraction of oil timum (Sharma et al., 2002a). Fig. 2 shows the effectfrom Jatropha curcas L. of varying the ratio of volumes of t-butanol to Jat- ropha slurry. Decreasing the t-butanol volume by half Three phase partitioning was carried out as de- decreases the oil yield whereas increasing t-butanolscribed earlier (Sharma et al., 2002a). Jatropha seeds to twice the volume did not lead to any significantwere cracked, the shells carefully removed and the increase. Thus, 1:1 ratio was employed in furtherkernels thus obtained were used for slurry prepara- studies.tion. The slurry was prepared by grinding Jatropha It is well established that oil bodies in plantsseed kernels (5g/30ml) in distilled water. The pH are trapped in the meshwork of proteins and cel-of the slurry was adjusted to the desired value with lulose/hemicellulose structures. Thus, the treatment0.1 N NaOH or 0.1 N HCl. The appropriate amount with enzymes is an integral part of an approach calledof ammonium sulphate was added and vortexed gen- aqueous enzymatic oil extraction (AEOE) (Rosenthaltly, followed by addition of appropriate amount of et al., 1996). AEOE has been successfully used in ex-t-butanol. The slurry was then incubated at 25 °C for traction of oils from rice bran (Sharma et al., 2001b)1 h for the three phase formation. The three phases and peanut (Sharma et al., 2002b). Thus, it was con-were then separated by centrifugation at 2000 x g for sidered worthwhile to attempt pretreatment of the10min. The upper organic layer was collected and Jatropha slurry by enzymes before carrying out TPP.evaporated on rotary evaporater (under reduced pres- In order to distinguish it from other approaches, wesure at 50 °C, for 5min) to obtain oil. The amounts would like to call this approach enzyme assistedof oil recovered were calculated as percentages of three phase partitioning (EATPP).total oil present in Jatropha seed kernels. The latter In an earlier case (Sharma et al., 2002b), a com-was determined by soxhlet extraction using hexane mercial preparation called ProtizymeTM had givenas a solvent as per the standard AOAC procedure good results with AEOE. ProtizymeTM is a mixture(Horowitz, 1984). The FT-IR of oil obtained by TPP of enzymes, its three main components are pro-was done by taking oil (10 jxl) on KBr disc, the teases with different pH optimum range of 3-4, 5-7spectra was recorded on Nicolet-Protégé-460 spec- and 7-10. Hence, treatment of Jatropha slurry withtrometer. ProtizymeTM before carrying out TPP was attempted Each extraction by three phase partitioning was run (Fig. 3). Extraction of oil was carried out at three dif-in duplicate and the yields were found to agree be- ferent pH values: 4.0, 7.0 and 9.0, since the enzymetween duplicates within 3%. preparation has three proteases with these pH optima.
S. Shah et al. /Industrial Crops and Products 20 (2004) 275-279 277 40 50 Ammonium sulphate (%, w/v)Fig. 1. Effect of varying amount of ammonium sulphate in TPP on oil extraction. Slurry was prepared by dispersing ground Jatrophaseeds (5g/30ml distilled water, pH 7.0). Varying amount of ammonium sulphate (%, w/v) was added to this slurry followed by additionof t-butanol (30 ml). The oil was recovered from upper t-butanol layer by following the procedure described in the Section 2.By enzyme pretreatment at pH 9.0 followed by TPP, higher yields (Rosenthal et al., 2001). Sonication is92% oil could be obtained (Fig. 3). It has been re- an efficient way for breaking up bigger particles intocently shown that the particle size of plant material smaller ones (Szentmihalyi et al., 2002). It was foundplays an important role in giving optimum oil yield that using sonication before enzyme pretreatmentby AEOE. Smaller particle size was found to give increased the oil yield upto 97% (Fig. 4). 1:0.5 1:1 Ratio of slurry:t-butanolFig. 2. Effect of varying the ratio of t-butanol to the Jatropha slurry in TPP on oil extraction. Ammonium sulphate (30%, w/v) was addedto the slurry and vortexed gently followed by addition of t-butanol to achieve different volumetric ratios of slurry to t-butanol. The oilwas recovered as described in the Section 2.
278 S. Shah et al. / Industrial Crops and Products 20 (2004) 275-279 7.0 9.0 pHFig. 3. Effect of enzyme pretreatment in TPP on oil extraction. The pH of the slurry was adjusted to 4.0, 7.0 and 9.0 before addingProtizymeTM (250mg). The slurry was incubated at 50 °C with constant stirring at 100rpm for 1h. Thereafter, ammonium sulphate (30%,w/v) and t-butanol in a ratio of 1:1 (v/v) were added to the slurry. The oil was recovered from upper t-butanol layer as described inthe Section 2. A control was also carried out by omitting the addition of enzymes. Control (dotted), enzyme treated TPP (verticallyhatched). 100 1 97 92 82 80 - 60 - £ 40 1 20 - TPP EA-TPP Sonication+EATPP ProcessFig. 4. Effect of sonication in TPP on oil extraction. The pH of the slurry was adjusted to 4.0, 7.0 and 9.0 followed by sonication for 5min.After addition of 250 mg ProtizymeTM, the slurry was incubated at 50 °C with constant stirring at 100 rpm for 1 h. After this pretreatment,ammonium sulphate (30%, w/v) and t-butanol in the ratio of 1:1 (v/v) (ratio of slurry to t-butanol) were added. The oil was recoveredfrom upper t-butanol layer as described in Section 2. (TPP, three phase partitioning; EA-TPP, TPP in presence of enzyme; sonication+ EA-TPP; sonication for 5 min followed by TPP in presence of enzymes). The oil obtained was further analysed by FT-IR partitioning (and removal of t-butanol after evapora-spectroscopy to detect the presence of t-butanol in the tion at reduced pressure).oil. The characteristics hydroxyl peak at 3368 cm" 1was absent in the IR spectra of Jatropha oil, con-firming the absence of t-butanol. The detection limit 4. Conclusionof FT-IR (as determined with standard solutions oft-butanol) was found to be 1%. Besides, no smell of The novel approach of combining a recently de-t-butanol was found in the oil obtained by three phase veloped technique of TPP with enzyme pretreatment
S. Shah et al. /Industrial Crops and Products 20 (2004) 275-279 279and sonication constitute an efficient procedure for Haas, W., Mittelbach, M., 2000. Detoxification experiments withobtaining oil from Jatropha seed kernels. It should the seed oil from Jatropha curcas L. Ind. Crops Prod. 12, 111-118.be added that unlike the soxhlet extraction, which is Horowitz, W., 1984. Methods of Analysis of the Association ofcarried out for 24 h, the method developed here takes Official Analytical Chemists. Washington, DC.only about 2 h. Makkar, H.P.S., Becker, K., Sporen, F., Wink, M., 1997. Studies on nutritive potential and toxic constituents of different provenances of Jatropha curcas L. J. Agric. Food Chem. 45, 3152-3157.Acknowledgements Openshaw, K., 2000. A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioenerg. 19, 1-15. This work was partially supported by project funds Rosenthal, A., Pyle, D.L., Niranjan, K., 1996. Aqueous andfrom Technology Mission on Oilseeds, Pulses and enzymatic processes for edible oil extraction. Enzyme Microb.Maize (TMOP&M), Council for Scientific and In- Technol. 19, 400^120. Rosenthal, A., Pyle, D.L., Niranjan, K., Gilmour, S., Trinca, L.,dustrial Research (CSIR) and Department of Science 2001. Combined effect of operational variables and enzymeand Technology (DST), Department of Biotechnology activity in aqueous enzymatic extraction of oil and protein(DBT), all Government of India organisations. from soybean. Enzyme Microb. Technol. 28, 499-509. Sharma, A., Gupta, M.N., 2001a. Purification of pectinases by three phase partitioning. Biotechnol. Lett. 23, 1625-1627. Sharma, A., Khare, S.K., Gupta, M.N., 2001b. Enzyme assistedReferences aqueous extraction of rice bran oil. J. Am. Oil Chem. Soc. 78, 949-951.Dennison, C., Lovrein, R., 1997. Three phase partitioning: Sharma, A., Khare, S.K., Gupta, M.N., 2002a. Three phase concentration and purification of proteins. Protein Exp. Purif. partitioning for extraction of oil from soybean. Bioresour. 11, 149-161. Technol. 85, 327-329.Foidl, N., Foidl, G., Sanchez, M., Mittelbach, M., Hackel, S., Sharma, A., Khare, S.K., Gupta, M.N., 2002b. Enzyme assisted 1996. Jatropha curcas L. as a source for the production aqueous extraction of peanut oil. J. Am. Oil Chem. Soc. 79, of biofuel in Nicaragua. Bioresour. Technol. 58, 77- 215-218. 82. Szentmihalyi, K., Vinkler, P., Lakatos, B., Vendel, I., Then, M.,Gubitz, G.M., Mittelbach, M., Trabi, M., 1998. Exploitation of the 2002. Rose hip (Rosa canina L) oil obtained from waste hip tropical oil seed plant Jatropha curcas L. Bioresour. Technol. seeds by different extraction methods. Bioresour. Technol. 82, 67, 73-82. 195-201.