ORIGINAL PAPER Enzyme-Assisted Aqueous Extraction of Oil from Isolated Oleosomes of Soybean Flour Lili T. Towa • Virginie N. Kapchie • Catherine Hauck • Patricia A. Murphy Received: 21 August 2009 / Revised: 21 October 2009 / Accepted: 23 October 2009 / Published online: 10 December 2009 Ó AOCS 2009 Abstract Enzyme-assisted aqueous extraction of oil from isolated soybean oleosomes was evaluated as an alternative to the conventional organic solvent extraction. Three dif- ferent processes: hydrolysis of oleosomes, thermal demul- sification of the skim or the slurry, and destabilization of the cream by the churning butter process were examined to enhance the release of free oil from isolated oleosomes. The oil extraction involved incubating the oleosomes with either 0, 2.5 or 5% protease (Protex 6L Ò ) at 60 °C, pH 9 for 18 h, destabilizing the slurry by three thermal strategies: freeze/thaw, freeze/thaw and heating, and destabilizing the cream by the churning butter process without and with 5% of phospholipase A 2 (Multifect L1 10L Ò ), at 40 °C, pH 8 for 4 h. The best total free oil yield was 83–88% by hydrolyzing oleosomes with 2.5 or 5% Protex 6L Ò , destabilizing the slurries by heating and destabilizing the resulting cream by the churning butter process. The oleo- somes treated with 2.5 and 5% proteases generated hydrolyzed soybean storage proteins at 18–20% degree of hydrolysis, with all the storage proteins hydrolyzed to peptides smaller than 6.5 kDa, compared to the oleosomes disrupted without proteases. Keywords Soy oleosomes Á Soy oil Á Aqueous-extraction Á Hydrolysis Á Destabilization Introduction The standard practice of oil extraction from seeds in the industry is the organic solvent extraction leading to a cake with residual oil content below 1%, but with inherent disadvantages of high investment and energy requirement [1]. Hexane, the common organic solvent used represents a fire and explosion hazard as well as neurological and respiratory disorders of the workers from prolonged exposure [1, 2]. These economic, safety, environmental and health concerns propelled the need to find an alterna- tive safe and efficient oil extraction process from seeds. Oleosomes or oil bodies are discrete subcellular organelles mainly found in oilseeds, which consist of a lipid core that is surrounded by a phospholipid monolayer in which oleosins are embedded which are proteins that stabilize the oleosome structure and prevent coalescence of the oil in the cell cytoplasm [3, 4]. Oleosome purification from oilseeds and oleosome characterization for different purposes have been reported [5–12]. Recently, a laboratory process for isolating oleosomes from 25 g of soybean flour [13] was evaluated on a mass balance basis. This successful process was scaled up to a pilot plant scale for 75 kg of soybean flour resulting in improvement in the oil yield, as extracted oleosomes, to 93 ± 0.88% (Towa et al. submitted). This research provided a strategy to isolate oleosomes from soybean flour on a large scale and suggested the feasibility of the process for industry. Based on the structure of oleosomes, we hypothesized that free oil could be released from those organelles by disrupting the membrane with specific enzymes during a new process of enzyme-assisted aqueous extraction (EAAE). EAAE has already been studied as an environmentally clean technology to simul- taneously extract free oil and protein from oilseeds including soybean [1, 14–19]. Particularly for the soybean, L. T. Towa (&) Á V. N. Kapchie Á C. Hauck Á P. A. Murphy Department of Food Science and Human Nutrition, Center for Crops Utilization Research, Iowa State University, Ames, IA 50011, USA e-mail: ltowa@iastate.edu 123 J Am Oil Chem Soc (2010) 87:347–354 DOI 10.1007/s11746-009-1503-3