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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.
2011 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.
Further characterization of the metastable radicals found in both laboratory and commercial soy protein products were performed using power saturation studies with the electron paramagnetic resonance (EPR) spectrometer. Determining microwave power saturation levels is critical for characterization of the primary type of radicals in a material.
Published studies involving nitrogen-centered radicals in food proteins have used microwave power levels of 10-to-20 mW. Because microwave power levels of less than one-half that of the power saturation should be used in EPR experiments, the corresponding power saturation of nitrogen radicals in food proteins would be greater than, or equal to, 40mW. Power saturation curves for all soy protein samples (including commercial isolated soy protein (ISP), defatted soy flour, and laboratory ISP) exhibited maximum signal amplitudes at about 4 mW of power. Further increases in microwave power levels resulted in decreases in signal amplitude. These findings provide a further indication that the metastable radicals in soy protein products are carbon-centered radicals.
A primary objective of this project is to investigate the reactions and factors that contribute to the formation of these radicals in the powdered protein. Incremental increases in the moisture content of powdered soy protein products from 4.4 to 13.4 percent were found to produced an inverse effect on the ability of soy proteins to maintain metastable free radicals. The corresponding reduction in electron paramagnetic resonance signals was shown not to be due to dielectric loss in the range of moisture contents examined.
Because of the strong influence of moisture contents on the levels of metastable radicals in ISP, subsequent evaluations of various treatments were conducted after drying soy proteins with molecular sieve to a water activity below 0.085 in order to minimize the influence from variations in moisture. Isolated soy protein (ISP) samples, prepared with "defatted flour" that had been further extracted with chloroform/methanol (2:1), had a 96 percent reduction in total lipids compared to the control ISP samples. The initial rate of radical accumulation in the "reduced-lipid" ISP during the first three weeks was not significantly different from the initial rate of radical increases in the control ISP. After three weeks, radical accumulation in the "reduced-lipid" ISP continued to increase, but at a rate that was less than the control.
These findings demonstrate that the initial reactions contributing to the formation of metastable radicals in the powdered ISP are not strongly dependent on associated lipids. Also, blocking sulfhydryl groups during ISP preparation with N-ethylmaleimide did not significantly slow the rate of radical accumulation compared to the control ISP. Blocking arginine residues in ISP samples with phenylglyoxal caused an increased rate of radical accumulation during the first four weeks.
Liebold CM, Q. Lei, W.L. Boatright and M.S. Jahan, 2011. Metastable Radicals & Intrinsic Chemiluminescence from Soy Proteins, Journal of Food Science, 76(7):C1101-1107.
Liebold C.M., Q. Lei, W.L. Boatright and M.S. Jahan, Metastable Radicals & Intrinsic Chemiluminescence from Soy Proteins, Institute of Food Technologists Annual Meeting Technical Program Book of Abstracts, New Orleans, June 2011.