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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.
2010 Project Description
In 2010, five graduate students (4 Ph.D., 1 M.S.) participated in this project. All these students have obtained significant results from their individual studies, and have presented their research findings at national meetings or published papers in scientific journals. Progressive results were summarized in one refereed journal article published and in six abstracts presented at the 2010 Institute of Food Technologists (IFT) annual meeting in Chicago.
The project director (PD) attended several domestic conferences (IFT; Reciprocal of Meat Conference of the American Meat Science Association) where he gave presentations and communicated with delegates and audiences representing various food companies, consumer groups, and other universities to share major discoveries from this research project. As a part of outreach and promotion of the research, the PD visited several universities in China where he presented seminars and talks to students and faculty on subjects related to this project (peptides and proteins). Through these scholarly activities, he was able to recruit several outstanding domestic and international students to the graduate research program at the University of Kentucky to increase its diversity.
Two experiments were completed. In experiment 1, soy protein hydrolysates (SPHs, or mixed peptides) of 1-5% degrees of hydrolysis, all capable of inhibiting lipid oxidation (TBARS), were prepared with Alcalase. SPHs and nonhydrolyzed soy protein (SP) were reacted with hydroxyl radicals generated from 10 uM FeCl2/0.1 mM ascorbate/2 mM H2O2 for 1 h at 37C. Amino acids profile was obtained using HPLC. Protein/peptide structure was analyzed as surface hydrophobicity, intrinsic tryptophan fluorescence, and UV absorption.
Lysine, leucine and methionine residues in SPHs decreased markedly (P < 0.05) after reaction with hydroxyl radicals. Surface hydrophobicity of SP decreased 2 to 4-fold after hydrolysis and slightly (13%) when oxidatively stressed. On the other hand, surface hydrophobicity of SPHs increased (25%) when reacted with hydroxyl radicals. Enzymatic hydrolysis destroyed protein tertiary structure shown by a red shift in the maximum wavelength of intrinsic tryptophan fluorescence and a blue shift of the first derivative of SP UV spectra in the range of 280-290 nm. However, no shift of intrinsic tryptophan fluorescence or UV spectra was detected in SPHs or SP after reaction with hydroxyl radicals.
The results suggested that although most amino acid residues in SPHs were modified following hydroxyl radical attack, lysine, leucine and methionine were destructed the most, suggesting their important role in neutralizing the free radicals. Structural changes in SPHs, normally indicated by the exposure of aromatic amino acid residues, were not a main consequence of hydroxyl radical oxidation.
In experiment 2, soy protein isolate (SPI) and its polypeptide fractions (7S, 11S) were unfolded at pH 1.5 or 12.0 (1 h), followed by refolding at pH 7.0. Protein solubility was determined at 0, 0.1, and 0.6 M NaCl in pH 2.0-8.0, and compared with native protein samples. Moreover, treated samples were heated from 30 to 97C, and thermal aggregation and insolubilization were monitored (turbidity at 600 nm). SDS-PAGE was applied to detect protein cross-linking and identify the contributing components. Thermal transition characteristics [temperature (Tmax) and enthalpy delta H)] of treated samples were analyzed by differential scanning calorimetry.
The pH 12-shifting resulted in substantial increases (up to 2.5-fold, P < 0.05) in SPI solubility in the pH 6.0-7.0 range; the solubility enhancement became less remarkable at increasing salt concentrations. The pH 1.5-shifting had a lesser effect. The 11S exhibited a similar solubility pattern to that of SPI under varying pH and NaCl concentration conditions, but the solubility of 7S was largely unaffected except for samples in 0.6 M NaCl. The pH-shifting, notably at pH 12.0, produced soluble, disulfide-linked polymers from 11S and decreased its delta H by 92% (P < 0.05) without affecting Tmax. Structurally-altered soy proteins by pH-shifting had a reduced sensitivity to thermal aggregation.
The results indicate that extreme pH treatments can greatly improve soy polypeptide solubility and stability against aggregation. Hence, it has practical implications for beverage and other liquid-type food product applications.
Jiang J., Xiong Y.L., and Chen J. 2010. pH shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions. J. Agric. Food Chem. 58:8835-8842.
Shang, Y. and Xiong, Y.L. 2010. Xanthan enhances water binding and gel formation of transglutaminase-treated porcine myofibrillar protein. J. Food Sci. 75:178-185.
Xiong, Y.L. 2010. Antioxidant peptides. In Bioactive Proteins and Peptides as Functional Foods and Neutraceuticals. Mine, Y., Jiang, B., and Li-Chan, E. (eds.), Wiley-Blackwell, Hoboken, NJ. pp. 29-42.
Zhao, J. and Xiong, Y.L. 2010. Structural changes of antioxidative soy protein hydrolysates resulting from inactivation of hydroxyl radicals. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 099-04).
Zhao, J. and Xiong, Y.L. 2010. The influence of calcium ion and pH on cured pork: color and water-holding capacity. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 235-05).
Liu, C., Xiong, Y.L., and Rentfrow, G.K. 2010. Effect of kiwifruit protease extract on tenderness of pork subjected to freeze-thaw abuse. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 235-08).
Greene, J.R., Xiong, Y.L., Liu, Z., and Mims, S. 2010. Brine absorption and microstructural properties of paddlefish (Polyodon spathula) meat treated with four brine solutions and hot smoked. Book of Abstracts. Annual Meeting of the Institute of Food Technologists. (Abst. No. 143-03).