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Characterization of Carbon-Centered Free Radicals in Food Proteins
Department of Animal and Food Sciences
Free-radicals in food products can catalyze deteriorative type reactions, both in foods during storage and in the consumer of foods that contain high levels of free radicals. In 2008, Boatright and others were the first to quantify the levels of carbon-centered free radicals in commercial soy protein products and retail products made from isolated soy proteins (ISP). These levels ranged from 2.96 x 1014 to 4.10 x1015 free radicals per gram of soy protein. The higher radical contents were found in the powered drink mixes. The one-electron oxidation potentials for the alpha-carbon centered radicals of glycine anhydride, L-alanine anhydride, and DL-alanine anhydride have been shown to be similar to the alkyl peroxyl radicals, and likely contribute to numerous reactions once the protein is hydrated including the generation of hydroxyl radials from the reaction with molecular oxygen.
Because high levels of free-radicals in food proteins can be harmful to the consumer of foods containing them, it is important to understand the mechanisms that led to the production and stabilization of these radicals in the food protein. As the second largest food crop in the U.S.A., with about 87 million metric tons produced in 2006, soy protein can have a strong economic impact.
Since first commercially isolated in 1941, soy proteins have primary been used in non-human food applications; in large part because of their objectionable taste. This rather low usage level in the past may explain why there has been no strong connection reported between the consumption of soy proteins and human disease.
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.
The primary objective of this project is to develop techniques that can be applied to the characterization of carbon-centered radicals in food proteins.
The majority of work in this area during 2011 was focused on the development of chemical luminescence techniques. Chemiluminescence is a phenomenon that results from the release of energy, caused by the reaction of two or more molecules, in the form of a light emission. In the current study, no external oxidants have been added. The chemiluminescence methods measured light produced from protein samples as a result of the release, or production, of free radicals native to the protein samples. Intrinsic chemiluminescence is the measure of the production of light from protein samples without the addition of any external luminescence agents (e.g., luminol). Previous investigations have attributed this type of chemiluminescence to intramolecular free radical reactions (such as the formation of excited carbonyls on tryptophan residues).
Chemiluminescence was read on a Tri-Carb 2900 TR Liquid Scintillation Analyzer set to record data as light counts per minute (CPM) with a count time set for 1 minute. The chemiluminescence produced from soy protein samples was compared with other chemical analyses of the proteins including protein carbonyl, various electron paramagnetic resonance spectroscopy techniques, free radicals quenching techniques, and protein solubility analyses.
In addition to intrinsic luminescence analyses, we also developed techniques to measure the production of reactive oxygen radicals produced from the same protein samples. This allowed us to correlate the ability of carbon-radicals in the protein samples to produce oxygen radical capable of reacting with adjacent molecules on the protein was hydrated. The method developed in our lab involved a 3.5 mM stock luminol solution in DMSO. The reaction buffer was a 0.1 M carbonate buffer, pH 10.2. The amount of light emitted was measured with a Tri-Carb 2900 TR Liquid Scintillation.
Upon hydration, soy proteins produced from 4- to 8-times more intrinsic luminescence than whey protein isolates (the second highest level among all the different types of proteins examined). This indicates that a portion of the energy released from the metastable carbon-radicals when powdered soy proteins are hydrated is consumed in the generation of intrinsic chemical luminescence. Comparison of the intrinsic luminescence (without luminol) and the luminol-enhanced luminescence produced from ISP samples revealed little difference.
This indicates that most of the energy released from the carbon-centered radicals of soy protein upon hydration was absorbed in internal reactions within the protein. The oxidative burst from individual proteins (both soy and whey) occurred almost immediately, and then rapidly declined in the first 5 minutes. An unexpected finding was the ability of certain high protein drink mixes (which contained numerous other ingredients) to generate and sustain high levels of reactive oxygen radicals.
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.