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Formation & Reactivity of Carbon-Centered Radicals in Isolated Soy Proteins
W.L. Boatwright, M.S. Jahan
Department of Animal and Food Sciences
Soybeans are the second largest food crop in the U.S.A., with about 87 million metric tons produced in 2006. Soybeans provide high-quality protein, and in 1999 the Food and Drug Administration gave food manufacturers permission to put labels on products high in soy protein indicating that these foods may help lower the risk of heart disease. Generally, high-fat foods that have been exposed to excessive levels of free radical activity smell rancid, and are not considered acceptable for human consumption. However, purified proteins are capable of acting as free-radical traps and thus can lead to the unintentional ingestion of high levels of free-radicals, as well as poor shelf-life and loss of nutritional qualities.
Using solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of isolated soy proteins (ISP) we found a symmetrical free-radicals signal typical of carbon-centered radicals (g=2.005) ranging from 296 trillion to 642 trillion spins per gram (Boatright and others, 2008). The level of free-radicals in ISP was 14 times greater than similar radicals in sodium caseinate, 29 times greater than egg albumin and about 100 times greater levels than casein. Nine soy protein powdered drink mixes contained similar types of free-radicals, but at levels up to 4,100 trillion spins per gram of drink mix, or up to 6.4 times greater than the highest free radical content found in commercial ISP. Laboratory prepared ISP samples contained trapped radicals similar to the levels in commercial ISP samples.
Preliminary stability studies of the ISP stored in the dark at 23°C in sealed containers found that the level of free-radicals increase by as much as 35 fold during the first 9 weeks of storage. This indicates a mechanism for the formation of these radicals in ISP that differs from previously proposed mechanisms.
The high levels of free radicals in the commercial soy protein products examined can contribute to numerous degradation reactions once the protein is hydrated, and consuming such high levels of free radicals may have harmful consequences in humans. This is the first time the levels of free radicals in commercial ISP, and food products high in ISP, has been reported. It is also the first time the large increase in free radicals during ISP storage (which appears to account for the majority of free radicals) has been reported.
The goal of this project is to investigate the mechanism that free-radicals in ISP are formed and stabilized within the "dry" protein, and to investigate the reactions catalyzed by carbon-centered radicals in ISP once the protein is hydrated. Results from these findings should allow us to minimize the occurrence of the free-radicals in commercial soy protein products, and to begin to determine how harmful these free-radicals can be when consumed.
2010 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 meetings to interested parties including the annual meeting of the Institute of Food Technologists (where attendees from both industry and academia were present) and at the NRI/AFRI project directors meeting. Furthermore, we have been in direct contact with largest U.S. soybean processor (the Archer Daniels Midland Co.) 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 provide in advance by as much as 9 months.
Carbon-centered free radicals ranging from 6.12 x 1014 to 9.10 x 1015 per gram of protein have been reported in retail food products containing soy protein. The level of these metastable radicals in soy proteins are from to 10 to 100 times higher than free radicals from other food protein sources. A primary objective of this project is to investigate the reactions catalyzed by the released of these radicals when the soy proteins is hydrated.
The release of the naturally occurring metastable free radicals from powdered soy proteins upon hydration corresponded to an oxidative burst and production of intrinsic chemiluminescence from about 4- to 8-times greater than other source proteins. Based on the production of chemiluminescence, these radicals were largely released from soy proteins within 30 minutes of hydration at 23 degrees C. Elevating the hydration temperatures increased chemiluminescence by as much as 280% at 70 degrees C, and decreased the half-life of the light-emitting reaction by about 9-fold.
All sources of proteins examined emitted low levels of chemiluminescence in the solid state, but only soy protein, with its elevated levels of trapped radicals produce elevated chemiluminescence upon hydration. By quenching the radicals in powdered soy proteins with hydrogen sulfide (well-known to quench carbon-centered radicals), the electron paramagnetic resonance signals of the naturally-occurring radicals in soy protein samples were quenched below detectable levels. The chemiluminescence produced from the hydrogen sulfide treated ISP when hydrated was about 65% lower than the non-treated control soy proteins.
Through the use of antioxidants and luminol, it was demonstrated that the hydration of soy proteins, and subsequent release of trapped radicals, generated reactive oxygen species. The release of free radicals when powdered soy protein is hydrated also caused further oxidation of the protein itself as evident by an elevation in the protein carbonyls. These findings are consistent with the hypothesis that free radicals released when powdered soy proteins are hydrated can catalyzed various degradative-type reactions.
Further characterization of powdered soy proteins using thermally stimulated luminescence (TSL) revealed two levels of activation energies for the metastable radicals. One group of radicals had activation energies ranging from 0.8 to 1.1 eV and a more stable group of radicals had activation energies from 1.34 to 3.5 eV.
Lei Q., C.M. Liebold, W.L. Boatright and M.S. Jahan, 2010. Distribution of Stable Free Radicals among Amino Acids of Isolated Soy Proteins, Journal of Food Science, 75(7):C633-640.
Boatright W.L. and M.S. Jahan, 2010. Carbon-Centered Radicals in Soy Protein Products. In K.R. Cadwallader and S.K.C. Chang (Eds.), Chemistry Texture and Flavor of Soy (pp in press). American Chemical Society, Washington, DC.
Lei Q., C.M. Liebold, W.L. Boatright and M.S. Jahan, Distribution of Stable Free Radicals among Amino Acids of Isolated Soy Proteins, Institute of Food Technologists Annual Meeting Technical Program Book of Abstracts, Chicago, IL, July 2010.
Liebold C.M., Q. Lei, W.L. Boatright and M.S. Jahan, Hydration Induced Chemiluminescence and Protein Structure Modifications in ISP, Institute of Food Technologists Annual Meeting Technical Program Book of Abstracts, Chicago, IL, July 2010.