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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.
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 and the Institute of Food Technologists 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.
Carbon-centered free radicals ranging from 6.12 x 1014 to 9.10 x 1015 per gram of soy protein were found in retail food products, which corresponds to 10 to 100 times more free radicals than other food protein sources. Levels of carbon-centered radicals in newly prepared samples of ISP were much lower than levels in the retail soy protein products, and levels reported for commercial ISP and laboratory ISP samples. The levels of radicals in ISP samples were found to increased over a 12-to-25 week period of storage in the dark at 22 C and exposed to air from ca 8.00 x 1013 spins/g immediately after preparation up to 9.95 x 1014 spins/g ISP. Storing the ISP samples under nitrogen at 22 C greatly reduced the increase in radical content while storing the ISP in 99.9 percent oxygen at 40 C accelerated the formation of stable carbon-centered radicals. ISP samples hydrated at either 22 C or 92 C, rapidly frozen, and dried lost ca. 92 percent of the trapped radicals. The level of carbon-centered radicals in these same ISP samples immediately began to increase during subsequent storage exposed to the air and gradually returned back to similar levels obtained before they were hydrated. Small changes in the moisture content of powdered soy protein products (ranging from 4.4 to 13.4 percent) produced a pronounced inverse effect on the ability of soy proteins to trap radicals. Attempts to elevate the trapped radicals in ISP samples (by storing samples at 50 C in an oxygen atmosphere with or without exposure to a free radical initiator) had limited success until the ISP was dried to 0.45 percent moisture (0.025 water activity) using a molecular sieve. Spiking the protein samples with niobate crystal doped with K3CrO8 demonstrated that the decreased electron paramagnetic resonance (EPR) signals with elevated moisture contents was not due to dielectric lose in the range of moisture contents examined. Thermally Stimulated Luminescence (TSL) glow curves obtained from commercial and laboratory ISP samples exhibited two major peaks. The first peak, from 140 to 165 C, represents less stable trapped charged-centers, and a second peak at about 300 C corresponds to more stable charged-centers. Treating soy protein with the free radical quencher hydrogen sulfide reduced both the EPR signal and the TSL peaks by greater than 90 percent. TSL analyses demonstrated that the 11S soy protein fraction contains primarily the more stable trapped radicals, while the less stable radicals are primarily found in the 7S protein fraction, along with a substantial portion of the more stable radicals. In order to determine if the location of trapped radicals in the dry soy protein corresponded to specific amino acids, a unique deuterium labeling process and gas chromatography-mass spectrometry (GC/MS) technique was developed. Distribution of the radicals among amino acids was determined by measuring the deuterium isotope ratio. Isotope enrichment data showed that the trapped radicals were not ubiquitously distributed among amino acids of soy proteins. Positively identified radical-bearing amino acids only included Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln) and Trp.
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.