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Characterization of Enzyme(s) Associated with Sulfur Assimilation Type Reactions in Soy Protein Products
Department of Animal and Food Sciences
The undesirable flavor of soy protein products is a key limiting factor to there increased use as human food. This proposal is designed to isolate and characterize a key enzyme(s) associated with the development of undesirable odors in soy protein food products. Understanding key enzymes is essential to understanding the factors that effect the development of undesirable odors/flavors in soy products and preventing their occurrence.
2009 Project Description
Results from this project have been disseminated to stakeholders through three primary avenues. The first is through scientific publications in peer reviewed journals. Secondly, results have been presented at national meeting to interested parties including annual meetings of the American Chemical Society, the Institute of Food Technologists and the American Oil Chemists Society where attendees from both industry and academia were present. Furthermore, we have been in contact with major soy protein processors such as Archer Daniels Midland and Solae, where we have disseminated information directly by means of telephone conversations and e-mails. The information disseminated directly to soy processors is primarily the same information presented at annual meetings and in publications, but provided in advance by as much as 9 months.
Hexane defatted soy flour was found to contain 106 ppm cysteine-S-sulfonate, which is the precursor of free sulfite and thus the early stages of the sulfate assimilation reactions are not necessary for the formation of free sulfite. We also demonstrated that alkaline extracts of defatted flour contained 329-579 ppm cysteine-S-sulfonate and ISP contained 0-43 ppm. Neither sulfite nor methanethiol were detected until after the soluble components at pH 4.6 were separated from the precipitated soy proteins. The formation of sulfite during the final processing stages of isolated soy proteins (ISP) corresponds to a rapid decrease in cysteine-S-sulfonate, and rapid increase in methanethiol levels. When S34-labeled sodium sulfite was added to aqueous slurries of ISP (as with unlabeled sulfite) methanethiol levels greatly increased but there was no incorporation of the stable isotope into methanethiol. This demonstrates that sulfite is not the sulfur source for methanethiol synthesis. Mass spectra of methanethiol formed with addition of methyl-C13-labeled L-methionine and unlabeled sulfite showed that the carbon-13 labeled methyl group was integrated into methanethiol. Thus, the carbon and sulfur of methanethiol originates from the methyl-carbon and sulfur of methionine.
Sulfite free-radicals are spontaneously formed from sulfite, manganese and oxygen in aqueous solutions. Further reaction with oxygen can also generate sulfate free-radicals which have a one-electron reducing power similar to the hydroxyl radical. Free sulfites formed during the manufacturing of ISP can react with the naturally high levels of manganese that occur in soybeans and oxygen to degrade methionine into methanethiol. Similar aqueous mixtures of sulfite, manganese and oxygen also produce sufficient levels of free radicals that are capable of degrading fluorescein.
The degradation of methionine by free radicals generated in the sulfite, manganese and oxygen reaction mixture is inhibited by the free radical spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide. Processing ISP with either L cystine or potassium iodate reduces the free sulfite content of ISP and reduces the headspace methanethiol from aqueous ISP slurries to non-detectable levels. ISP processed without additives contained sufficient levels of free-radicals to generate methanethiol from the oxidation of added methionine. There were no detectable levels of methanethiol produced when methionine was added to ISP processed with iodate.
This investigation also demonstrated the existence of lipoxygenase independent hexanal formation induced by reducing agents in ISP and the potential requirement of iron as a catalyst. Furthermore, solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of ISP revealed a symmetrical free-radicals signal typical of carbon-centered radicals (g=2.005) ranging from 6.12 x 1014 to 9.10 x 1015 per gram of soy protein in retail food products. These levels of free radicals already present in the "dry" soy protein are from 10 to 100 times higher than levels found in other food protein sources.
Boatright, W.L. and C.J. Stine, Sulfur-Assimilation Type Reactions During Processing of Isolated Soy Proteins: Sulfate to Sulfite , Institute of Food Technologists Annual Meeting Technical Program Book of Abstracts, New Orleans, LA, July 2005.
Boatright, W.L., Q. Lei and J.C. Stine, 2006. Sulfite Formation in Isolated Soy Proteins, Journal of Food Science, 71(3):115-119.
Boatright, W.L. and G. Lu, 2006. A greater than 100,000 MW Soybean Protein Fraction that Inhibits the Formation of Methanethiol and Hydrogen Sulfide in Aqueous Slurries of Isolated Soy Proteins with Added L-Cysteine, Journal of Food Science, 71(3):185-189.
Lei, Q. and W.L. Boatright, 2006. Methionine is the Methyl Group Donor for Sulfite-Associated Methanethiol Formation in Isolated Soy Proteins, Journal of Food Science, 71(9):C527-531.
Boatright, W.L. and G. Lu, Hexanal Synthesis in Isolated Soy Proteins, American Oil Chemists Society Annual Meeting Technical Program Book of Abstracts, Louis, MO, July 2006.
Boatright, W.L. and Lu, G,. 2007. Hexanal Synthesis in Isolated Soy Proteins, Journal of the American Oil Chemists' Society, 84(3):249-257.
Lei, Q. and Boatright, W.L., 2007. Sulfite Radical Anions in Isolated Soy Proteins, Journal of Food Science, 72(5):C302-307.
Boatright W.L., Q. Lei and M.S. Jahan, 2009. Effect of Storage Conditions on Carbon-Centered Radicals in Soy Protein Products, Journal of Agricultural and Food Chemistry, 57(17):7969-7973.