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Fate of Antioxidant Peptides and Proteins in Food Processing
Department of Animal and Food Sciences
When proteins are partially hydrolyzed with appropriate enzymes, many of the resulting products (protein fragments, known as peptides) show antioxidant activity. Compared with synthetic antioxidants, antioxidative peptides have the main advantages of exerting no known health risks and can offer additional functionality. Despite the demonstrated antioxidant potential, the fate of antioxidative protein hydrolysates or peptides in foods during processing and subsequent storage is poorly understood.
This project seeks to investigate the physicochemical changes in antioxidative peptides as a result of exposure to reactive oxygen species (objective 1), interactions of antioxidative peptides with various food components under different food processing pH, temperature and salt conditions (objective 2), and properties of selected foods treated with antioxidative peptides (objective 3). Mixed antioxidative peptides (protein hydrolysates) will be prepared from soy, milk whey, potato and buckwheat proteins that have proven to be excellent sources of antioxidant peptides from previous research. Antioxidative peptides and proteins in 0.1-0.6 M NaCl at pH 5-7 will be oxidatively stressed by exposures to hydroxyl radicals produced from FeCl3/ascorbate/H2O2 reactions, or auto-oxidized at different temperatures (5-80C). Oxidatively modified peptides and proteins will be evaluated for structural characteristics, physicochemical changes, tendency to interact with major food components, and influence on the quality of selected foods (meat batters, pork loins, and gravies).
We expect that in the process of neutralizing small-sized radials, for example, hydroxyl radicals, antioxidant peptides and proteins will undergo major structural changes and form aggregates among themselves and with other chemical components in food, and ultimately, alter the physicochemical characteristics of final food products. Furthermore, we expect peptides to degrade into fragments when exposed to strong oxidizing environments while serving as antioxidants. It is likely that many of the antioxidative peptides not only can protect foods from oxidative destruction, but they also can enhance the functional attributes of final food products.
With an annual output of 46.92 million bushels (1.28 M tons) of soybeans, Kentucky ranks 17th in the nation in total soybean production. Through identification of antioxidant activity and physical and chemical stability of soy peptides during processing, the proposed research may lead to novel utilization of soybeans or soy proteins thereby stimulating the economy of the state.
Results obtained from this study will be disseminated to the stakeholders through presentations at local, regional and national meetings and publication in technical and non-technical journals. The output of the research will be evaluated by the progress (annual) publications and presentations at meetings, the number of graduate students trained from the project, and patents (if applicable) filed.
2011 Project Description
In 2011, three graduate students and one postdoctoral associate participated in this project. Major finding from their individual studies were presented at national Institute of Food Technologists' annual conference or published in scientific journals.
The national meeting provided the opportunity for all researchers to not only present original findings from the project but also communicate and interact with delegates and audiences representing various food companies, consumer groups, and other universities.
As a part of outreach and promotion of the research, the PD visited a university in France (AgroSup Dijon) several universities in China where he presented seminars and talks to students and faculty on subjects related to this project (peptides and proteins).
Two experiments were completed. In experiment 1, oil-in-water emulsions were prepared with 10% soybean oil, 10 mg/mL Tween 20, and 2% SP, SPH, or their hydroxyl radical-oxidized forms (oxi-SP, oxi-SPH) in a pH 7.4 phosphate buffer. Emulsion stability, particle size, and oxidative stability (TBARS) were monitored during storage at 37C up to 14 days. Morphology of emulsion droplets was observed with confocal laser scanning, transmission electron, and phase contrast light microscopy. Absorbed proteins/peptides and those remaining in the continuous phase were separated by centrifugation, quantified, and analyzed with electrophoresis and reverse phase-HPLC.
Results showed that emulsion stability gradually decreased during storage (P<0.05) due to flocculation not coalescence as the emulsion droplet size (330 nm) never changed. Oxi-SP and oxi-SPH, and more significantly, SP and SPH, exhibited strong protection against lipid oxidation compared to the emulsions stabilized with Tween 20 alone (P<0.05). Intact proteins adsorbed more effectively at the interface than hydrolyzed peptides while nonoxidized proteins/peptides were more abundant at the membrane compared to their oxidized counterparts. Oxi-SPH had a low peptide adsorption selectivity while SPH showed a preferred adsorption of nonpolar peptides at the interface.
Hence, oxidative stress affects the distribution of radical-scavenging soy peptides at the emulsion interface but does not diminish their effectiveness as antioxidants, making SPH a promising ingredient in food application.
In experiment 2, native and preheated (90C for 5 min) pea protein isolate (PPI) was hydrolyzed for 0.5 and 1 h using four pure (trypsin, chymotrypsin, papain, and pepsin) and three crude (Alcalase, Flavourzyme, and Protamex) proteases to produce hydrolysates (PPHs).
Degree of hydrolysis (DH), protein fragmentation (SDS-PAGE), and protein solubility were determined. Antioxidant activity was measured using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydroxyl (OH) radical-scavenging activity (RSA) assays, and the TBARS test in a liposome emulsion subjected to oxidation with 4 uM FeCl3/40 uM ascorbate.
Generally, increasing DH (4-26%) led to an improved protein solubility except for PPHs prepared by Flavourzyme and Protamex that showed nearly 4-fold loss in solubility. RSA of PPHs against ABTS and OH, regardless of enzymes, was markedly improved (up to 2-fold, P<0.05) compared to PPI. PPHs from heated PPI showed slightly higher RSA than those from nonheated PPI, but no appreciable difference was noted between 0.5 and 1 h hydrolysis. The combination of PPHs (1 mg/mL) and licorice extract (0.0125 mg/mL) had significant synergistic effects (P<0.05) on inhibiting TBARS formation in liposome, most notably for PPHs prepared from Flavorzyme and Protamex-treated preheated PPI.
Hence, limited proteolysis, especially with Flavourzyme and Protamex (producing intermediate fragments), can generate antioxidative peptide mixtures from pea proteins that are effective and synergistic with licorice in inhibiting oxidation of food emulsions.
Ma, L. and Xiong, Y.L. 2011. Textural attributes and oxidative stability of pork longissimus muscle injected with marbling-like emulsified lipids. Meat Science 89:209-216.
Zhao, J. and Xiong, Y.L. 2011. Thermal aggregation of hydroxyl radical-stressed antioxidative soy peptides. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 236-37).
Jiang, J., Xiong, Y.L., Newman, M.C., and Rentfrow, G.K. 2011. Film-forming properties of alkaline and acidic pH-shifting-treated soy proteins. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 150-06).
Wang, Y., Xiong, Y.L., Rentfrow, G.K., and Newman, M.C. 2011. Hydroxyl radical oxidation promotes cross-linking but reduces film-forming properties of whey proteins. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 236-71).