An aqueous enzymatic procedure for oleosome fractionation from 25 g of soy flour was developed in our laboratory. This fractionation procedure was evaluated with 75 kg using pilot plant equipment to evaluate the effect of the scale-up on the recovery, proximate composition, soybean storage protein profiles, and subcellular microstructure of oleosome fractions. The process included enzymatic hydrolysis, grinding, and centrifugation, respectively. Pilot-scale grinding and centrifugation of the slurry were accomplished with a Stephan Ò Microcut mill grinder and a three phase decanter. A blender and swinging bucket rotor were used for the laboratory-scale fractionation. The oleosome fractions recovered in the pilot plant were similar in oil and protein content to those obtained in the laboratory. The pilot-scale process resulted in a significantly higher oil yield of 93.40% as total oleosomes compared to that of 76.83% achieved in the laboratory. Urea-SDS gel electrophoresis of proteins extracted from the oleosomes and supernatant from the pilot-scale fractionation had similar profiles to those obtained in the laboratory. Electron microscopy verified that the structure of isolated oleosomes was virtually identical with that of in situ oleosomes. This work confirms that large-scale fractionation of oleosomes from full fat soybean flour can be accomplished.

Oleosome extractions from soybean flour typically generate significant quantities of aqueous sucroseand sodium chloride-rich supernatant which could be recycled. To determine the feasibility of recycling the oleosome process aqueous supernatants, three extraction protocols were evaluated. The first extraction used the original extraction solution, 0.1 M fresh potassium acetate pH 4.6 containing 0.4 M sucrose and 0.5 M NaCl. The second protocol reused the aqueous supernatant obtained from the first extraction. The third protocol reused the aqueous supernatant obtained from the second protocol. Oleosome extraction yields were significantly higher in the first extraction with enzymes (Multifect Ò Pectinase FE, Multifect Ò GC, and Multifect Ò CX B, 1% each, v/w) compared to the yield when the supernatant was reused with no additional enzymes (81.41 ± 2.24 vs. 73.09 ± 3.39%, respectively). Oil yields from oleosome fractions were not statistically different when extractions were made with 0 or 3% enzymes in the third protocol. Protein was the predominant constituent in the supernatant in addition to mineral and carbohydrate. Soybean storage protein profile from recycled supernatants obtained without adding enzyme were similar to a traditional soy protein water extract but with a decrease of intensity of the b-conglycinin bands. Addition of 3% enzymes in both recycling protocols resulted in the disappearance of the a 0 and a subunits of the b-conglycinin due to a protease contaminant in Multifect Ò Pectinase FE. Results from this work revealed essential information for a promising possibility of the future industrial application of this technology.

Enzyme-assisted aqueous extraction of oil from isolated soybean oleosomes was evaluated as an alternative to the conventional organic solvent extraction. Three different processes: hydrolysis of oleosomes, thermal demulsification 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 oleosomes 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.

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The operational variables impacting the quantity and quality of free oil recovered from isolated soybean oleosomes by enzymatic extraction were evaluated to optimize this process. Those variables were: protease concentration (0-2.5%), protease hydrolysis time (3 vs. 18 h), and slurry destabilization time (30 min vs. 3 h). Analysis of interactions between these variables and the yield of free oil revealed that the protease concentration was the most significant variable. The quantity of free oil extracted by using 3 h of oleosomes hydrolysis and 30 min of slurry destabilization was not significantly different from that using 18 h of oleosomes hydrolysis and 3 h of slurry destabilization. The optimum conditions, 0.5% Protex 6L, 3 h of hydrolysis, and 30 min of destabilization, resulted in 90% free oil. Oils extracted by the aqueous process had a fatty acid composition similar to conventional hexane-based process with oxidative stability indices ranging from 9 to 12 h. Enzyme assisted aqueous extraction resulted in a high quality oil which has 88% less free fatty acids than hexane-extracted oil. The optimal conditions generated 85.5% soybean storage proteins in skim with peptides smaller than 6.5 kDa and the degree of hydrolysis of 19.5%. The present study demonstrates that oil can be extracted from soybeans efficiently without hexane or other petroleum solvents.