Biochemical Engineering Journal 29 (2006) 220–226 Optimization of enzymatic hydrolysis of triglycerides in soy deodorized distillate with supercritical carbon dioxide Nagesha K. Guthalugu, Manohar Balaraman, Udaya Sankar Kadimi ∗ Department of Food Engineering, Central Food Technological Research Institute, Mysore 570020, India Received 4 August 2005; received in revised form 2 December 2005; accepted 3 December 2005 Abstract Enzymatic hydrolysis of triglycerides of soy deodorized distillate (DOD), using immobilized Candida rugosa lipase under supercritical carbon dioxide (SC-CO 2 ) medium, was carried out. Optimization of the reaction parameters using response surface methodology based on Box-Behnken model at three levels of pressure (120–180 bar), temperature (40–60 ◦ C) and moisture content (40–80% of triglyceride content) for maximum hydrolysis of triglycerides was arrived by multilinear regression of the experimental results. The optimum conditions for maximum degree of triglyceride hydrolysis (94%) were found to be: pressure of 180 bar, temperature of 43 ◦ C and moisture content of 40% to the triglyceride content. Maximum degree of hydrolysis was achieved with short incubation time of 1.5 h under SC-CO 2 . Whereas conventional method of hydrolysis in hexane under similar reaction conditions of temperature, moisture and enzyme concentration, needs 5 h to achieve 88% of triglyceride hydrolysis. © 2005 Elsevier B.V. All rights reserved. Keywords: Soy deodorized distillate (DOD); Supercritical carbon dioxide (SC-CO 2 ); Lipase; Immobilization; Zeolite; Optimization 1. Introduction The soy deodorized distillate (DOD) is a by-product (0.3–0.5%) of soybean oil refining process. Soy DOD is a com- plex mixture of free fatty acids (FFA), triglycerides, sterols, sterile esters, tocopherols and hydrocarbons [1]. Tocopherols and sterols find extensive applications in cosmetic, pharmaceu- tical industries and as natural antioxidants in processed foods. Tocopherol content of the soy DOD varies widely (0.5–15%) depending on the raw material source and the conditions employ- ing during deodorization process and soy DOD is one of the principle source for the manufacture of natural tocopherols [2]. Preparation of tocopherol concentrates using deodorized dis- tillates involves a series of physical and chemical processes, by separating FFA from the distillate in the initial step, followed by separation of sterols to yield a rich tocopherol concentrate. Some of the processes established for the manufacture of tocopherols from the DOD of different vegetable oil sources are urea adduct formation, liquid–liquid extraction with polar and non-polar sol- vent pairs, double distillation, alkali saponification, molecular distillation and supercritical fluid extraction [3–7]. Among these, ∗ Corresponding author. Tel.: +91 821 2514874; fax: +91 821 2517233. E-mail address: udaya@cftri.res.in (U.S. Kadimi). supercritical carbon dioxide (SC-CO 2 ) extraction is found to be as a potential technology for the enrichment of tocopherols from deodorized distillates of various vegetable oils sources [8–11]. Soy DOD as such will not be feasible to work with SC-CO 2 for the tocopherol enrichment, owing to its poor selective solubility for the components of soy DOD. Hence, soy DOD has to be modified to obtain fatty acid esters (FAME) from FFA and fatty acids associated with triglycerides, to improve the selective sol- ubility and preferential extraction of fatty acids in the form of fatty acid esters with SC-CO 2 . For that, triglycerides the major component (55–60 wt%) of soy DOD need to be hydrolyzed to obtain FFA. Attempts have been made to successfully hydrolyse the triglycerides of DOD both chemically, i.e., with acid hydrol- ysis [8] and enzymatically, using commercially available lipases with shake flask method [12]. Our objective is the enzymatic modification of soy DOD using lipases under SC-CO 2 medium, as a preliminary step towards the recovery of tocopherols. Enzymatic modification of soy DOD involves both the hydrolysis and the esterifcation reac- tions using suitable lipases. The present work covers first part of the modification process of soy DOD that is, enzymatic hydrol- ysis of triglycerides under SC-CO 2 medium. SC-CO 2 with a critical pressure of 7.4 MPa and temperature of 31 ◦ C has been used for the extraction and fractionation various food and phar- maceutical components, particularly lipids [13–18]. Recently, 1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.12.001