Progress 11/01/03 to 10/31/06
Outputs
Because hexane defatted soybean flour contains 106 ppm cysteine-S-sulfonate (the precursor to free sulfite), the early sulfate assimilation reactions do not appear to be significant to the formation of free sulfite and methanethiol in isolated soy proteins (ISP). 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 stages of ISP processing 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 the unlabeled sulfite) methanethiol levels greatly increased but there was no incorporation of the stable isotope into methanethiol. This conclusively demonstrates that sulfite is not 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. 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 occurring manganese and oxygen to degrade methionine into methanethiol. In aqueous solutions, sulfites and manganese, at levels found in ISP (17-33 ppm and 17 ppm, respectively), react with oxygen to produce sufficient free radicals to degrade methionine into methanethiol and other compounds. The increase in methanethiol with the addition of cysteine appears to be caused by a different mechanism than the increases from added DTT (release of bound sulfite). DTT, but
not cysteine, causes large increases in methanethiol from aqueous-defatted flour. There is little hydrogen sulfide produced with added DTT while cysteine causes large increases in hydrogen sulfide. Also, the pyridoxal phosphate inhibitor, amino oxyacetic acid (AOA), had a greater inhibitory effect on methanethiol formation from added cysteine than added DTT. AOA also caused hydrogen sulfide formation with added DTT, which did not occur without AOA. A different pathway for cysteine would be consistent with the proposed mechanism outlined in the sulfur assimilation pathway. Another important indicator is our discovery of a protein in defatted flour, with a MW of >100,000 and a pI of 5.9, that prevents the conversion of added cysteine to hydrogen sulfide and methanethiol in aqueous commercial ISP. We found that the majority of this protein is soluble at pH 4.6, and is largely separated from ISP during isoelectric precipitation. The portion of this protein not lost during isoelectric
precipitation is inactivated during the temperatures used in commercial ISP processing.
Impacts
Soybeans are the second largest food-crop grown in the US with 85 million metric tons produced in 2004 with a value in excess of $17 billion. Only about 1.5 percent of soy proteins are used in human foods, largely because of flavor problems. Soy protein products with improved flavor will promote their consumption by humans and add value to the US soybean crop. Sulfur-containing compounds (e.g., methanethiol) and lipid oxidation products (e.g., hexanal) are the most potent odorants found in soy protein products. As a result of our research, we have found that certain additives that are generally used to reduce lipid oxidation in foods (reducing agents such as ascorbic acid and sodium erythrobate) cause large increases in both hexanal and the sulfur-containing odorants when added to aqueous soy proteins. The largest soy processing companies in the United States have already applied this simple but important finding to their soy products. Furthermore, as a result of the
findings of this research, commercial processes are being designed to minimize the occurrence of free sulfites in soy protein products and thus reduce the level of sulfur-containing odorants.
Publications
- 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 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.
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Progress 01/01/05 to 12/31/05
Outputs
Defatted soybean flour contained 2,820 parts per million (ppm) inorganic sulfate. The corresponding laboratory isolated soy proteins (ISP) contained 1,364 ppm sulfate bound to a greater than 3,000 molecular weight fraction. Defatted soy flour contained 0.98 nmoles of adenosine triphosphate (ATP) per 0.1 g. ATP levels decreased when the defatted flour was hydrated under a variety of conditions. Neither adenosine-5'-phosphosulfate or 3'-phosphoadenosine-5'-phosphosulfate could be detected in defatted soybean flour or in aqueous extracts of defatted flour. Defatted soy flour contained 1034 ppm reduced glutathione and 647 ppm reduced homoglutathione. These levels dropped to 62 and 13 ppm, respectively, immediately after the isoelectric precipitation step of ISP processing. No oxidized glutathione or homoglutathione were detected. Defatted flour contained 106 ppm cysteine-S-sulfonate, alkaline extracts of defatted flour contained 329-579 ppm 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. These findings indicate that cysteine-S-sulfonate is present in defatted flour and increases during ISP processing. Also, the formation of sulfite during the final stages of ISP processing corresponds to elevated methanethiol levels. Also, common food grade reducing agents (such as ascorbic acid and sodium erythrobate) cause large increases in both hexanal and the sulfur-containing odorants when added to aqueous soy proteins.
Impacts
Soybeans are the second largest food-crop in the US and are grown primarily as a source of edible oil. Only about two percent of soy proteins are used in human foods, largely because of their undesirable taste. Sulfur containing compounds (methanethiol and dimethyl trisulfide) and lipid oxidation products (e.g., hexanal) are the most potent odorants found in soy protein products. As a result of our research, we have found that certain additives that are generally used to reduce lipid oxidation in foods (reducing agents such as ascorbic acid and sodium erythrobate) cause large increases in both hexanal and the sulfur-containing odorants when added to aqueous soy proteins. The largest soy processing companies in the United States have already applied this simple but important finding to their soy products.
Publications
- Boatright, W.L. and Stine, C. J. 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.
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Progress 01/01/04 to 12/31/04
Outputs
Hexane-defatted soybean flour was found to contain 1,480 ppm total sulfate (0.15%) using an acid hydrolysis/HPLC method. Adenosine-5'-triphosaphe (ATP), which is required by ATP sulfurylase to convert inorganic sulfate to an active form of organic sulfate, was present in defatted flour at 980 pmol per 0.1g, similar to the values found for dry soybean cotyledons. Unlike the ATP increases upon germination of intact codyledons, the ATP level in defatted flour decreased rapidly upon hydration of the defatted flour. The addition of magnesium, ammonium or sodium sulfate during ISP processing had no significant effect on the sulfite or methanethiol content of ISP. Using perchloric acid and alkaline extracts, the common forms of activated (organic) sulfate found in plants (adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine-5'-phosphosulfate (PAPS)) were not found in defatted flour or samples obtained at various stages of ISP processing. Adenosine-5'-monophosphate (the
degradation product of APS and PAPS) was the predominate adenylate compound. Significant quantities a previously unreported adenosine compound was found. The sulfite content of hexane-defatted soybean flour was about 1 ppm, and ISP contained 21-33 ppm sulfite. Analyses of samples obtained during various stages of ISP processing revealed that no sulfites were formed until immediately after the isolated soy proteins were separated from an unknown component in the pH 4.6 supernatant during protein isoelectric precipitation. Another important finding was that the reducing agents erythrobate and nitrite, which are commonly added to food products as reducing agents/antioxidants, caused pronounced increases in headspace methanethiol in aqueous ISP. Several observations indicate that the increase in methanethiol with the addition of cysteine is caused by a different mechanism than the increases from added dithiothreitol (DTT). DTT, but not cysteine, causes large increases in methanethiol from
aqueous defatted flour. There is little hydrogen sulfide produced with added DTT while cysteine causes large increases in hydrogen sulfide. Also, the pyridoxal phosphate inhibitor, amino oxyacetic acid (AOA), had a greater inhibitory effect on methanethiol formation from added cysteine than added DTT. AOA also caused hydrogen sulfide formation with added DTT, which did not occur without AOA. A different pathway for cysteine would be consistent with the proposed mechanism outlined in the sulfur assimilation pathway. Another important indicator is our discovery of a protein in defatted flour, with a MW of >100,000 and a pI of 5.9, that prevents the conversion of added cysteine to hydrogen sulfide and methanethiol in aqueous commercial ISP. We found that the majority of this protein is soluble at pH 4.6, and is separated from ISP during isoelectric precipitation. The portion of this protein not lost during isoelectric precipitation is inactivated during the temperatures used in
commercial ISP processing. Amino acid analyses indicated that free amino acids (e.g., cysteine) are not the primary source of methanethiol in aqueous ISP without additives.
Impacts
This research will lay the foundation for novel processes to produce soy protein products with improved flavor.
Publications
- Boatright, W.L. and Stine, J., 2004. Residual Sulfur Metabolites in Isolated Soy proteins: Sulfite to Cysteine, Journal of Food Science, 69(3):200-205.
- Stine, C.J. and Boatright, W., 2004. Intrinsic Sulfite Content of Isolated Soy Proteins, American Oil Chemists' Society Annual Meeting Technical Program Book of Abstracts, p 114, Cincinnati OH.
- Stine, C.J., Boatright, W., and Lu, G. 2004. Intrinsic Sulfite Content of Isolated Soy Proteins, Journal of the American Oil Chemists' Society, 81(9):829-833.
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