LLoouuiissiiaannaa SSttaattee UUnniivveerrssiittyy LLSSUU DDiiggiittaall CCoommmmoonnss LSU Master's Theses Graduate School 2006 PPhheennoolliicc ccoommppoouunnddss aanndd aannttiiooxxiiddaanntt aaccttiivviittyy ooff ooaatt bbrraann bbyy vvaarriioouuss eexxttrraaccttiioonn mmeetthhooddss Darryl Lourey Holliday Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Life Sciences Commons RReeccoommmmeennddeedd CCiittaattiioonn Holliday, Darryl Lourey, "Phenolic compounds and antioxidant activity of oat bran by various extraction methods" (2006). LSU Master's Theses. 757. https://digitalcommons.lsu.edu/gradschool_theses/757 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY OF OAT BRAN BY VARIOUS EXTRACTION METHODS A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the Requirements for the degree of Master of Science in The Department of Food Science By Darryl Lourey Holliday B.S., Nicholls State University, 2005 December 2006 DEDICATION First, I want to thank God for what he has given me. Then, thanks to my mom, Claire Holliday, I will never understand how much she sacrificed for me. I want to thank my brother and sister for pushing me to follow my dreams. Also, I want to recognize my teachers and friends throughout my life who have guided me through my different stages. Specifically, I want to thank Bill Koren, Michael Nolen, Douglas Harrison, George Charlet, George Kaslow, Gavin Estes and the faculty of the Food Science Department of Louisiana State University and the Chef John Folse Culinary Institute at Nicholls State University. ii ACKNOWLEDGEMENTS I would like to convey my gratitude towards my major professor and advisor, Dr. Zhimin Xu, for his guidance, consideration, and, in particular, the patience he has had with me throughout this research. I have taken the teaching and support to heart and enjoyed being his student and working with him. I would also like to thank the other members of my committee, Dr. Witoon Prinwayiwatkul, Dr. Kerry Dooley and Dr. Daryl McKee for all their help. I would like to thank Ting Sun, Sandeep Bhale, and Mandy Mayeaux for their incredible help throughout my research. I could not have done it without them. I would also like to thank the Chemical Engineering Department and the Department of Agricultural and Biological Engineering for the use of their labs and equipment. I would like to express my gratitude to the General Mills Corporation for their generosity in funding the research along with providing the oat samples. I would like to extend my thanks to Terri Gilmer, Dr. Jack Losso, Dr. Joan King, and everyone else in the Food Science Department for all their help and support throughout the research period and for making my graduate experience at LSU a memorable one. iii TABLE OF CONTENTS DEDICATION…………………………………………………………………………...ii ACKNOWLEDGEMENTS…………………………………………………………….iii LIST OF TABLES……………………………………………………………………....vi LIST OF FIGURES…………………………………………………………………….vii ABSTRACT……………………………………………………………………………viii CHAPTER 1. INTRODUCTION……………………………………………………….1 CHAPTER 2. LITERATURE REVIEW……………………………………………….3 2.1 Introduction………………………………………………………………………..3 2.2 Oats………………………………………………………………………………..3 2.2.1 Oat History…………………………………………………………………..3 2.2.2 Oat Bran Health Aspects…………………………………………………….6 2.2.3 Antioxidants in Oat Bran……………………………………………………8 2.3 Lipid Oxidation…………………………………………………………………..9 2.3.1 Basic Lipid Oxidation……………………………………………………...9 2.3.2 Autoxidation ………………………………………………………………10 2.3.3 Singlet Oxidation…………………………………………………………..10 2.3.4 Enzymatic Oxidation………………………………………………………10 2.3.5 Rancidity Due To Lipid in Oats……………………………………………11 2.3.6 Negative Aspects of Lipid Oxidation……………………………………...11 2.3.7 Preventing Lipid Oxidation…………………………………………...........11 2.4 Oxidation of Oat Bran and Its Effects………………...………………………….12 2.5 Solvents and Solvent Extraction………………………………………………...13 2.6 Extraction Techniques…………………………………………………………...14 2.7 Analysis Methods…………………………………………………………….…..17 CHAPTER 3. EXTRACTIONS AND ANALYSES……………………….………….20 3.1 Materials………………………………………………………………………….20 3.2 Methods…………………………………………………………………………..20 3.2.1 Extraction Methods………………………………………………………...20 3.2.1.1 Traditional Solvent Extraction (TSE).…………………………….20 3.2.1.2 Microwave-Assisted Solvent Extraction (MAS)...………………..21 3.2.1.3 Supercritical Fluid Treatment (SFT) ……………………………..21 3.2.2 Extract Analysis……………………………………………………………22 3.2.2.1 DPPH Method……………………………………………………..23 3.2.2.2 Total Phenolics/Folin-Ciocalteau Method……………………..….23 3.2.2.3 DHA Oxidation………..…………………………………………..24 3.2.2.4 Cholesterol Oxidation……………………..………………………25 3.3 Statistical Analysis Method….........................................................................25 CHAPTER 4. RESULTS AND DISCUSSION………………………………………..27 iv 4.1 Temperature Increase Rate of Different Extract Treatments………………...27 4.2 Extract Yields from Different Extraction Conditions………………………..27 4.3 Antioxidative Capabilities and Total Phenolic Contents in Different Extracts………………………………………………………………………28 4.4 Inhibition of the Extracts on Cholesterol and Long-Chain Fatty Acid Oxidation……………………………………………………………………..32 CHAPTER 5. SUMMARY AND CONCLUSIONS…………………………………..35 REFERENCES………………………………………………………………………….36 APPENDIX: STATISTICAL ANALYSIS FOR RESEARCH DATA…………...42 1. ANOVA Tables…………………………………………………………..…...42 2. SAS Coding For Running ANOVA………………………………………...…45 VITA…………………………………………………………………………………….46 v LIST OF TABLES Table 1: Top Oats Producers………………………………………………………………3 Table 2: Nutritional Breakdown for Oats per 100g..……………………………………...7 Table 3: Extract Volumes And Yield…………………………………………………….28 vi LIST OF FIGURES Figure 1: Two Major U.S. Oat Production Regions………………………………………4 Figure 2: Breakdown of an Oat Groat (Entire Seed Kernel)…...........................................5 Figure 3: Steps in Basic Lipid Oxidation………………………………………………...9 Figure 4: Microwave-Assisted Extraction Process………………………………………15 Figure 5: Different States Leading to Supercritical Fluid………………………………..16 Figure 6: Phase Diagram of Supercritical CO …………………………………………..17 2 Figure 7: SFT Process……………………………………………………………………22 Figure 8: Comparison of Extraction Temperatures……………………………………...27 Figure 9: Measurement of Antioxidant Activity Using DPPH for TSE and MAS……....29 Figure 10: Measurement of Antioxidant Activity Using DPPH for MAS and SFT……..30 Figure 11: Results of DPPH inhibition for TSE, MAS-100°C, and SFT-75°C………....30 Figure 12: Catechin Equivalency for Total Phenolics…………………………………...31 Figure 13: Total Phenolics per Gram of Extract…………………………………………32 Figure 14: Percent Cholesterol Oxidized………………………………………………...33 Figure 15: Un-reacted DHA Determined by GC-FID………………………………...…34 vii ABSTRACT Recent studies have suggested that the health promoting capabilities of oats are due to its antioxidants (tocopherols, tocotrienols, and sterols) found within the bran along with phenolic compounds, such as avenanthramides, p-hydroxybenoic acid and vanillic acid. Long-chain fatty acid oxidation is directly responsible for most off-flavors in food. Since oat bran is a good source of antioxidants, a concentrated extract could be used as a natural preservative, for foods rich in unsaturated long-chain fatty acids. Three methods, traditional solvent (TSE), microwave-assisted solvent (MAS), and supercritical fluid treatment (SFT), were used to obtain the extracts. One extraction temperature in TSE, 60ºC, and two extraction temperatures in MAS, 60ºC and 100ºC, were tested. The DPPH (2, 2’-diphenyl-1-picrylhydrazyl) method demonstrated that the MAS-100ºC was the most efficient extraction in the group, thereby serving as MAS sample against the TSE and supercritical-treated samples. For the treated samples, oat bran was exposed to supercritical CO before extraction. 2 Three different temperatures of CO were tested, 25ºC, 50ºC, and 75ºC. The treated samples 2 then underwent MAS-100ºC to gather extracts for analysis. The experimental results for the DPPH test favored the SFT-75ºC treatment at a 40µl concentration. Therefore, SFT-75ºC served as the treated sample in the final three experiments. Antioxidant activity was further tested using two other methods: cholesterol oxidation and the DHA model. The total phenolic content was determined using Folin-Ciocalteau Method. The SFT-75ºC treatment showed statistically higher results for antioxidant activity in both the cholesterol oxidation and DHA oxidation experiments over the TSE-60ºC or MAS-100ºC. In terms of total phenolics, the SFT-75ºC treatment showed statistically higher results than TSE or MAS-100ºC in terms of catechin equivalency, but no statistical difference was seen among the viii treatments when compared on the basis of total phenolics per gram of original oat bran sample. However, extraction techniques can be evaluated based on extract yield, which this research demonstrated would be SFE-75ºC. In conclusion, the SFT-75ºC treatment was the optimal extraction based on antioxidant activity, catechin equivalency for total phenolics, and sample yield. This information could be used in the future development of food products as a natural antioxidant source. ix