Research Accomplishment Reports 2010

Characterization of Carbon-Centered Free Radicals in Food Proteins

W.L. Boatwright
Department of Animal and Food Sciences

 

Non-Technical Summary

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.

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 provided in advance by as much as 9 months.

2010 Impact

Characterization of the metastable radicals in powdered proteins requires very different techniques than those developed for protein in an aqueous medium. An important distinction is that most published studies on radicals in food proteins involve artificially creating relatively high levels of radicals using techniques such as the Fenton reaction or Azo compounds. The radicals being investigated in this project are naturally occurring and are found in "off-the-shelf" food products.

A second important consideration is that once powdered soy proteins are hydrated, most of the metastable radicals within the protein are released, so the techniques used have to be implemented in the "dry" state or within a very short period of time after the initial hydration. To complicate matters further, the reactions catalyzed by the oxidative burst following the release of the metastable radicals from soy proteins are occurring in a slurry that contains not only the soy protein, but other components normally found associated with soy protein products including about 4.5 percent lipids (mainly phospholipids), 2 percent carbohydrates, 0.75 percent nucleic, isoflavones, toccopherols and relatively high concentration of transition metals.

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals (which was determined by electron paramagnetic resonance spectroscopy) and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate.

The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln) and Trp but not on Val, Met, Phe, Lys and His. Due to the low abundance of Ser, Thr and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. Isotope ratio increase for Tyr was also observed but further investigation using nuclear magnetic resonance (NMR) spectroscopy revealed that the increase for Tyr was mainly from non-specific deuterium-hydrogen exchange not free radical quenching.

The development of thermally stimulated luminescence (TSL) techniques to evaluate metastable radicals in powdered food proteins was also accomplished.

These techniques revealed two major levels of activation energies for the metastable radicals in soy proteins. 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. Each technique used in the investigation (free-radical quenching with deuterium sulfide, GC/MS of labeled amino acid derivatives, NMR and TSL) required considerable method development.

2010 Publications

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.

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.

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.