Source: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY submitted to
OXIDATIVE AND FREE RADICAL REACTIONS IN FOODS AND BIOLOGICAL SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0183471
Grant No.
(N/A)
Project No.
NJ10134
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Schaich, K. M.
Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
Food Science
Non Technical Summary
Oxidation in food leads to rapid deterioration of eating quality with development of off-flavors, off-odors, some browning, and texture changes; it also results in destruction of nutrients and potential formation of toxic compounds. Oxidation is the only deteriorative reaction that occurs in very dry foods. Traditional stabilization tools of the food industry are proving to be ineffective in foods formulated for health with highly oxidizable n-3 and essential n-6 fatty acids. Additional stresses are imposed by increasing demand for international distribution and storage of foods for emergency food aid, particularly in high temperatures. Lipid and protein oxidation are also a problem in vivo, contributing to a long list of pathologies and diseases. Thus, there is clear and critical need for new information to understand the reactions involved in oxidation of foods and guide improvements in formulation, processing, and packaging to protect the world food supply against oxidation loss. This project addresses these issues by combining fundamental studies of lipid and protein oxidation in model systems to identify chemical details with applied studies in real foods to determine how the reactions change in complex systems. Patterns of oxidation in actual foods will be used to design chemical and instrumental analyses to measure the rate and extent of oxidation in foods and monitor the quality and safety of foods during transport and storage. Since lipid oxidation is a dynamic process that constantly changes and generates many different products over time, combinations of tests that most accurately portray the total state of oxidation in foods will be developed for research, on-line industrial process control, product quality control, and field screening. Analyzing foods with these tools will provide information critically needed to identify problems in production and develop more effective ways to limit oxidation in foods. Recognition that oxidation is involved in aging and cancer has stimulated intense research to identify natural antioxidants that prevent or inhibit these pathologies when foods are consumed, and hundreds of fruits and vegetables exhibiting strong radical scavenging have been identified. Considering security of world food supplies, it seems wasteful to divert edible food to antioxidant production, so antioxidant assays are being redirected from in vivo protection to food stabilization, and new natural antioxidants are being isolated from waste food materials for use in preventing oxidation in foods. This approach can protect and extend world food supplies while creating opportunities for value-added agricultural products. Natural antioxidants are also being added to multilayer controlled release packaging for continual delivery during extended storage. Integrating all these results will provide the first new understanding of lipid oxidation in several decades, the most complete and detailed picture of lipid oxidation and its footprints in foods ever obtained, and dynamic new stabilization approaches that should greatly reduce food loss during storage and distribution of regular and emergency food supplies world-wide.
Animal Health Component
30%
Research Effort Categories
Basic
45%
Applied
30%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5025010200020%
5025010202010%
5027299200010%
5027410202010%
5035010200020%
5037299100010%
5115010200010%
5117299200010%
Goals / Objectives
Addressing the priority goal Global Food Security and Hunger, this research program aims to provide new information about the basic chemistry responsible for oxidative degradation of foods and actions of natural antioxidants, and to use that information to more effectively stabilize foods during world-wide distribution and long-term storage under a wide range of climatic conditions. Oxidation in foods imposes considerable costs to consumers in loss of edibility, diminished nutritive value, potential formation of toxic products, loss of health support, and outright food destruction as oxidation progresses. Development of rancidity also causes significant loss of emergency food aid products, particularly those targeted to regions with high ambient temperatures. To prevent these losses and increase food security, this program coordinates six fundamental and applied sub-projects that address different aspects of oxidative degradation in foods, develop strategies to counteract and prevent oxidative degradation, and protect food quality and nutrition: 1) alternate reactions that compete with hydrogen abstraction in lipid oxidation, thereby changing degradation kinetics, products, and consequences to quality; 2) lipid co-oxidation of proteins and other food molecules to broadcast oxidation potential and amplify damage; 3) analytical methodologies for assessing oxidative degradation and antioxidant stabilization strategies; 4) isolation, analysis, and applications of natural antioxidants from food wastes to conserve edible foods for consumption while stabilizing food supplies and providing value-added products; 5) development of controlled release packaging for long-term food stabilization, and 6) degradation processes in frying oils. Together, these studies will coordinate measurements of volatile and non-volatile lipid oxidation products with co-oxidation changes of proteins in model systems and real foods to provide the first new understanding of lipid oxidation in several decades and the most complete and detailed picture of lipid oxidation and its footprints in foods ever obtained. Results will show how oxidation reactions shift under different conditions, which factors have strongest directing effects or most negative impact on product quality, and which oxidation products best reflect oxidation under various conditions. This information will be used to develop improved analytical methods to accurately determine extent of oxidation in foods, and to specifically design food formulations, processing methods, packaging, and distribution practices to effectively limit degradation of food flavors, textures, and nutritional quality in all environments, thereby reducing food loss. From the stabilization side, the project will reorient antioxidant activity assays to elucidate how natural antioxidants will react in foods (rather than biological systems); it will also isolate and test antioxidants with a range of solubilities from food wastes (thereby conserving edible foods), then adapt these new materials to stabilize oils and emulsions as well as semisolid and solid complex foods.
Project Methods
Lipid oxidation, the major chemical failure mode of foods, causes considerable food loss during storage. This research program seeks to reduce food loss and protect food quality and nutrition in coordinated studies of how lipids oxidize, how oxidizing lipids damage proteins, how lipid and protein oxidation can best be detected in foods, and how foods can be stabilized by controlled release antioxidant packaging and natural antioxidants from food wastes and non-food sources. Integration of multiple aspects of oxidation will provide a unique and comprehensive new view of oxidation processes and ways to prevent and counteract them in foods. Sub-projects combine basic research in model systems of pure lipids and proteins (to identify processes at the molecular level) with applied research to observe how oxidation reactions manifest in real foods. To understand how processing and storage conditions affect oxidation, reactions are conducted at temperatures ranging from ambient to frying (185C). Comprehensive product analyses cover early products (oxygen consumption, free radicals, conjugated dienes, hydroperoxides) through those that develop over time (epoxides, aldehydes, dimers), identifying general classes of lipid products by chemical assays and individual products by GC, HPLC and EPR. Protein analyses determine solubility, fragmentation and crosslinking by electrophoresis and HPLC, oxidation by antibody reactions and individual amino acid oxidation products. Integrating lipid and protein results in models systems and intact foods provides molecular-level understanding of how oxidation reactions, kinetics, and product distributions change with food system conditions, identify key compounds that accurately track oxidation progress and effects, and reveal conditions that enhance co-oxidations. To more accurately measure oxidation in foods, manual extractions of lipids and proteins are being converted to accelerated solvent extractions (ASE) that reduce time and solvents required, protect samples from artifactual oxidation, and increase yields. Traditional chemical assays are being re-evaluated and new electron paramagnetic resonance, chemiluminescence, and near infra-red methods are being developed to identify combinations of assays that provide most accurate information in research, on-line process and quality control. Two approaches to limiting oxidation in foods are being developed. To develop natural antioxidant replacements for BHT, DPPH, ORAC, and lipid oxidation antioxidant activity assays are being re-oriented from in vivo modeling to food stabilization. Together, these assays will identify natural antioxidants with strong ability to prevent lipid and protein oxidation in different phases of foods and different food applications. These assays will be used to analyze antioxidants isolated from waste food components such as potato peels and spent coffee grounds to develop value-added additives that do not divert edible foods. Also, specialized controlled release packaging is being developed to deliver volatile and non-volatile antioxidants to packaged foods slowly over time to extend the period of active protection and stability.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: This project had target audiences for research, education, and outreach and applications. Target Audiences for research results were academia and industries for which oxidation is a problem – e.g. food, cosmetics, soaps and detergents, nutraceuticals, pharmaceuticals, and more. Research results and new thinking in oxidation developed in this project were also major foci for consulting in the food and related industries. In education, target audiences were undergraduate and graduate students in course materials and research experiences, and scientists in general with research publications and presentations. In outreach and applications, target audiences were the food industry; personal products, soaps and detergents industries; and health/medicine. Changes/Problems: No changes to approach anticipated. Some aspects will be less active until new research support is obtained. What opportunities for training and professional development has the project provided? Research training and profesional development were provided to eighteen gradúate students in the following areas: 1) Alternate pathways of lipid oxidation - chemistry: Denis Xie (PhD, supported hourly by USDA grant), non-volatile products; Xin Tian (PhD, hourly support from USDA contract), technical support in analysis of non-volatile products; Brandon Bogusz (PhD, supported 8 months hourly by USDA), volatile products. 2) Development of improved lipid oxidation analyses for tracking alternate pathways: Linhong Yao (PhD, supported 4 months as graduate assistant on industry contract, 8 months hourly on USDA grant), accelerated solvent extractions and HPLC-DNPH assays for phospholipids and triacylglycerols; Chen-Hsiang Liao (MS, supported 4 months as graduate assistant on industry contract, 5 months on own funds), epoxides assays; Teng Peng (PhD, supported 4 months as graduate assistant on industry contract, 8 months hourly on USDA grant), HPLC separation of total lipid classes with electrochemical detection of oxidized products; Brandon Bogusz (PhD, supported 4 months as graduate assistant on industry contract, 8 months hourly on USDA grant), static headspace, SPME, and thermal desorption analyses of volatile oxidation products; Chris Izzo (MS, self-supported), FT-NIR analyses of lipid and protein oxidation in intact foods; Weiyue Wang (MS, self-supported), FT-IR analyses of lipid oxidation in intact foods. 3) Integration of assays to track multiple pathways in food products: Emily Nering (MS, self-supported, some hourly support from USDA contract collaboration), integration of multiple assays to track oxidation in a novel fat blend designed to enhance fat metabolism; 4) Evaluation of natural antioxidants and development of predictive assays for food applications: Karen Chang (PhD, supported 4 months as graduate assistant on industry contract, 8 months hourly on USDA grant), evaluation of potato peel phenols as food antioxidant, development of three-phase approach to predict natural antioxidant effectiveness in foods; Gaoyuan Wu (MS, self-supported), evaluation of antioxidant interactions in activity assays and potential role in foods; Guarav Patel (MS, self-supported), evaluation of novel oregano extract for stabilization of citrus flavors; Anna Puganen (MS, exchange student from Finland, scholarship from Turku University, 4 months), evaluation of antioxidant components and activities from four Finnish berries. 5) Protein co-oxidation mechanisms and chemistry: Wan Zunair Wan Ibadullah, (PhD, Malaysian Government Fellowship), protein oxidation in peanut butter; Greg Resch (MS, self-supported), protein co-oxidation and functional property degradation in stored wheat flour. 6) Thermal degradation mechanisms in food oils: Xin Tian (PhD, self-supported), oxygen bomb analyses and chemical assays of thermal decomposition in heated oils, determination of degradation mechanisms; Ilona Repko (MS, self-supported), thermal desorption analysis of volátiles released from oils heated in oxygen bombs, determination of degradation mechanisms. Research experiences providing training in fundamental laboratory techniques used to study lipid oxidation were provided to two undergraduates: Eric Mendez (focus on FT-IR analyses) and Katherine Malvetti (spectrophotometric analyses of conjugated dienes and antioxidant assays). How have the results been disseminated to communities of interest? Results of this project have been disseminated in three chapters in research books and one chapter in a food biochemistry textbook, in two journal articles, in six oral presentations and two posters presented at the American Chemical Society Annual Meeting in Indianapolis, and two oral presentations and two posters presented at the American Oil Chemists Society Annual Meeting in Montreal. Results have been presented in departmental seminars and discussed in Dr. Schaich’s Lipid Chemistry and undergraduate classes, and have been discussed in consulting contracts in industry. What do you plan to do during the next reporting period to accomplish the goals? 1) Alternate pathways of lipid oxidation – chemistry: focus on detailed identification of both non-volatile and volatile products by mass spectrometry, integrate volatile and non-volatile results to derive pathways active under different conditions, identify products with stability problems that limit detection by specific assays and determine protocols to ensure inclusion of those products. 2) Development of improved lipid oxidation analyses for tracking alternate pathways: Complete validation of HPLC gradient procedures for detection and quantitation of epoxides and carbonyls in phospholipids and triacylglycerols and apply these procedures in routine analyses of oils and extracts. Add evaporative light scattering detection to HPLC analyses of total lipid classes to provide quantitation of fractions; coordinate this with electrochemical detection for quantitation of oxidation products. Develop sample clean-up procedures to protect column in analyses of oxidized lipids. Test pelleting and press procedures developed for miniaturizing samples for FT-NIR analyses and increasing reproducibility. Apply procedures to test sensitivity and accuracy of FT-NIR for detecting and quantitating lipid oxidation in intact food materials. Extend thermal desorption studies of oxidation volatile products to increase reproducibility and determine optimum collection times and temperatures for various endpoints. Complete evaluation of a solid state FT-IR reflectance instrument designed for rapid on-site analyses. 3) Integration of assays to track multiple pathways in food products: Integrate product results from various methods to derive kinetics and relative time courses of various competing reaction pathways. Apply statistical analyses to facilitate organization of results into a new model of lipid oxidation. 4) Evaluation of natural antioxidants and development of predictive assays for food applications: complete analyses of phenol-phenol and phenol-protein interactions and effects on antioxidant activity in complex systems such as foods; test natural antioxidants in lipid assays (fluid and lyophilized emulsions), coordinate with mechanistic information from aqueous assays to explain antioxidant behavior; begin tests of antioxidants in complex model foods to determine synergistic and antagonistic effects of food components and food structure. 5) Protein co-oxidation mechanisms and chemistry: General behaviors have been identified in several food systems. New investigations will now focus on identifying specific points of amino acid and protein modification using advanced LC-MS analyses following enzymatic digestion of modified proteins.

Impacts
What was accomplished under these goals? Impact: Rancidity, the chemical reaction most limiting shelf life of foods, is well-recognized by consumers as characteristic oily or stale off-flavors and odors that arise from oxidation of lipids (fats and oils), but it also causes extensive texture changes, browning, loss of nutritional value, and formation of toxic products in foods by damaging other molecules, particularly proteins. In the past, the food industry controlled oxidation largely by limiting lipid content and by formulating with saturated fats that are more stable. However, as the nutritional requirements for unsaturated fatty acids and higher dietary lipids have become increasingly recognized, challenges in stabilizing foods have re-emerged. Ineffectiveness of traditional approaches (such as just adding antioxidants) in preventing food degradation have shown that our current understanding of lipid oxidation chemistry is incomplete or inaccurate. Base on observations in model systems, this program has proposed a revised reaction scheme that integrates concurrent competing alternate pathways (e.g. internal rearrangement, addition, scission) with hydrogen abstractions in the traditional free radical chain reaction -- the first new thinking about lipid oxidation in over 50 years – and our research results document that these alternate pathways occur also in foods or biological tissues. In foods, alternate pathways have significant consequences to degradation kinetics, product quality and safety, and analyses that must be used to detect oxidation. In vivo, alternate reactions of lipids have a major impact on pathological processes and damage to cells. Previous understanding of lipid oxidation mechanisms placed major emphasis on formation of hydroperoxides, with secondary products only arising after decomposition of hydroperoxides. Thus lipid oxidation is usually determined only by hydroperoxides, perhaps with hexanal also as a decomposition product. Our studies of alternate pathways raise serious questions about the accuracy of this understanding and show that measuring only hydroperoxides or only one product can grossly underestimate lipid oxidation and give a very misleading picture of the oxidation state. In addition, lipid oxidation occurs earlier and faster than previously thought, and most current analytical methods are too insensitive to detect the products. Thus, determining how fast lipid oxidation occurs, how far it progresses, and how lipid oxidation products may be connected to damage occurring in foods and biological tissues requires analysis of products in all pathways over short time intervals using improved analytical methods that can detect products at very low levels (e.g. micromolar or lower). Monitoring multiple oxidation pathways will provide critical new understanding of lipid oxidation processes that will make possible development more effective approaches to stabilization, protection of sensory quality, and prevention of toxic product formation in foods, and reduction of pathological effects in vivo. This project addresses all these issues. Activities and Results: 1) alternate reactions that compete with hydrogen abstraction in lipid oxidation: Parallel studies of non-volatile and volatile products of methyl linoleate oxidation under a broad range of conditions have verified existence of competing alternate oxidation pathways and raised serious questions about existing dogma regarding oxidation processes. Epoxides from rearrangements, in particular, are as important as hydroperoxides, and decomposition sequences are different and more varied than have been previously described. Results are being integrated to propose new pathways. 2) Lipid co-oxidation of proteins to broadcast oxidation potential and amplify damage: Protein modifications from lipid co-oxidation were studied in baked vs fried corn tortillas, peanut butter, and wheat flour to determine how protein co-oxidation varies with protein composition and food matrix. The pattern of co-oxidation damage – disulfide vs free radical crosslinking, fragmentation, peptide rearrangements, surface modification and oxidation, thiol modifications -- changed dramatically with each protein and reaction matrix. Details of chemical changes are being investigated. 3) Analytical methodologies for assessing oxidative degradation and antioxidant stabilization strategies: Major improvements have been made in four areas: extraction methods, chemical assays of specific oxidation products in lipid extracts, instrumental (physical) assays of oxidation in intact foods, and tests to facilitate natural antioxidants to foods. Accelerated solvent extraction (ASE) was validated as the method of choice when lipid oxidation analyses are to be performed, extracting oil with minimum new oxidation and without decomposing existing products (except epoxides, which were instable even in solvent) and providing quantitative extraction of lipids even in difficult matrices such as extruded foods. Chemical assays were re-evaluated for chemical accuracy, stoichiometry, reproducibility, and handling requirements in analyses of lipid hydroperoxide, epoxide, and carbonyl products. Protocols were developed for recommended assays (traditional iodometric titration and PeroxySafe test for hydoperoxides, DETC-HPLC and NMR assays for epoxides, and dinitrophenylhydrazine-HPLC assay of carbonyls); discontinuation of other methods based on inaccurate chemistry was recommended (ferric thiocyanate assays of hydroperoxides, hydrogen bromide and nitrobenzyl pyridine assaysfor epoxides). Instrumental analyses of intact food materials has focused on gas chromatography (GC) of volatiles, Fourier transform infra-red and near infra-red spectroscopy, HPLC of total lipid classes with electrochemical detection of oxidized products. Static headspace, solid phase microextraction (SPME) and thermal desorption (TD) methods for collection of volatiles vary tremendously in sensitivity of detection and pattern of products detected, so give very different pictures of lipid decomposition and reaction pathways. HPLC protocols for separation of total lipid classes with selective detection of oxidation products by electrochemical detections are being developed. Modifications of instrument sample handling have been made to reduce sample size requirements and detection variability in Near Infra-Red analyses of lipid oxidation. A three-phase approach to elucidation natural antioxidant mechanisms and predicting their effectiveness in foods has been developed: standard antioxidant assays to distinguish active mechanisms, lipid-based assays (oils, fluid emulsions, lyophilized emulsions) to assess lipid interactions, and incorporation into model foods to determine antioxidant dispersion and interactions with other molecules in complex matrices. 4) Isolation, analysis, and applications of natural antioxidants from food wastes: Phenol compositions of extracts from potato peels were determined and are being used to develop antioxidant applications of these extracts in foods. 5) Development of controlled release packaging for long-term food stabilization: Inactive. 6) Degradation processes in frying oils: Degradation of oils during frying was shown to proceed primarily by thermal scission of fatty acids at specific positions on the acyl chains, yielding a broad range of products differing from normal oxidation. Degradation was limited under inert atmosphere; when oxygen was present, oxygen added to scission radicals at the ends of chains, resulting in levels of aldehydes orders of magnitude higher than observed in room temperature oxidations. Standard oxidation catalysts such as metals cannot compete with thermal scissions at high temperatures and interact more with secondary oxidation products than native lipids. Results show why antioxidants are rapidly consumed during frying and suggest that the best stabilization approach is elimination of oxygen.

Publications

  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Schaich, K.M, Shahidi, F., Zhong, Y., and Eskin, N.A.M. 2013. Lipid Oxidation. In: Biochemistry of Foods, Third Edition, Ed. Eskin, N.A.M., Elsevier, pp. 419-478
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Schaich, K.M. 2013. Challenges in elucidating lipid oxidation mechanisms: When, where, and how do products arise? In: Lipid Oxidation: Challenges in Food Systems, Ed. Nienaber, U., Logan, A., and Pan, X., American Oil Chemists Society, Champaign, IL, pp.1-52.
  • Type: Book Chapters Status: Accepted Year Published: 2013 Citation: Schaich, K.M. 2013. Challenges in analyzing lipid oxidation: Are one product and one sample concentration enough? In: Lipid Oxidation: Challenges in Food Systems, Ed. Nienaber, U., Logan, A., and Pan, X., American Oil Chemists Society, Champaign, IL, pp. 53-128.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tian, X. and Schaich, K.M. 2013. Effects of molecular structure on kinetics and dynamics of the Trolox Equivalent Antioxidant Capacity assay with ABTS+", J. Agric. Food Chem., 61(23): 55115519.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Apak, R., Gorinstein, S., B�hm, V., Schaich, K.M., �zy�rek, M., and G��l�, K. 2013. Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report), Pure Appl. Chem., ASAP Article, http://dx.doi.org/10.1351/PAC-REP-12-07-15.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Schaich, K.M. 2013. Thinking beyond traditional theory in lipid oxidation. Symposium on Reconsideration of Lipid Oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Schaich, K.M. 2013. Proteins as co-oxidation targets for lipid oxyl radicals, hydroperoxides, epoxides, and aldehydes, Symposium on Lipid Oxidation, Thermal Degradation, and Co-oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Schaich, K.M. 2013. Differential co-oxidation of proteins in tortilla chips and peanut butter by lipids. Symposium on Lipid Oxidation, Thermal Degradation, and Co-oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Xie, J. and Schaich, K.M. 2013. Evidence for multiple oxidation pathways from non-volatile products of methyl linoleate. Symposium on Reconsideration of Lipid Oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Bogusz, B. and Schaich, K.M. 2013. Monitoring volatile product formation to track lipid oxidation pathways in methyl linoleate, Symposium on Reconsideration of Lipid Oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Tian, X. and Schaich, K.M. 2013. Degradation of lipids at high temperatures: Thermal scission vs accelerated autoxidation. Symposium on Lipid Oxidation, Thermal Degradation, and Co-oxidation Mechanisms, American Chemical Society National Meeting, Indianapolis, IN, Sept. 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Yao, L. and Schaich, K.M. 2013. Accelerated solvent extraction of lipids: A highly efficient method preferred for lipid oxidation studies. American Oil Chemists Society National Meeting, Montreal, Canada, April 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Wan Zunairah Wan Ibadullah and Schaich, K.M. 2013. Lipid co-oxidation of proteins in peanut butter. American Oil Chemists Society National Meeting, Montreal, Canada, April 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Schaich K.M. and Steltzer, E. 2013. Evaluation of thiosulfate, xylenol orange, ferric thiocyanate, and trophenyl phosphine methods for quantitating lipid hydroperoxides, American Oil Chemists Society National Meeting, Montreal, Canada, April 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Liao, Chen-Hsiang and Schaich, K.M. 2013. Evaluation of HBr, nitrobenzyl pyridine, diethyl dithiocarbamate, and 1NMR methods for quantitating lipid epoxides. American Oil Chemists Society National Meeting, Montreal, Canada, April 2013.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: This project studies fundamental mechanisms and consequences of oxidative degradation of foods, focusing on free radical reactions in lipid oxidation, protein co-oxidation, and antioxidant actions. Research areas include 1) elucidating multiple pathways of lipid oxidation and mechanisms of thermal degradation of oils, lipid co-oxidations of proteins, and anti-and pro-oxidant actions of natural antioxidants; 2) improving analyses for determining extent and characteristics of lipid and protein oxidation in foods, applying these to track oxidation in foods and identify problem points; 3) improving food stabilization through natural antioxidants, packaging, and processing; 4) isolation, analysis, and applications of natural antioxidants. RESEARCH ACTIVITIES:1) Chemical assays of lipid oxidation product classes, HPLC analyses of specific non-volatile products, and GC analyses of volatile products were integrated to track product pathways and determine controlling factors in oxidation of methyl linoleate, food oils and novel spreads, extruded pet foods, and food oils heated in an OxipresTM oxygen bomb with various catalysts and oxidation inhibitors. 2) Chemical (conjugated dienes, hydroperoxides, epoxides, carbonyls) and physical (chemiluminescence, EPR, FT-NIR, HPLC, GC) analyses of lipid oxidation were evaluated to determine detection limits, response linearity and stoichiometry, conditions required for accuracy and reproducibility, and appropriate applications. Reversed phase HPLC separation and quantitation of total lipid classes with electrochemical identification of oxidation products was developed. Integration of these analyses in shelf life studies is providing detailed information about degradation of lipids in foods during processing and storage. 3) Chemical changes (solubility loss, browning, fragmentation and crosslinking, formation of protein carbonyls, surface charge modifications) were monitored in peanut butter stored at different temperatures to determine mechanisms of damage responsible for textural hardening, moisture loss, and quality degradation. A collaborative study of protein oxidation in flour was initiated. 4) Ability of tocopherol controlled release packaging to stabilize a creamed soup-cheese spread MRE product during storage was investigated. 5) Antioxidants isolated from potato peels were analyzed for polyphenol content and composition, and ability to inhibit lipid oxidation in foods was tested in oils and model systems. 6) Evaluation of novel processing methods to stabilize anthocyanin color and antioxidant activity in cranberry juice continued. SERVICES: Consulting on free radical reactions, lipid oxidation, and/or protein oxidation was provided to four companies. One chapter on lipid oxidation was written for a textbook; chapters on lipid oxidation mechanisms and analyses, and a chapter on antioxidants and health effects of dried cranberries were provided for reference books. EVENTS: A short course on lipid oxidation was presented to industry. PRODUCTS: Research training provided to 8 PhD, 14 MS, 5 undergraduate students; 4 MS theses completed. Dissemination: 7 research papers presented at national meetings. PARTICIPANTS: K.M. Schaich, PI: designed and supervised projects, trained students, wrote reports. Xin Tian, PhD student: conducted oxygen bomb studies on thermal degradation of frying oils and stability of novel margarine blends. Jia Xie, PhD student: conducted chemical assays and HPLC analyses of methyl linoleate oxidation. Brandon Bogusz, PhD student: analyzed volatiles in oxidizing methyl linoleate and foods by gas chromatography. Lynhong Yao, PhD student: developed and optimized accelerated solvent extractions of lipids from food materials, tested stability ot oxidation. Teng Peng, PhD student: developed RP-HPLC method for separating total lipid fractions in oils and extracts. Wan Zunair Wan Ibadullah, PhD student: conducted studies of protein oxidation in peanut butter. Karen Chang, PhD student: developed procedures for predicting effectiveness of natural phenols as food antioxidants. Ed Pappas, PhD student: tested approaches for stabilizing anthocyanins in cranberry juice. Elah Steltzer, MS student: evaluation and optimization of lipid hydroperoxide assays. Chen-shiang Liao, MS student: evaluated and optimized assays to detect epoxides in oxidized lipids. Chris Izzo, MS student: developed and validated FT-NIR methods for detecting and quantitating lipid oxidation in foods. Spurti Ravi, MS student: conducted controlled release packaging studies with cheese spread. Ilona Repko, MS student: analyzed volatiles released during thermal oxidation of frying oils. Hayley Schultz, MS student: evaluated capillary electrophoresis for elucidation of changes in oxidized proteins. Gaoyuan Wu, MS student: investigated effects of molecular interactions on antioxidant activity assays. Greg Resch, MS student: studied protein oxidation in aged flour. Emily Nering, MS student: analyzed oxidative and thermal stability of novel margarine and cooking blends. Partner Organizations: Government of Malaysia and Center for Advanced Food Technology, Rutgers University -- co-oxidation of peanut proteins. Bakto Flavors - development, analysis, and applications of natural antioxidants. Pappas Cranberry Distributors -- anthocyanin stabilization. TARGET AUDIENCES: Target audience for research results is academia and any industry for which oxidation is a problem, although main focus is the food industry. Research results have become a major focus for consulting in the food industry. Research results are also integrated into formal classroom instruction: Undergraduate students - Principles of Food Science. Graduate students - Food Chemistry Fundamentals and Lipid Chemistry. Practicum experiences in oxidative degradation: Provided research experiences for four undergraduates, working on accelerated solvent extraction of lipids from extruded foods, amylase pre-digestion for facilitation of lipid extraction, and FT-IR and FT-NIR analyses of lipid oxidation. Extension and Outreach: Presented short course on radical reactions and lipid oxidation to industry. Research Presentations at professional meetings: Schaich, K.M., Bogusz, B., Xie,J., and Hartman, T.G. 2012. Alternate lipid oxidation pathways: directing factors, products, and consequences in foods, USDA Program Directors Annual Meeting, Las Vegas, NV. Yao, L. and Schaich, K.M. 2012. Accelerated solvent extraction of lipids from extruded pet foods, American Oil Chemists' Society National Meeting, Long Beach, CA. Xie, Jia and Schaich, K.M. 2012. Looking beyond hydrogen abstraction in lipid oxidation: Evidence of alternate pathways in non-volatile products, American Oil Chemists' Society National Meeting, Long Beach, CA. Bogusz, B, and Schaich, K.M. 2012. Monitoring Volatile Products to Track Alternate Pathways in Lipid Oxidation, American Oil Chemists' Society National Meeting, Long Beach, CA. Bogusz, B, and Schaich, K.M. 2012. Alternate pathways beyond hydrogen abstraction in lipid oxidation: GC studies of volatile products from methyl linoleate, American Chemical Society National Meeting, March, San Diego, CA. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Oxidation in foods imposes considerable costs in loss of edibility and diminished nutritive value at low levels plus potential formation of toxic products, loss of health support, and outright food destruction as oxidation progresses. Preventing oxidation in foods formulated with highly unsaturated fatty acids, especially omega-3 fatty acids, is a critical challenge for the food industry, military rations, and emergency food aid products. This project provides a new base of information about the chemistry underlying oxidative degradation of lipids and proteins and how this oxidation can be controlled. Theoretically, results of this project show that lipid oxidation reactions are much more complex than the simple free radical chain reaction traditionally described. A proposed Integrated Reaction Scheme that includes alternate rearrangement, addition, and scission reactions that compete with classical hydrogen abstractions to propagate chains and generate products is the first rethinking of lipid oxidation in fifty years. Practically, learning about how oxidation reactions shift under different conditions makes it possible to deliberately tailor food formulations, processing methods, packaging, and distribution practices to limit degradation of food flavors, textures, and nutritional quality, as well as reduce food loss. Learning about factors with strongest directing effects or most negative impact on product quality provides invaluable guidance for redesigning industrial processing, formulations, and packaging to stabilize highly oxidizable lipids. Knowing what oxidation products to look for under various conditions has guided development of improved analytical methods to accurately determine rates, extent, and detailed products of oxidation in foods during manufacturing, storage, and distribution. These outcomes should greatly reduce food loss, improve sensory quality, and maintain nutritional value of foods at all stages. Optimized sensitive methods for quantitating oxidation in foods integrated in a battery of physical and chemical analyses aim to provide critically-needed tools for quality control in labs and on-line in the food industry as well as to generate the most accurate picture of degradation yet available. A third major impact arises from focus on natural antioxidants as replacements for BHT. Refocusing antioxidant activity assays to determine mechanisms, interactions with other food molecules, antagonisms and synergisms is providing critical new information that can be used to predict how natural antioxidants will behave in foods and to determine how best to use them in stabilizing food systems. Lastly, this project initiates a paradigm shift to identify and harvest industrially useful antioxidants from plant wastes. Successful development can significantly reduce biological waste while at the same time creating value-added products that create new industry opportunities in the depressed agricultural region. Isolation of active antioxidants from potato peel has been achieved and appears to be commercially feasible.

Publications

  • Schaich, K.M. 2012. Functional characteristics of dried cranberries, In Dried Fruits: Phytochemicals and Health Effects, Ed. Alasalvar, C.and Shahidi, F., Wiley-Blackwell, New York, pp.101-132.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: This project studies fundamental mechanisms and consequences of oxidative degradation of foods, focusing on free radical reactions in lipid oxidation, protein co-oxidation, and antioxidant actions. Research areas include 1) elucidating multiple pathways of lipid oxidation and mechanisms of thermal degradation of oils, lipid co-oxidations of proteins, and anti-and pro-oxidant actions of natural antioxidants; 2) improving analyses for determining extent and characteristics of lipid and protein oxidation in foods, applying these to track oxidation in foods and identify problem points; 3) improving food stabilization through natural antioxidants, novel processing, and controlled release packaging; 4) isolation, analysis, and applications of natural antioxidants, particularly from food wastes. ACTIVITIES:1) Chemical assays of lipid oxidation product classes, HPLC analysis of specific non-volatile products, and GC analysis of volatile products were integrated to track product pathways and determine controlling factors in oxidation of methyl linoleate. Thermal vs oxidative degradation mechanisms in food oils were studied in high oleic sunflower-corn oil blends heated in an Oxipres oxygen bomb with various catalysts and oxidation inhibitors. Coordination of initiation rates with volatile and non-volatile oxidation products plus hydrolysis products determines reaction mechanisms and distinguishes thermal degradation from autoxidation. Thermal degradation mechanisms and progression as a function of time and temperature were tracked in corn oil heated in thermal desorption tubes, trapping volatile products sequentially in different time windows. Differences in thermal vs oxidative degradation of proteins were determined in baked vs fried tortilla chips and in peanut butter. Protein fragmentation, crosslinking, and oxidation were analyzed by polyacrylamide gel electrophoresis and Western blots with antibodies to oxidized proteins; browning and texture changes were monitored as oxidation consequences to quality. 2) Chemical (conjugated dienes, hydroperoxides, epoxides, carbonyls) and physical (chemiluminescence, EPR, FT-NIR,GC) analyses of lipid oxidation were re-evaluated to determine detection limits, response linearity and stoichiometry, conditions required for accuracy and reproducibility, and appropriate applications. Integration of these analyses in shelf life studies is providing detailed information about degradation of lipids in foods during processing and storage. 3) Stabilization of anthocyanin color and antioxidant activity was investigated in cranberry juice. Design, functionality, and applications of packaging for controlled release of antioxidants into food remain under development. Key projects included adding [edible] contact layer films to slow antioxidant release, using antioxidant diffusivities in packaging films to predict target release rates required for food stabilization, and testing effectiveness of controlled release packaging with mixed tocopherols, quercetin, or a mixture in extending shelf life and protecting quality in a cheese spread. 4) Antioxidants effective in limiting lipid oxidation were isolated from potato peels. PARTICIPANTS: K.M. Schaich, PI: designed and supervised projects, trained students, wrote reports. Xin Tian, graduate student: conducted oxygen bomb studies, thermal degradation of corn oil: sunflower oil blend. Xiaotian Qin, graduate student: conducted thermal desorption studies, thermal degradation of corn oil; headspace analyses of oxidizing foods. Jia Xie, graduate student: conducted chemical assays and HPLC analyses of methyl linoleate oxidation. Brandon Bogusz, graduate student: analyzed volatiles in oxidizing methyl linoleate and foods by gas chromatography. Lynhong Yao, graduate student: developed and optimized accelerated solvent extractions of lipids from food materials. Chenshiang Liao, graduate student: evaluated and optimized assays to detect epoxides in oxidized lipids. Chris Izzo, graduate student: developed and validated FT-NIR methods for detecting and quantitating lipid oxidation in foods. Karen Chang, undergrad Honors student and grad student: isolated, identified, and tested antioxidants from potato peels; developing antioxidant activity comparison to predict natural antioxidant effectiveness in foods. Yuan Dong, graduate student: conducted studies of protein oxidation in tortilla chips. Wan Zunair Wan Ibadullah, graduate student: conducted studies of protein oxidation in peanut butter. Spurti Ravi, graduate student: conducted controlled release packaging studies with cheese spread. Partner Organizations: Pepsico International -- thermal degradation. Natick Army Research Center -- controlled release packaging. Berry Plastics -- controlled release packaging. Government of Malaysia -- co-oxidation of peanut proteins. Center for Advanced Food Technology, Rutgers University Bakto Flavors -- natural antioxidants Pappas Cranberry Distributors -- anthocyanin stabilization Collaborators: Kit Yam, Dept of Food Science -- controlled release packaging. Daphna Havkin-Frenkel and Chaim Frenkel, Plant Science -- natural antioxidants Training and professional development: 7 PhD dissertations in progress 9 MS theses 1 BS Honors thesis 4 undergraduate students - research experiences. TARGET AUDIENCES: Graduate and undergraduate students, food and allied industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Oxidation in foods imposes considerable costs in loss of edibility and diminished nutritive value at low levels plus potential formation of toxic products, loss of health support, and outright food destruction as oxidation progresses. Rancidity also causes significant loss of emergency food aid products, particularly those targeted to regions with high ambient temperatures. Results of this project provide a new base of information about the chemistry involved in oxidative degradation of lipids and proteins, from which more effective measures to prevent this oxidation can be developed. Learning how oxidation reactions shift under different conditions make it possible to deliberately tailor food formulations, processing methods, packaging, and distribution practices to limit degradation of food flavors, textures, and nutritional quality, as well as reduce food loss. Learning which factors have strongest directing effects or most negative impact on product quality provides invaluable guidance for redesigning industrial processing, formulations, and packaging to stabilize highly oxidizable lipids. Learning what oxidation products to look for under various conditions guides development of improved analytical methods to accurately determine extent oxidation in both foods and tissues during manufacturing, storage, and distribution of foods. These outcomes should greatly reduce food loss during storage and distribution of regular and emergency food supplies world-wide, which addresses a new NIFA priority. Optimized methods for quantitating oxidation in foods integrated in a battery of tests will generate the most accurate picture of degradation yet available and provide critically-needed tools for quality control in labs and on-line. A third major impact: stabilization approaches using natural antioxidants and packaging that releases antioxidants slowly into food over time offers tremendous opportunities to extend shelf life and protect sensory and nutritional quality for very long periods. Identification of ways to produce polymer films with varied release rates, calculation of effective target release rates for individual foods, and demonstration of long-term stabilization of real foods is bringing this new technology closer to commercialization. Refocusing antioxidant activity assays to determine mechanisms, interactions with other food molecules, antagonisms and synergisms is providing critical new information that can be used to predict how natural antioxidants will behave in foods and to determine how best to use them in stabilizing food systems, e.g. alone, in antioxidant combinations as ingredients, or by combining whole foods with complementary activity. Lastly, this project initiates a paradigm shift to identify and harvest industrially useful antioxidants from food wastes such as peels, outer leaves of cruciferous plants, and coffee grounds. Successful development can significantly reduce biological waste while at the same time creating value-added products that create new industry opportunities in the depressed agricultural region. Isolation of active antioxidants from potato peel has been achieved and appears to be commercially feasible.

Publications

  • Schaich, K.M. 2012. Lipid Oxidation. In: Biochemistry of Foods, Third Edition, Ed. Eskin, N.A.M., Elsevier, in press.
  • Schaich, K.M. 2012. Thinking outside the classical chain reaction box of lipid oxidation, Lipid Technology, 24(2), 1-4.
  • Zhu, X., Schaich, K.M., Chen, X., Chung, D., and Yam, K. 2012. Target release rate of antioxidants to extend induction period of lipid oxidation, Food Research International, accepted, in press, doi: 10.1016/j.foodres.2012.01.002.
  • Zhu, X., Schaich, K.M., Finnigan, B., and Yam, K. 2012. Antioxidant effects of sesamol released from polymeric films on lipid oxidation in linoleic acid and oat cereal, Food Packaging Technology, accepted, in press.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This research focuses on oxidative degradation in foods and biological systems. Six currently active research areas are: 1) alternate pathways of lipid oxidation; 2) lipid co-oxidation of proteins; 3) lipid oxidation processes, analytical methodologies, and stabilization strategies in foods; 4) development of controlled release antioxidant packaging for long-term food stabilization; 5) isolation, analysis, and applications of natural antioxidants; 6) degradation processes in frying oils. ACTIVITIES:1) Lipid oxidation pathways were studied in methyl linoleate incubated at 25, 40, and 60 C and in corn oil heated at 100-1235 C in thermal desorption tubes under air and nitrogen, with GC-MS analysis of volatile products. 2) Lipid co-oxidation of proteins was studied in baked vs fried tortilla chips and in peanut butter incubated at 25, 40, and 60 C. Lipid and protein degradation were followed over 8 weeks. 3) Four methods for measuring lipid hydroperoxides were re-evaluated for accuracy, range of detection, and procedural requirements. A solid phase extraction-DNPH-HPLC method for determining lipid carbonyls was developed. Headspace and purge-and-trap methods were compared to optimize collection of volatile products. 4) Peanut butter and processed cheese spread packaged in controlled release packaging with tocopherols and tocopherol plus quercetin were obtained for long term testing of packaging effectiveness. 5) Methods and calculations for ABTS and DPPH antioxidant assays were modified to provide kinetic and mechanistic information rather than simple stoichiometry. Phenolic compounds extracted from potato peels and coffee grounds were quantified and tested for antioxidant activity and mechanisms using ORAC and DPPH assays. 6) Fresh, stripped, and steady-state oils were heated at 180 C in an oxygen bomb alone and with added catalytic and antioxidant factors to elucidate thermal degradation processes and kinetics. Lipid oxidation products in the oils were measured every 15 minutes and headspace volatiles were trapped after heating to determine degradation pathways and kinetics. SERVICES: Mentored a junior professor, consulted for several companies re lipid and protein oxidation processes and analyses in foods, antioxidant testing. PRODUCTS:This program is developing a broad base of new and detailed information about oxidation processes and consequences in foods. Initial results suggest that current portrayal of lipid oxidation is inaccurate and that commonly measured products such as hexanal often are not formed. Reaction mechanisms, not kinetics, change as temperature increases. Above 120 C, thermal scission reactions rather than lipid autoxidation chains dominate during the first 3 hours of heating. Antioxidants delivered via packaging effectively inhibit lipid oxidation and dramatically reduce protein co-oxidation in peanut butter and cheese over more than one year. Many food wastes that are normally discarded may offer value-added sources of antioxidants for food use. Antioxidant yields and activity in potato peels are high enough to suggest commercial applications are feasible. DISSEMINATION: 9 professional presentations, 3 journal articles. PARTICIPANTS: (1) Principal investigator: Karen Schaich,provided overall project direction, maintain instrumentation, participated in many experiments (2) Graduate students: Xin Tian - oxygen bomb and lipid oxidation product analyses in frying studies, tests of various factors affecting lipid degradation Teng Peng - development and testing of oxygen bomb methodology at high temperatures, analyses of oxygen consumption and lipid oxidation products in oils Ilona Repko - GC-MS analysis of headspace volatiles in oxygen bomb studies Xiaotian Qin - Purge and trap/thermal desorption GC-MS studies of degradation processes in oils heated to high temperatures Denis Xie - development of SPE-DNPH-HPLC assay for lipid carbonyls, coordinated assays of multiple oxidation products in oxidation studies Brandon Bogusz - volatile products of lipid oxidation at moderate temperatures Elah Steltzer - evaluation and optimization of assays for lipid hydroperoxides Yuan Dong - studies of protein co-oxidation in tortilla chips Wan Ibadullah Wan Zunair - studies of protein co-oxidation in peanut butter Spurti Ravi -lipid oxidation in cheese spread stored in controlled release antioxidant packaging Karen Chang - isolation, testing, and food applications of antioxidants extracted from potato peels Jonathan Baldasare - isolation and testing of antioxidants from coffee grounds Edward Pappas - stabilization of anthocyanin activity and color in cranberry juice Partner Organizations: Pepsico International Natick Army Research Center Berry Plastics Government of Malaysia Center for Advanced Food Technology, Rutgers University Bakto Flavors Pappas Cranberry Distributors National Starch Collaborators and contacts are others within the recipient's organization: Kit Yam, Dept of Food Science, controlled release packaging Daphna Havkin-Frenkel and Chaim Frenkel, Plant Science, natural antioxidants TARGET AUDIENCES: Target audiences: a) researchers in food science, nutrition, nutraceuticals, and medicine b) the food industry, particularly companies with oil-based products and those developing antioxidant extracts from natural products c) food science students, both undergraduate and graduate. Efforts: Results have been incorporated into three courses taught by PI Schaich - Principles of Food Science (undergraduate), Food Science Fundamentals I and Lipid Chemistry (graduate), into a short course on frying chemistry, industry symposia on natural antioxidants, an IUPAC taskforce evaluation and report on antioxidant assays, standardization of two antioxidant assays, and 11 professional presentations. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1) Results in three subprojects raise serious questions about currently accepted reactions written for lipid oxidation and thermal degradation. We do not find expected and predicted products from methyl linoleate oxidized at low to moderate temperatures, corn oil (high in linoleic acid) dispersed on Celite and heated to 100-235 C for up to three hours under air or nitrogen, or sunflower-corn oil blends heated in a closed oxygen bomb at 180 C for up to 3 hours. In particular, it has long been accepted that C-9 and C-13 hydroperoxides form in equal proportions in linoleic acid, and that the alpha and beta scission products deriving from these are also equal. This is why hexanal (from 13-OOH) and 2,4-decadienal (from 9-OOH) are so commonly analyzed as lipid oxidation markers. Instead, we find pentane and derivatives to be the major initial products at lower temperatures, and hexane derivatives (including hexanal) grow in over time. The rate of hexanal production increases with temperature and becomes substantial at > 100C, but still does not contribute the predicted 25% of the products. 2,4-decadienal is a minor product. Initial products at high temperatures are mainly pentane derivatives, then hexane, heptane, and longer alkanes and derivatives. The distribution between alkanes, alkenes, and corresponding oxidation products (aldehydes, ketones, carboxylic acids) is determined by the available oxygen in the system. These apparent differences may arise from a) increased sensitivity and resolution in capillary GC columns and GC and MS instrumentation, and b) earlier and more frequent analysis of oxidation products. A major practical impact of these results is analytical - use of hexanal and decadienal as a quality control measure to detect rancidity may severely underestimate the true extent of lipid oxidation. Similarly, the product distributions after heating short times at high temperatures are distinctly different than those expected from hydrogen abstractions of autoxidation, and show clearly that thermal scission reactions dominate in short time heating. These scissions form fragment products that then add oxygen to form terminal peroxides which decompose to aldehydes. Both of these products are detected in standard assays but are interpreted as arising from C9 and C13 hydroperoxides and derivative aldehydes. A major practical impact of thermal scissions is to create a constant background of radical reactions that overwhelm common autoxidation catalysts and antioxidant during heating and must be counteracted to stabilize the oils. 2) Co-oxidation studies show quite dramatically that oxidizing lipids transfer radicals and oxidation potential to proteins in foods. Transfer of radicals stops oxidation chains, so lipid oxidation measured by peroxide values may appear to be negligible while proteins in the food are highly oxidized and crosslinked. Maize proteins from corn are particularly susceptible to radical crosslinking induced by lipids. A major practical impact of these observations is that protein oxidation must be monitored along with lipid oxidation in complex foods to accurately determine the extent and effects of oxidation.

Publications

  • Zhu, X., Schaich, K.M., Chen, X., and Yam, K. 2010. Target release rate of antioxidants to extend induction period of lipid oxidation, submitted, Food Research International.
  • Zhu, X., Schaich, K.M., Finnigan, B., and Yam, K. 2010. Antioxidant effectiveness of sesamol released from polymeric films to linoleic acid or oat cereal. In press, Food Packaging Technol.
  • Farris, S., Schaich, K.M., Liu, L-S., Cooke, P.H., Piergiovanni, L., and Yam, K.L. 2010. Gelatine-pectin composite films from polyion-complex hydrogels. Food Hydrocolloids online doi:10.1016/j.foodhyd.2010.05.006, in print 2011, 25(1): 61-70.
  • Schaich, K.M. 2010. Can natural pigments also be antioxidants Guest lecture, Food colors graduate course, Dept of Food Science, Rutgers University, New Brunswick, NJ, November 2010.
  • Schaich, K.M. 2010. In vitro antioxidant activity assays: Sorting your ORAC from your FRAP, NutraIngredients Short course on antioxidant assays for the food industry, Brussels, Belgium, June, 2010.
  • Schaich, K.M. 2010. Facts and Myths in Teaching Lipid Chemistry, Presented in Hot Topics Forum: Challenges and Opportunities in Lipids and Oils/Fats Education and Curriculum Development, American Oil Chemists Society National Meeting, Phoenix, May 2010.
  • Schaich, K.M. and Dong, Y. 2010. Protein Oxidation in Cereal Products: Footprint of Lipid Oxidation or Reflection of Processing Stress American Oil Chemists Society National Meeting, Phoenix, May 2010.
  • Schaich, K.M. 2010. Critical considerations in ORAC, TRAP, ABTS/TEAC, and DPPH assays of antiradical action. IUPAC Taskforce Report and Symposium, Methods of Measurement and Evaluation of Natural Antioxidant Capacity/Activity, Istanbul, Turkey, April, 2010.
  • Schaich, K.M. 2010. Lipid co-oxidation of proteins, Brigham Young University, Dept. of Food Science, January, 2010.
  • Schaich, K.M. 2010. Revisiting and redirecting the chemistry of antioxidant (antiradical) activity assays, Annual Meeting of the American Chemical Society, Boston, August, 2010.
  • Dong, Y. and Schaich, K.M. 2010. Lipid co-oxidation of proteins in food systems, National Meeting of the Institute of Food Technologists, Chicago, IL, July, 2010.
  • Schaich, K.M., Xie, D., and Bogusz, B. 2010. Alternate lipid oxidation pathways: Directing factors, products, and consequences in foods, USDA Annual Project Directors Meeting, Chicago, IL, July, 2010.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project focuses on free radical reactions in foods and biological systems. Four currently active research areas are: 1) mechanisms of lipid oxidation, 2) lipid co-oxidations of proteins and starches, 3) development of antioxidant controlled release packaging, and 4) evaluation and application of natural antioxidants. ACTIVITIES: 1) Mechanisms of thermal vs oxidative degradation of food oils was studied using high oleic sunflower-corn oil blends. Oxipres oxygen bomb methodology was refined and validated for following oxygen consumption and determining initiation rates from ambient to frying temperatures (180 C); oxygen consumption was coordinated with volatile and non-volatile oxidation products and hydrolysis products to determine reaction mechanisms and distinguish thermal degradation from autoxidation. Pro-oxidant actions of metals and metal stearates, free fatty acids, phospholipids, oxygen, oxidation state of the oil, and water were tested. 2) Lipid co-oxidation of proteins was determined in baked vs fried tortilla chips using polyacrylamide gel electrophoresis with differential staining and Western blots with antibodies to oxidized proteins. Protein co-oxidation in packaged peanut butter and processed cheese spread incubated at 40 and 60 C was observed as crosslinking and fragmentation, browning, and changes in odors and flavors; degradation was prevented by inclusion of tocopherol and quercetin in controlled release antioxidant packaging. 3) Controlled release films were produced with volatile sesamol as well as non-volatile oregano extract, quercetin, quercetin-tocopherol mixtures, and mixed tocopherols and tested for antioxidant release rates, film properties, and effectiveness in stabilizing peanut butter, processed cheese, and chicken breast. 4) Standard assay protocols were developed from detailed studies of radical reaction chemistries involved in TEAC (ABTS radical cation) and DPPH assays. Antioxidant effectiveness of an oregano extract shown to have unusually high ORAC and TEAC activity was tested in citrus oils. Improved methods for stabilization of color, anthocyanins, other flavonoids, and ascorbic acid in processed cranberry products are being investigated. EVENTS: Eight presentations of results and concepts were made to professional workshops, symposia, and continuing education courses. PRODUCTS: 1) New methodology for differentiating thermal from oxidative degradation of oils at high temperatures, new information about lipid oxidation mechanisms at high temperatures; short-course on frying chemistry for Culinology Continuing Education. 2) Data documenting difference in thermal and lipid co-oxidation of maize proteins, new recognition of protein oxidation effects in processed foods. 3) Standard protocols for ABTS and DPPH antioxidant assays, book of standard protocols, application of natural antioxidants for stabilizing cranberry and citrus juice products. 4) Successful production of controlled release antioxidant films with several antioxidants, demonstration of effective use with peanut butter and processed cheese spread; one MS thesis. DISSEMINATION: professional presentations, review article, book chapters. PARTICIPANTS: Principal Investigator: K.M. Schaich Role: Primary source of ideas and expertise; guide overall project development; decide goals; train and supervise students and post docs working on project; write reports, publications, and new grant proposals. 1) Lipid oxidation mechanisms: Post-doctoral associate: Traian Popa - Developing capillary electrophoresis analyses for free fatty acids and lipid oxidation products in heated oils; analyzed formation of metal carboxylates. Graduate students on stipends: Xin Tian (PhD) - oxygen bomb and lipid oxidation product analyses in frying studies, tests of various factors affecting lipid degradation. Teng Peng (MS) - development and testing of oxygen bomb methodology at high temperatures, analyses of oxygen consumption and lipid oxidation products in oils. Brandon Bogusz (PhD) - volatile products of lipid oxidation. Partner organizations - Pepsico International, USDA, Rutgers University. Collaborators - T.G. Hartman, Dept. of Food Science, Rutgers University. Training and professional development: one post-doc trained, 3 MS and 2 PhD projects, one undergrad honors thesis. 2) Lipid co-oxidation reactions: Graduate students on stipends: Yuan Dong (MS) - studies of protein co-oxidation in tortilla chips. Wan Ibadullah Wan Zunair (PhD) - studies of protein co-oxidation in tortilla chips, model system studies of lipid-protein co-oxidation. Alejandro J. Perez Gonzales (PhD) - lipid co-oxidations of starch. Partner organizations - Center for Advanced Food Technology, Rutgers University, National Starch, government of Malaysia. Training and professional development: 1 MS and 2 PhD projects. 3) Controlled release antioxidant packaging: Graduate students on stipends: Spurti Ravi - thermal stability of antioxidants; analyzed lipid oxidation in peanut butter and cheese spread stored in controlled release antioxidant packaging. Partner organizations - Pliant Corporation, Natick Army Research and Engineering Center. Collaborators - K.V. Yam, Dept of Food Science, Rutgers University; D. Zumbrunnen, Dept of Mechanical Eng., Clemson University. Training and professional development: 1 MS project. 4) Assays and applications of natural antioxidants: Graduate students on stipends: Xin Tian - studies of ABTS reactions. Denis Xie - studies of DPPH reactions. Partner organizations - Bakto Flavors, Pappas Cranberry Distributors. Collaborators - Daphna Havkin-Frenkel, Chaim Frenkel, Plant Science. Training and professional development: 3 MS and 1 PhD projects. TARGET AUDIENCES: Target audiences: researchers in food science, nutrition, nutraceuticals, and medicine; the food industry, particularly companies with oil-based products and those developing antioxidant extracts from natural products; food science students. Efforts: Results have been incorporated into three courses taught by PI Schaich - Principles of Food Science (undergraduate), Food Science Fundamentals I and Lipid Chemistry (graduate), into a short course on frying chemistry, an industry symposium on new perspectives in food ingredients, and several professional presentations. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1) Oxygen bomb methodology combined with lipid oxidation product analysis has been adapted to study degradation of polyunsaturated oils at high temperatures, in particular to distinguish thermal vs oxidative reactions under a range of conditions. Thermal scissions dominate at temperatures above 150 C; products are determined by degree of agitation in the fryer and oxygen in the atmosphere. Dimers and polymers are major products in oil heated with limited stirring under low oxygen; increasing agitation disrupts radical recombinations and shifts products to fragmentation species (alkanes and alkenes). Oxygenation of scission species forms peroxyl radicals, peroxides and aldehydes that are different from traditional autoxidation products. Conjugated dienes indicative of hydrogen abstractions remain low while aldehydes increase rapidly. Thermal scissions obscure catalytic effects of metals, phospholipids, free fatty acids, and preformed hydroperoxides in heated fresh oils; autoxidation chains and expected catalyses become active only during secondary stages of frying and in the presence of water. These reaction patterns have been integrated into strategies for stabilizing unsaturated frying oils. 2) Studies of baked vs fried tortilla chips differentiate thermal damage from lipid co-oxidation of proteins. Although there is little protein solubility difference in fresh samples, polyacrylamide gel electrophoresis reveals significant loss of Coomassie blue dye binding in fried chips, indicating damage to lysine, arginine, tryptophan, and histidine residues. Silver staining shows loss of some protein fractions in fried chips. In contrast, disulfide crosslinking was the only structural change evident in proteins from baked chips. Western blotting and reaction with antibodies revealed extensive presence of carbonyl oxidation products in proteins from both products. Changes in these patterns during storage is under investigation. 3) Controlled release packaging was produced with sesamol, mixed tocopherols, and quercetin as antioxidants. Sesamol was as nearly as effective as BHT in preventing lipid oxidation of cereal and protected flavor over more than a year of storage at 40 C. Tocopherol in packaging limited oxidation in peanut butter, and tocopherol-quercetin mixtures stabilized the cheese spread at 40 and 60 C. Antioxidants maintained fresh flavors and also prevented browning and textural changes, suggesting that protein oxidation was also limited. 4) TEAC and DPPH assays of antioxidant activity have been studied extensively to determine optimum reaction conditions and determine factors controlling antioxidant reactivity. Both reactions are complete within milliseconds and depend first on molecular size and accessibility and only secondarily on number and placement of phenol groups. ABTS reacts very rapidly with superoxide anion (an oxygen reduction product) so overestimates antioxidants such as ascorbic acid that autoxidize. Testing at a range of pH and different methanol:water proportions reveals antioxidants that transfer hydrogens vs electrons. Results have been written into new standard protocols to be tested and adopted internationally.

Publications

  • Schaich, K.M. 2009. Lipid oxidation: A chemical stabilization challenge for packaging, Encyclopedia of Packaging, K. Yam, ed., John Wiley, New York, pp. 659-674.
  • Farris, S., Schaich, K.M., Liu, L-S., Piergiovanni, L., and Yam, K.L. 2009. Development of polyion-complex hydrogels as bio-based polymers for food packaging applications: a review. Trends in Food Science and Technology 20: 316-332.
  • Pappas, E. and Schaich, K.M. 2009. Phytochemicals of cranberries and cranberry products: characterization, potential health effects, and processing stability, Critical Reviews in Food Science and Nutrition, 49:552-592.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This project focuses on free radical reactions in foods and biological systems. Three currently active research areas are: 1) mechanisms of lipid oxidation, 2) development of antioxidant controlled release packaging, and 3) evaluation and application of natural antioxidants. ACTIVITIES: 1) Factors affecting thermal vs oxidative degradation of food oils were studied using fresh, stripped, and steady state (used in frying for 4-8 hours) oils. Methodology was developed to follow oxygen consumption and determine initiation rates from ambient to frying temperatures (180 C) using Oxipres oxygen bombs, and data was coordinated with analysis of conjugated diene, hydroperoxide, and alkenal oxidation products and free fatty acid hydrolysis products to determine mechanisms. Pro-oxidant action of ferric chloride and antioxidant effects of mixed tocopherols, quercetin, and an oregano extract in frying oils were tested. 2) Controlled release films developed to deliver mixed tocopherols to food over time were proven effective with peanut butter and processed cheese. To demonstrate safety of the tocopherols as required for FDA approval of the applications, mixed tocopherols incorporated in polyethylene and polystyrene films were extracted and analyzed by gas and liquid chromatography to determine decomposition products during production and storage. Product structures were verified by mass spectrometry. Factors influencing release of active compounds from packaging (nature of compounds, polymer composition, film processing, film morphology, film properties, and food characteristics), were integrated into a Conceptual Framework that will guide systematic study and development of controlled release packaging. Procedures were developed for determining "target release rates" at which antioxidants must be released from packaging to stabilize individual foods. 3) Procedures for ORAC, TEAC (ABTS radical cation), and DPPH assays of antioxidant activity were evaluated and standard protocols were developed for TEAC assays. Antioxidant effectiveness of an oregano extract shown to have unusually high ORAC and TEAC activity was tested in frying oils, refrigerated hamburger, and fish meal processed with added fish oils. Improved methods for stabilization of color, anthocyanins, other flavonoids, and ascorbic acid in processed cranberry products are being investigated. A book of standardized antioxidant protocols is being compiled. EVENTS: Four presentations of results and concepts were made to professional workshops, symposia, and continuing education courses. SERVICES: Consulting services were provided to three companies. PRODUCTS: 1) new methodology for oxidation studies at high temperature, new information about lipid oxidation mechanisms at high temperatures; 2) data to support application for FDA approval of controlled release films with mixed tocopherols; integrated conceptual framework for systematic development of controlled release packaging; one MS thesis, one PhD thesis; 3) standard protocols for ABTS and DPPH antioxidant assays, book of standard protocols, one MS thesis. DISSEMINATION: professional presentations, review article, book chapters, book. PARTICIPANTS: Principal Investigator: K.M. Schaich Role: Primary source of ideas and expertise; guide overall project development; decide goals; train and supervise students and post docs working on project; write reports, publications, and new grant proposals. Post-doctoral associate: Traian Popa Role: determine mechanisms of metal catalysis of lipid oxidation in frying oils, develop a capillary electrophoresis method for separating total lipid oxidation products in oils or extracts. Part 1, Lipid oxidation mechanisms: 0.45 GSY; Partner organization -- Pepsico International; Collaborators - T.G. Hartman, Dept. of Food Science, Rutgers University; Training and professional development -- Two students and one post-doc trained, continuing education sessions presented to industry professionals. Part 2, Development of controlled release packaging: 1 GSY; Partner organizations -- Pliant Corporation, U.S. Army Natick Research & Engineering Center; Collaborators - K.V. Yam, Dept of Food Science, Rutgers University; D. Zumbrunnen, Dept of Mechanical Eng., Clemson University; Training and professional development: 1 MS, 1 PhD thesis completed, 2 additional students, 1 post doc trained. Part 3, Antioxidant assays and development of natural antioxidan: 1.15 GSY; Partner Organizations -- Batko Flavors, LLC Clement Pappas & Co.; Collaborators - Daphna Havkin-Frenkel, Plant Biotechnology, Rutgers University,and Bakto Flavors; Training and professional development -- 2 MS students trained. TARGET AUDIENCES: Target audiences: researchers in food science, nutrition, nutraceuticals, and medicine; the food industry, particularly companies with oil-based products and those developing antioxidant extracts from natural products; and food science students. Efforts: Results have been incorporated into three courses taught by PI Schaich - Principles of Food Science (undergraduate), Food Science Fundamentals I and Lipid Chemistry (graduate), into a short course on frying chemistry, an industry symposium on shelf life extension, and an industry symposium on new perspectives in food ingredients. New information is being written into research articles and several book chapters, and applied in consulting for the food industry. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1) Reformulating oils/foods with high unsaturated and low saturated fatty acids greatly increases sensitivity to oxidation and degradation, particularly in frying. At T>150 C, bond scissions generate radicals that consume more oxygen than can be accounted for by simple lipid autoxidation. At low oxygen pressures radicals are released as alkane fragments or recombine to form dimers, but under oxygen form peroxyl radicals that become dominant autoxidation initiators, decompose to form unusually high levels of aldehydes, and overwhelm the catalytic effects of metals and antioxidant actions of phenols. Improved approaches to stabilizing frying oils thus must counteract radicals from thermal processes before oxidation can be inhibited in bulk oils and in surface layers adsorbed on foods. Concurrent analysis of multiple lipid oxidation products, including dimers and short chain alkanes, aldehydes, and ketones, is critical to distinguish between thermal degradation and autoxidation. 2) Safety of mixed tocopherols in controlled release packaging has been demonstrated. Tocopherols extruded in polymer films were available fully in low density polyethylene (LDPE) and LDPE/PP blends and at 90% in polypropylene (PP) films. No volatile tocopherol degradation products were detected. Trace amounts of non-volatile tocopherol oxidation products in LDPE and low levels in PP films were normal products found in all foods. FDA petitions have been submitted. 3) Lack of standardized assay protocols causes inconsistent reporting of natural product antioxidant capacity and improper use of assay "numbers" to market nutraceutical products. ORAC, a widely used antioxidant assay, has at least five major procedural flaws: excessively high fluorescein levels cause fluorescence self-quenching, azide initiator and oxygen levels are too low to drive efficient reaction, poor plate reader design gives temperatures lower than expected and uneven across plates, and complexation between natural compounds and fluorescein stabilizes emissions and erroneously indicates extraordinarily high antioxidant activity. TEAC assays measure reactivity of antioxidants with ABTS radicals. In contrast to previous claims, reactions with ABTS radicals are complete within milliseconds and depend first on molecular size and accessibility and only secondarily on number and placement of phenol groups. ABTS reacts very rapidly with superoxide anion (an oxygen reduction product) and with electrons as well as hydrogen atoms. Thus, for accurate representation of antioxidant activity TEAC reactions must be conducted a) measuring loss of absorbance constantly from mixing through six minutes (no longer ) to determine initial reaction rate and presence of steric hindrance, b) with a range of antioxidant concentrations, c) both with normal aeration and with argon sparging, and d) over a range of pH to distinguish antioxidants that act by electron transfer rather than H transfer. The DPPH assay shows similar steric effects but opposite oxygen effects (DPPH does not react with superoxide anion). These recommendations have been written into new standard protocols to be tested and adopted internationally.

Publications

  • No publications reported this period


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: ACTIVITIES: Controlled Release Packaging. Developed a conceptual framework relating polymer composition, processing, film morphologies, and film properties (physical and active compound release) as a basis for systematic development of controlled release packaging; in collaboration with Pliant Corporation, developed a smart blending prototype line to produce functional polymer blend films with morphologies and release properties not attainable by conventional cast or blown films; produced and tested LDPE/PP or PS films for stabilization of oxidation in foods: mixed tocopherols or sesamol in cast films for a cereal product, mixed tocopherols in laminate films for processed cheese and peanut butter, tests with peanut butter are in progress; determined target release rates necessary to stabilize oil-based foods. Oxidative degradation in foods. Integrated chemiluminescence (CL) analyses of early excited state species of lipid oxidation reactions with electron paramagnetic resonance (EPR) analyses of free radicals and chemical analyses of lipid oxidation products to improve sensitivity in determining extent of oxidative damage in foods; tested procedures in peanuts, peanut butter, processed cheese food, and potato chips. Antioxidant activity assays. Investigated antioxidant activity in oregano and other natural extracts; identified several problems with ORAC, TEAC, and DPPH assays; modified procedures for these assays. EVENTS/DISSEMINATION: Presented one week laboratory short course on analysis of antioxidant activity; students presented four papers on controlled release packaging at IFT national meeting. SERVICES: Provided product testing and consulting on analytical methods and improved strategies for stabilization of lipids in foods. PRODUCTS: Controlled Release Packaging. New information about production of packaging films with a range of release properties; production of food packaging antioxidant films with conventional extrusion and with smart blending; successful stabilization of cereal and processed cheese. Oxidative degradation in foods: New fundamental information about multiple pathways of lipid oxidation and oxidation processes in foods, validation of new approach to monitoring oxidative degradation in foods. Antioxidant activity assays: Established new collaboration with faculty in Plant Biotechnology and a small entrepreneurial company to study actions of natural antioxidants and develop food applications; established laboratory capabilities for testing antioxidant capacity in natural products for industry; improved procedures for ORAC, TEAC, and DPPH antioxidant assays. PARTICIPANTS: PI: Karen M. Schaich. Individuals working on project: No individuals paid by NJAES. Individuals paid from USDA packaging grant: Post docs Songmai Liang, Xing Jin. Graduate students paid from DOD-Pliant controlled release packaging grant:Rohini Marathe, Aishwarya Balasubramanian. Undergraduate student paid from DOD-Pliant controlled release packaging grant, worked on both packaging and measurements of oxidation: Niti Lathia. Collaborators - controlled release packaging: Pliant Corp., Cargill Health and Food Technologies, Dr. Linshu Liu, USDA-ARS-ERRC, Dr. Nazir Mir, Perftech Inc. High school students (Project SEED: Andrea Huerta, Chentan Rajput, Lindsay McLannen, Samir Ra. Collaborators - natural antioxidant activity and applications: Dr. Daphna Havkin-Frenkel, Bakto Corp.; Dr Chaim Frenkel, Plant Biology and Biochemistry, Rutgers University. TARGET AUDIENCES: Target audience is food industry, packaging industry, scientific community at large, USDA-NRI. PROJECT MODIFICATIONS: The following components of this project have been discontinued: 1) Large-scale isolation of flavonoids from cranberries for tissue culture and animal testing of bioavailability and bioactivity, chemical and electron paramagnetic resonance determination of chemical reactivities of various fractions, and evaluation of their potential nutraceutical/pharmaceutical uses (collaboration with N. Vorsa, NIH funding ended, not replaced). This has been replaced with the project on development of natural antioxidant isolation and applications, collaboration with D. Havkin-Frenkel, Bakto Corp. 2) Development of a sensitive screening method for early detection of atherosclerosis based on blood redox status (collaboration with T. Sanikidze, Tbilisi State Medical University (TSMU), Tbilisi, Georgia, outside funding not obtained, TMSU changed programmatic directions and withdrew support).

Impacts
New knowledge: When incorporated into packaging films, volatile sesamol was as effective as BHT in protecting cereal against oxidation over six months storage at 25, 40 and 60 deg C; non-volatile tocopherols were ineffective in this application. However, tocopherols significantly reduced browning and flavor degradation in processed cheese during accelerated shelf life analyses at 25, 40 and 60 deg C. Determinations of target release rates necessary to stabilize oil-based foods indicate that linoleic acid as a food stimulant requires 40 ppm per day at 30 deg C and 80 ppm per day at 40 deg C. Initial tocopherol release from conventional cast films is still faster than this, resulting in very rapid initial tocopherol degradation in food simulants and potential pro-oxidant activity in foods. New film morphologies with slower release must be developed. Measurement of oxidative degradation in foods: In peanuts, peanut butter, processed cheese, cereal, and potato chips CL detected clear differences in oxidation well below detection limits for chemical analyses, in part because CL measures radical recombinations rather than products. EPR detects foot prints of lipid oxidation - radicals transferred to proteins and other molecules - rather than lipid radicals themselves. Again, clear differences are detected in samples before chemical analyses. Together, CL and EPR provide very sensitive early evidence of degradation, although the results are not yet quantifiable in conventional terms. Alternative corroborated data is being developed. Chemical analyses of lipid oxidation (conjugated dienes, peroxide values, and carbonyls) indicate that additional oxidation pathways (e.g. rearrangements to epoxides) are active and products are not all accounted for. Together, this data indicates that standard lipid oxidation analyses of peroxides and carbonyls probably do not accurately reflect the true extent of oxidation in most foods and new approaches are needed. Antioxidant activity assays: Investigations of antioxidant activity in oregano and other natural extracts have identified several problems with ORAC, TEAC, and DPPH assays. Reactions vary with oxygen, pH, reagent and sample concentrations, analysis timing, and also temperature for ORAC. For TEAC and DPPH, the reactions are complete in milliseconds and reflect molecular size and reducing power more than radical quenching ability. Importantly, components of natural extracts appear to interact with fluorescein in ORAC assays, preventing its degradation and thus yielding erroneously high ORAC values. Improved procedures are being developed and applied to evaluation of extracts for commercial applications.

Publications

  • Schaich, K.M. 2008. Lipid oxidation: A chemical stabilization challenge for packaging, Encyclopedia of Packaging, K. Yam, ed., John Wiley, New York, in press.
  • Schaich, K.M. 2008. Co-oxidations of oxidizing lipids: Reactions with proteins, in Lipid Oxidation Pathways, Chapter 8, Vol 2, ed. Kamal-Eldin, A. and Min, D., AOCS Press, pp. 167-258, in press.
  • Schaich, K.M., Obinata, N., and Yam, K. Delivering natural antioxidants via controlled release packaging, Proceedings of the Second International Symposium on Natural Preservatives in Food Systems (June 2006), D. Havkin-Frenkel, ed., May 2006, International Society for Horticultural Science, Brugge, Belgium, in press.


Progress 01/01/06 to 12/31/06

Outputs
This project studies processes of oxidative deterioration of foods and biological materials. Efforts focused on four areas: 1) Development of active packaging for controlled release of natural antioxidants into food. Films that release tocopherols at different rates have been generated from various blends of polyethylene, polystyrene, and polypropylene using conventional cast film technology and chaotic advection ("smart blending"). Release rates are controlled by polymer composition and film morphology. Maximum oxidation protection against linoleic acid oxidation is provided by low levels of tocopherol added directly to the oil plus tocopherols slowly released from packaging to maintain tocopherol levels at 50 mg/kg lipid. Tests with food showed that packaging with nonvolatile tocopherols requires contact with food containing an oil phase for effective antioxidant delivery; it does not protect dry solid foods or pre-oxidized foods. In contrast, volatile sesamol is almost as effective as BHT with dry cereal and appear to actively protect emulsions as well, so future experiments will investigate sesamol use in controlled release packaging in more detail. 2) Pathways of protein degradation during processing and oxidation. Proteins were hydrolyzed by immobilized proteases to release amino acids without damage. Immobilization on agarose (available commercially) both reduced exo- and endo-protease activity excessively and bound proteins and peptide fragments, preventing release of digested material. Proteases are being immobilized on different materials to identify methods that retain maximum activity, and these enzymes will then be tested with oxidized proteins. 3) Early detection of oxidation in intact foods by chemiluminescence (CL). CL analyses consistently detect differences in processed foods such as cereals, encapsulated oils, dry infant formula, flour, and processed cheese in the induction period before conjugated dienes, peroxides, or carbonyl products of lipid oxidation accumulate. Products in four spectral ranges are differentiated - excited oxygen, two types of oxyl radicals, and carbonyls. In accelerated shelf life studies, CL emissions decay rapidly at elevated temperatures so cannot be used as a sole measure of oxidation. However, CL decay parallels decomposition of preformed hydroperoxides in foods; combined with assays of conjugated dienes, peroxides, and carbonyls, CL is thus extremely useful for clarifying stages of lipid oxidation. Correlations of CL with oxygen incorporation (elemental analysis) and lipid and free radical production (electron paramagnetic resonance) are underway. 4) Absorption and metabolism of cranberry flavonoids. Nine anthocyanins and glycosides were isolated, fed to mice, and followed in plasma and tissues over time. Aglycones were not absorbed (<0.02%). Myricetin-3-galactoside, quercetin-3 galactoside, and quercetin-3-rhamnoside were absorbed at very low levels (1-3%) but were metabolized within minutes, and metabolites were detected in kidneys and liver. Other anthocyanins and glycosides were barely detectable. This raises serious questions about how dietary antioxidants mediate effects in vivo.

Impacts
Oxidation of lipids and proteins are the major chemical reactions limiting shelf life of foods; they also play critical roles in oxidative stress and pathologies in vivo. Despite the importance of these reactions, relatively little is yet known about specific reactions leading to molecular damage, loss of food quality, and disruption of cell function; methods for detecting and quantitating oxidation in early stages are problematic; and current approaches to antioxidant protection are not sufficient for stabilization during long term storage. To fill these research gaps, this program seeks to elucidate non-traditional reactions of lipid oxidation; determine sites and products of protein oxidation and their consequences to food properties; and develop more sensitive and faster methods for detecting and quantifying lipids and protein oxidation in intact foods. A second focus aims to control oxidation in foods and in vivo by improved use of antioxidants. Controlled release packaging is being designed to deliver antioxidants at rates sufficient to inhibit lipid and protein oxidation over extended periods. In addition, documentation of specificity and levels of cranberry flavonoid antioxidant absorption, pathways of metabolism, and kinetics of tissue distribution (specifically flavonoids from cranberries) will improve general understanding of how dietary antioxidants act in tissues. This information, in turn, will provide direction for development of cranberry varieties with specifically-enhanced bioactivity.

Publications

  • Schaich, K.M. 2006. Natural antioxidants. Food Ingredients and Analysis International 28(2): 6, 8, 10
  • Schaich, K.M. 2006. Developing a rational basis for selection of antioxidant screening methods, In: Proceedings of I International Conference on Natural Preservatives in Food, March 2005, ed. D. Havkin-Frenkel, C. Frenkel, N. Dudai, Acta Horticulturae 709:
  • Schaich, K.M. 2006. Lipid oxidation in specialty oils. In: Nutraceutical and Specialty Oils, Shahidi. F., ed., Marcel Dekker, New York, pp. 401-448.
  • Duarte, S.; Gregoire, S.; Singh, A.P.; Vorsa, N.; Schaich, K.; Bowen, W.H.; Koo, H. 2006. Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms. FEMS Microbiol Lett. 257: 50-56.
  • Schaich, K.M. 2007. Teaching lipid chemistry through creative problem solving, In: New and Novel Methods for Teaching Lipid Chemistry, Weselake, R., ed., American Oil Chemists Society, Champaign, IL, in press.


Progress 01/01/05 to 12/31/05

Outputs
It is too early in the life of the project to report on its progress.

Impacts
This research will improve understanding and control of oxidative deterioration processes in foods and biological systems.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
This project studies oxidative deterioration in food and living tissues and develops methods to limit the processes involved. Current research focuses on three areas. 1) Bioavailability and bioactivity of cranberry flavonoids. Novel flash chromatography methods have been developed for large-scale isolation of individual flavonoid compounds from cranberries for determining uptake and physiological actions in cultured cells and in mice. Our evidence indicates that absorption of flavonoids is generally low and specific, and facilitated by sugar and salt transport proteins. The preferred form for absorption is with a sugar attached (glycosides), rather than the aglycone parents; glucosides appear to be metabolized rapidly while galactosides are absorbed intact. However, cellular uptake of flavonoids is too low to account for activities attributed to them. Thus, the possibility that flavonoids act by maintaining intestinal redox tone or binding to membranes to initiate signal transduction cascades rather than scavenging free radicals is being investigated. Quercetin, quercetin-3-galactoside, and a benzoyl galactoside derivative are toxic to lung fibroblasts in cell culture, at least in part due to inhibition of COX-2 expression. These observations may partially explain antimicrobial activity proposed for flavonoids. 2) Development of packaging films for controlled release of active compounds (collaborator, D. Zumbrunnen, Clemson University). Food contact films in current use release additives very fast or not at all. We have shown that release rates of mixed tocopherols depend on the nanostructures of polymer films, and that manipulation of film morphology by polymer blend compositions (e.g. LDPE/EVA, PE/LDPE, LDPE/PS) and chaotic advection mixing before extrusion can provide a range of tocopherol release rates. Systematic elucidation of relationships between packaging properties and polymer processing, composition, and structure provides information critical for production of films with morphology specifically modified to increase or reduce tocopherols release rates. Results are being applied to industrial production of films for testing with consumer food products. 3) Protein oxidation during food processing. Electron paramagnetic resonance and selective antibody reactions have shown that protein free radicals are formed during nearly all types of food processing, accompanied by protein oxidation, fragmentation, and cross-linking. Heat and radiation form nitrogen-centered radicals localized on the peptide backbone, whereas shear forces produce predominantly sulfur radicals. Radical production is accompanied by appearance of fragmented and crosslinked proteins in gel electrophoresis and by an increase in protein oxidation. Protein oxidation products may contribute to degradation previously attributed to oxidizing lipids. Methods are being developed to enzymatically digest the proteins and determine modified amino acids by high pressure liquid chromatography / mass spectrometry detection. Results will be used to determine mechanisms and degree of protein damage, as well as production of flavor or off-flavor compounds and potential toxic products.

Impacts
This research will improve understanding and control of oxidative deterioration processes in foods and biological systems. The cranberry studies show that the antioxidant theory of fruits and vegetable nutrition may not be entirely accurate, and natural polyphenols may act physiologically entirely or primarily by other pathways. A long-term goal is to breed cranberry and blueberry varieties with enhanced levels of those flavonoids showing highest uptake and bioactivity. Development of food packaging that can provide controlled release of stabilizers such as antioxidants and antimicrobials as they are consumed during storage will significantly extend shelf life and enhance quality of stored foods. Systematic elucidation of how migration rates of active compounds are related to packaging film composition, structure, and processing will provide an extensive data base of fundamental information revealing how active compounds are bound in films, how they affect film properties, and how they are released -- information that will be critical for production of controlled release packaging effective for foods. Protein oxidation is a significant problem in foods and in vivo pathology. Elucidation of protein oxidation mechanisms and effects will quantitate essential amino acid loss that could lead to amino acid imbalances problematic in nutrition; identify specific amino acid oxidation products that may contribute to flavors, toxicity, or signal transduction; differentiate damage from different sources, and suggest approaches for degradation control in foods and in human pathology.

Publications

  • Schaich, K.M. 2005. Lipid oxidation in Fats and Oils: An integrated view. In: Baileys Industrial Fats and Oils, Shahidi, F., ed., John Wiley, New York, pp. 2681-2767.
  • Schaich, K.M. 2005. Lipid oxidation in specialty oils. In: Nutraceutical and Specialty Oils, Shahidi. F., ed., Marcel Dekker, New York, in press.
  • LaCoste, A., Schaich, K.M., Zumbrunnen, D., and Yam, K.L. 2005. Advancing controlled release packaging through smart blending, J. Packaging Technol. Sci. (in press).


Progress 01/01/03 to 12/31/03

Outputs
Extrusion is used to create a wide range of textures in food products, but relatively little is yet understood about the molecular rearrangements and chemistry responsible for texturization. Initial studies demonstrating a connection between protein free radicals and protein modifications during extrusion have led us to further analyze free radical damage in extruded wheat proteins. Proteins extracted from raw and extruded flour and analyzed by polyacrylamide gel electrophoresis (PAGE) revealed extensive protein crosslinking and also some fragmentation. Peptide bands migrating in normal positions could be tentatively identified by Rf values, but the source of peptides in new bands (polymers or fragments) could not be ascertained. To track modified peptides in extruded wheat flour, peptides separated on PAGE gels were transferred to membranes in Western blots and reacted with antibodies to individual wheat proteins, obtained in collaboration with the Grain Research Center at CSIRO in Australia. We expected direct information from antibody recognition of peptides and indirect information from bands that lost antibody recognition. Results indicated that some high molecular weight glutenins (HMW-GS) were polymerized or fragmented, but the changes were limited. B-Low molecular weight glutenins (B-LMW-GS) did not appear to be affected but there was near total loss of C-LMW-GS peptides. Albumins and globulins also showed extensive damage and loss of antibody recognition. Most interesting was the gliadin fraction. Sulfhydryl-containing a, b, and g gliadins were greatly decreased on PAGE gels and showed no recognition by antibodies. In contrast, w-gliadins with no cysteine residues were not altered and showed full antibody recognition. Further investigation revealed that a, b, and g gliadins were crosslinked by disulfide bonds and extracted and migrated with LMW-GS on PAGE gels. This is the first evidence of a specific protein fraction being selectively crosslinked during extrusion. The results also raise questions about texturization mechanisms since previous studies using nominally the same extrusion conditions revealed no significant disulfide crosslinking. EPR evidence that shear stress increases disulfide bond breakage and production of sulfur radicals is consistent the higher shear stress in this study leading to the unusual disulfide crosslinking observed. Antibodies to oxidized proteins and glycated proteins were used also to gain more information about the chemical changes leading to loss of antibody recognition. Oxidation arises from the high temperatures and reactions with oxidizing lipids during extrusion, while glycation results primarily from protein complexation with starch. Proteins soluble in SDS-2-mercaptoethanol showed glycation in nearly every peptide fraction after extrusion. Proteins released from the insoluble residue by amylase digestion were identified by antibodies and HMW-GS and LMW-GS and showed glycation primarily in the high polymer fractions. All proteins in both soluble and insoluble fractions were extensively oxidized. This is the first documentation of protein glycation and oxidation in extrusion.

Impacts
Even if it is argued that the American diet is so high in protein that loss of protein in extruded products would not affect protein nutrition, extrusion now is being used so extensively in a wide range of products that significant loss of essential amino acids could lead to problematic amino acid imbalances. In addition, the protein (amino acid) oxidation products need to be identified and then evaluated for toxicity.

Publications

  • Partridge, M.A.K., Jiang, Y., Skerritt, J.H., and Schaich, K.M. 2003. Immunochemical and electrophoretic analysis of soluble modified proteins in extruded wheat flour, Cereal Chemistry 80(6): 791-798.


Progress 01/01/02 to 12/31/02

Outputs
Processed foods have been attacked as the source of free radicals and oxidation products that may initiate pathological reactions in vivo. To test this theory, a variety of processed foods were analyzed for free radical contents using electron paramagnetic resonance (EPR). Room temperature measurements revealed simple single-line signals in all dry foods with g values 2.005-.0060 characteristic of nitrogen- centered radicals on proteins. Analyses in liquid nitrogen (77K) revealed additional structure and differences between the foods. Signal intensities correlated with the protein content of the foods, and even more, the extent of protein oxidation as determined by antibody reactions and lipid oxidation. To test whether free radicals would survive digestion, food samples were homogenized in water for two minutes, freeze-dried, and analyzed again by EPR. Free radical signals disappeared in all low-fat foods and were decreased by more than 95% in high-fat samples, e.g. sunflower seeds, indicating that mastication in the mouth will quench most radicals produced during processing. Further research is underway with gastric juice models to test effects of digestion and to identify oxidation products that may survive and have pathological potential if absorbed.

Impacts
Most safety issues and off-flavors arising in microbially-stable stored foods have been attributed to oxidized lipids (i.e. fat rancidity), and most antioxidant strategies have been focused solely on stopping lipid oxidation in foods. Results of this project suggest that oxidized proteins may be another important source of degradative changes (production of off-flavors, loss of natural flavors, browning, toughening of texture) in foods during storage, and call for new strategies to counteract protein oxidation as well as lipid oxidation in foods. One example is feeding animals high tocopherol diets to increase the levels of antioxidant in muscle tissue. This greatly decreases free radical production in processed meats. Use of non-phenolic free radical scavengers in dough-based foods may prevent production of stale off-flavors as well as texture changes from crosslinking, thereby increasing useful storage time. More work will be needed to determine the physiological/pathological impact of free radicals ingested in foods.

Publications

  • Schaich, K.M. 2002. NO production during thermal processing of beef: evidence for protein oxidation, in Free Radicals in Foods: Chemistry, Nutrition, and Health, American Chemical Soc., Washington, D.C., pp.151-161.


Progress 01/01/01 to 12/31/01

Outputs
This project studies free radical production, reaction, and involvement in oxidative degradation of food and biological materials. Varian E-12 and E-9 electron paramagnetic resonance (EPR) spectrometers with X-band and Q-band capabilities have been installed in a renovated laboratory. Data acquisition-storage-manipulation is being converted from minicomputer to PC. Electrochemical cells and continuous flow systems are being used to study short-lived radicals in antioxidants from blueberries, cranberries, spices, and other natural materials. These studies will provide new methods that can identify reactivity and reaction mechanisms of natural antioxidants. In pilot studies, neoandrographolides isolated as antioxidants from Chinese herbs showed apparent reactivity with superoxide radical anion by chemical analyses of products. However, EPR analyses revealed that the superoxide reaction was instead mediated by radicals in the dimethyl sulfoxide solvent. Other research focuses on radical production during food processing and storage. A survey of free radicals in commercial foods revealed stable protein radicals in all products tested, with high levels in high-lipid products (e.g. sunflower seeds and cookies) as well as aged or stale samples. EPR signals correlated with protein oxidation detected by specific antibodies, connecting the radicals with measurable molecular damage. However, radicals were destroyed by immersion in water, indicating that radicals in foods are not likely to survive digestion and propagate reactions in body tissues. Variable temperature analyses (minus 195 to 300 degrees C) for in situ detection of radicals formed during heating or freezing are being developed for detailed investigations of free radical production during food processing. Experiments following protein free radical formation and molecular modification during microwave-convection multimodal heating are in progress.

Impacts
The involvement of free radicals in living biological systems is well-recognized, but radicals in foods have received only limited attention despite claims by the medical community that they (primarily radicals from lipid oxidation) contribute to a wide range of pathological conditions. This project is providing the first documentation of radical production during food processing and its consequences for food quality. Previous work demonstrated how free radicals play an integral role in texturization of cereal products during extrusion. Current focus on heat generation of radicals is revealing patterns of protein modification and degradation during different forms of processing. The research resumed with natural antioxidants will provide critical information about radical reactivity and stability that will help determine effectiveness and elucidate how these compounds react in food systems and tissues. Information about molecular reaction mechanisms gained in this project will contribute greatly to controlling quality during food processing, limiting nutritional loss, and developing new approaches for extended shelf life.

Publications

  • Schaich, K. M. 2002. EPR methods for detecting and identifying free radicals in foods, in Free Radicals in Foods: Chemistry, Nutrition, and Health, American Chemical Soc., Washington, D.C.,12-34.
  • Schaich, K.M. 2002. Free radical generation during extrusion: a critical contributer to texturization, in Free Radicals in Foods: Chemistry, Nutrition, and Health, American Chemical Soc., Washington, D.C., 35-48.
  • Schaich, K.M. 2002. NO radical production during thermal processing of beef: evidence for protein oxidation, in Free Radicals in Foods: Chemistry, Nutrition, and Health, American Chemical Soc., Washington, D.C., 151-161.
  • Schaich, K.M. 2002. A spin label study of water binding and protein mobility in a lysozyme model system, in Free Radicals in Foods: Chemistry, Nutrition, and Health, American Chemical Soc., Washington, D.C., 98-113.


Progress 01/10/99 to 12/31/99

Outputs
After 10 years of delay, laboratory space has been renovated for a Biological EPR (electron paramagnetic resonance) Facility to study free radicals in foods and biological systems. Two EPR spectrometers have been moved out of storage and installed in the facility, and should be fully operational by February, 2000. Research has been initiated on free radical production in extruded cereal products, in vegetable oils and potato tissue during frying, in thermally-processed muscle foods, and in natural antioxidants (curcuminoids, tea catechins, and rosemary diphenols). Free radicals produced during extrusion play a critical role in the crosslinking of proteins and starches, leading to texturization, and proteins are the key mediators. EPR signals from radicals in wheat flour, as well as the associated chemistry, have been well-characterized. EPR signals from other extruded materials such as cornmeal, potato granules, and plantain are less well-defined spectrally and vary tremendously in intensity relative to wheat. Current focus is on determining why these differences occur and understanding what they reveal about extrusion chemistry in different materials. Chemical analyses have shown that frying oil degradation and crust formation in french-fried potatoes involve free radicals. This program is now using in situ heating in the EPR cavity to detect and identify free radicals in the oils under different frying conditions, and solid state analyses of french-fried potatoes to analyze free radicals involved in formation of the polysaccharide matrix of the crust. Pilot studies have shown that NO(.) (nitric oxide radicals) released by oxidation of arginine and lysine during thermal processing of beef can be trapped by reaction with the hemoglobin in the meat and detected by low-temperature EPR analyses. These studies are being extended to quantitate the NO(.) production and correlate it with protein oxidation leading to textural and flavor changes in the meat. Previous work with curcumin compounds showed that curcumins form redox-active complexes with iron, quench radicals preferentially in lipid or organic phases, and form very unstable phenolic radicals in the process (contrary to conventional antioxidant theory). Fast and slow flow EPR methods are being developed to detect phenolic radicals in natural antioxidants and study their reaction rates and specificities.

Impacts
Toxic effects of free radicals in foods have been invoked in the popular press despite the dearth of direct studies. Detailed information about free radicals in foods and the roles they play in food properties and food deterioration will counteract the undeserved popular hysteria, elucidate chemical reactions responsible for food characteristics and stability, and suggest new approaches to manipulate and control food properties and shelf-life.

Publications

  • Schaich, K.M. and Rebello, C.A. 1999. Extrusion Chemistry of Wheat Flour Proteins. I. Free radical production. Cereal Chem. 76: 748-755.
  • Rebello, C.A. and Schaich, K.M. 1999. Extrusion Chemistry of Wheat Flour Proteins. II. Sulfhydryl/Disulfide Content and Protein Structural Changes, Cereal Chem. 76: 756-763.