NOTE Method of phorbol ester degradation in Jatropha curcas L. seed cake using rice bran lipase Chusnul Hidayat, * Pudji Hastuti, Avita Kusuma Wardhani, and Lana Santika Nadia Graduate Program on Estate Crop Product Technology, Universitas Gadjah Mada, Jl. Flora, Bulaksumur, Yogyakarta 55281, Indonesia Received 18 February 2013; accepted 9 August 2013 Available online 4 October 2013 A novel enzymatic degradation of phorbol esters (PE) in the jatropha seed cake was developed using lipase. Cihera rice bran lipase had the highest ability to hydrolyze PE, and reduced PE to a safe level after 8 h of incubation. Enzymatic degradation may be a promising method for PE degradation. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Phorbol ester; Enzymatic degradation; Lipase; Rice bran; Jatropha curcas] The defatted pressed jatropha seed cake (DPJSC) has high pro- tein content and the essential amino acid contents are higher than the FAO amino acid reference except lysine (1). However, DPJSC has not been utilized as food or feed due to the content of phorbol esters (PE), which are very toxic (2,3). PE and its derivatives were also reported to potentially cause tumors (4). Most of the PE degradation treatments were not environmen- tally friendly or time consuming. PE degradations were performed using a combination of alkaline and high temperature treatments (1,5e7) and followed by solvent extraction (1,5). Both an alkaline treatment combined with an ozone treatment and the g-irradiation were also reported (7). Beside, biological treatment using Pseudo- monas aeruginosa PseA, Bjerkandera adusta, Phlebia rufa, and Aspergillus niger , all of which produced lipase, could degrade PE during fermentation (8e10). Co-composting DPJSC with rice straw also reduced PE content (11). But the degradation of PE due to lipase activity or any other reason was not explored yet. PE have ester bonds between phorbol moiety and its fatty acid, which are an analog to diacyl glycerol (2,12e14). Therefore, it may be hydrolyzed by lipase. Some researchers reported that the esterase of rat liver, rat plasma, and rat submandibular gland cells could degrade PE (13,15,16). However, data on the enzymatic PE degradation are very limited. In this research, a novel of the enzy- matic PE degradation in DPJSC was developed using rice bran lipase as biocatalyst. DPJSC powder was prepared according to the method previously described (17) with some modifications. Jatro- pha seeds were peeled. The kernel was separated and grinded using a homogenizer. It was dried at 40  C for 24 h. Kernel powder was pressed by a hydraulic press at a pressure of 450 kg/cm 2 . Further- more, pressed jatropha seed cake was grinded and sieved using a 40 mesh siever. Oil in the pressed jatropha seed cake powder (PJSC) was extracted using hexane to produce DPJSC. Crude, defatted rice bran lipase varieties of mentik susu, pepe, and cihera were prepared by dried acetone method according to the method previously described (18) with some modifications. Rice bran was defatted by adding 20 g of rice bran into 35 ml acetone (18  C) and mixed using a homogenizer at 5200 rpm for 10 min. Homogenization was stopped every 3 min for 1 min. Rice bran suspension was added into the column, then washed with acetone at 4  C. Defatted rice bran was further removed from the column and dried. It was used as crude defatted rice bran (DRB) lipase powder and stored at 18  C until use. Hydrolytic activity of various rice brain varieties on acyl glycerol was determined by adding DRB lipase (0.1 g) into the flask con- taining of 2 ml of olive oil in isooctane (60% v/v), and 2 ml of phosphate buffer pH 7. The mixture was incubated in a shaker water bath at 30  C and 120 strokes per min for 20 min. The reaction was stopped by placing the flask into the ice bath. About 200 mL of oil layer was added into a mixture of 1.8 ml isooctane and 0.4 ml Cu acetate pyridine pH 6. The mixture was then mixed using a vortex for 5 min and incubated for 10 min. Absorbance was measured at a wavelength of 715 nm. The unit of lipase activity is expressed as the amount of mmol free fatty acids that are hydrolyzed by lipase per min. A novel of the enzymatic PE degradation was developed as follow: (i) selection of the best rice bran lipase: one gram of DRB lipase (varieties of mentik susu, pepe, and cihera) were added into flasks containing a suspension of DPJSC (5 g) in a phosphate buffer of pH 7 (40 ml). The suspensions were incubated in a shaker water bath at 30  C and 120 strokes per min for 24 h; (ii) effect of DPJSC to phosphate buffer ratio on PE degradation: it was performed by adding 1 g of DRB lipase into flasks containing a suspension of various ratios of DPJSC to phosphate buffer (1:8, 1:10, 1:12, 1:14 and 1:16), and they were incubated in a shaker water bath at 30  C and 120 strokes per min for 24 h; (iii) effect of reaction time on PE degradation: it was performed by adding DRB lipase (1 g) into flasks containing of DPJSC (5 g) in a phosphate buffer of pH 7 (40 ml), and they were incubated in a shaker water bath at 30  C and 120 strokes * Corresponding author. Tel./fax: þ62 274 549650. E-mail address: chusnul@gadjahmada.edu (C. Hidayat). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. 117 No. 3, 372e374, 2014 1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.08.006