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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Aston Publications Explorer CHARACTERISATION AND OXIDATIVE STABILITY OF SPECIALITY PLANT SEED OILS ANUJ KUMAR CHANDER Doctor of Philosophy ASTON UNIVERSITY DECEMBER 2010 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without proper acknowledgement. 1 Summary The past decade has seen an influx of speciality plant seed oils arriving into the market place. The need to characterise these oils has become an important aspect of the oil industry. The characterisation of the oils allows for the physical and chemical properties of the oil to be determined. Speciality oils were characterised based on their lipid and fatty acid profiles and categorised as monounsaturated rich (oleic acid as the major acyl components e.g. Moringa and Marula oil), linoleic acid rich (Grape seed and Evening Primrose oil) or linolenic acid rich (Flaxseed and Kiwi oil). The quality of the oils was evaluated by determining the free fatty acid content, the peroxide value (that measures initial oxidation) and p-anisidine values (that determines secondary oxidation products containing the carbonyl function). A reference database was constructed for the oils in order to compare batches of oils for their overall quality including oxidative stability. For some of the speciality oils, the stereochemistry of the triacylglycerols was determined. Calophyllum, Coffee, Poppy and Sea Buckthorn oils stereochemistry was determined. The oils were enriched with saturated and/or a monounsaturated fatty acids at position sn-1 and sn-3. The sn-2 position of the four oils was esterified with a polyunsaturated and/or a monounsaturated fatty acid indicating that they follow a typical acylation pathway and no novel acylation activity was evident from these studies (e.g enrichment of saturates at the sn-2 position). The oxidative stability of the oils was evaluated at 18oC and 60oC and the effect of adding α-tocopherol at commercially used level i.e 750ppm was assessed. The addition of 750ppm of α-tocopherol at 18oC increased the oxidative stability of Brown flax, Moringa, 2 Wheat germ and Yangu oils. At 60oC Brown Flax, Manketti and Pomegranate oil polymerised after 48 hours. The addition of 750ppm α-tocopherol delayed the onset of polymerisation by up to 48 hours in Brown Flax seed oil. Pomegranate oil showed a high resistance to oxidation, and was blended into other speciality oils at 1%. Pomegranate oil increased the oxidative stability of Yangu oil at 18oC. The addition of Pomegranate oil to Wheat germ oil at 60oC, decreased the peroxide content by 10%. In Manketti and Brown Flaxseed oil at elevated temperatures, Pomegranate oil delayed the onset of polymerisation. Preliminary studies of Pomegranate oil blending to Moringa and Borage oil showed it to be more effective than α-tocopherol for certain oils. The antioxidant effects observed following the addition of Pomegranate oil may be due to its conjugated linolenic acid fatty acid, punicic acid. Key Words: Stereochemistry, Pomegranate, α-Tocopherol, Punicic acid 3 Dedication The thesis is dedicated to my late mother and father, Meena and Jagdish Chander. Years have passed since we were all together, you both are deeply missed. I hope I am making you proud. 4 Acknowledgements I would like to acknowledge the sponsorship from Earthoil Plantations and EPSRC. I would like to express my thanks to Andrew and Campbell Walter. I would like to thank Dr Gareth Griffiths for giving me the opportunity to undertake this research program. The input and support you have provided over the past four years has been invaluable. I really appreciate everything you have done for me, you have really influenced my life in a positive way. Thanks I would also like to acknowledge my colleagues, Simon Smith, Yi Zhou and Tulpesh Patel, for their friendship and support throughout the four years. I hope our friendship continues to grow as we all embark on new journeys in our lives. Simon Stevens our weekly sessions have made a great improvement to my health, your one in a million. I would like to thank my family Tony, Tina, and Tana Chander, and new arrival little Dudley. Thanks for all your love and support. Finally I would like to thank my beautiful wife Anuradha, for her continued love and support. 5 List of Contents Title Page 1 Summary 2 Dedication 4 Acknowledgements 5 List of Contents 6 List of Figures 11 List of Tables 14 List of Graphs 16 Abbreviations 16 Chapter 1 Introduction 20 1.1 The Chemistry of Fats and Oils 20 1.1.1 Lipids, Fatty Acids and Triacylglycerols 20 1.1.2 Fatty Acid Biosynthesis 24 1.1.3 Fatty Acid Derivatives 33 1.1.4 Triacylglycerol Biosynthesis and Assembly 47 1.1.5 Oil Bodies 50 1.1.6 Triacylglycerol Molecular Species 51 1.2 Fats and Oils Commercialisation and Speciality Products 53 1.2.1 Commercialization of Plant Seed Oils 53 1.2.2 Speciality Oils 54 1.3 Lipid Oxidation 61 6 1.3.1 Lipid Oxidation 61 1.3.2 Free Radical Oxidation 61 1.3.3 Hydroperoxide Formation 64 1.3.4 Lipid Oxidation Detection Methods 69 1.3.5 Oxidative Stability 75 1.4 Antioxidants 76 1.4.1 An Introduction to Antioxidants 76 1.4.2 Antioxidant Mechanism 77 1.4.3 Synthetic and Natural Antioxidants 79 1.5 Vitamin E 84 1.5.1 Vitamin E, Tocopherols and Tocotrienols 84 1.5.2 The Effects of Tocopherols on lipid Peroxidation: 86 Tocopherols As Free Radical scavengers. 1.5.3 Tocopherols as Proxidants 90 1.5.4 Major Factors Affecting Tocopherol Antioxidant Potency 90 Aims of PhD Research 93 Chapter 2 Materials and Methods 95 2.1 Materials 95 2.2 Methods 95 2.2.1 Fatty Acid Methyl Esters 95 2.2.2 Punicic Acid Fatty Acid Methyl Ester 96 2.2.3 Peroxide Value 96 2.2.4 Free Fatty Acid Value 97 7 2.2.5 Conjugated Diene 98 2.2.6 p-Anisidine 99 2.2.7 Saponification Value 99 2.2.8 Thin Layer Chromatography 100 2.2.9 Oxidative Stability 103 2.2.10 Stereochemistry 103 2.2.11 Bleaching 105 2.2.12 Oil Extraction 106 2.2.13 Punicic Acid Extraction 107 2.2.14 Removal of Natural Occurring Antioxidants and 107 Tocopherols Chapter 3 Characterisation of Plant Seed Oils 108 3.1 Introduction 108 3.2 Results 109 3.3 Results and Discussion 138 3.3.1 Mass Spectral Data 138 3.3.2 Oleic Acid rich Oils 140 3.3.3 Linoleic Rich Oils 142 3.3.4 Linolenic Rich Oils 143 3.3.5 Saturated Fats 143 3.3.6 Castor Oil 144 3.3.7 Oil Content of Seeds 144 3.3.8 Lipid Classes of Oils 144 8 3.3.9 Stereochemical Analysis 145 3.3.10 Punicic Acid Methylation 147 3.4 Conclusions 148 Chapter 4 Evaluating the Oxidative Stability of Plant Seed Oils 149 4.1 Introduction 149 4.2 Results 150 4.3 Results and Discussion 161 4.3.1 Oxidative Stability of Plant Seed Oils 161 4.3.2 Peroxide Accumulation in MUFA Rich Oil 162 4.3.3 Peroxide Accumulation in PUFA Rich Oils 163 4.3.4 Assays to Evaluate Oxidation 164 4.3.5 Oxidation of Oils Rich in Oleic Acid 166 4.3.6 Oxidation of Oils Rich in Linoleic Acid 168 4.3.7 Oxidation of Oils Rich in Linolenic Acid 170 4.4 Conclusions 173 Chapter 5 Preliminary Blending of Pomegranate Oil 174 5.1 Introduction 174 5.2 Results 176 5.3 Results and Discussion 180 5.3.1 Percentage Addition of Pomegranate Oil 180 5.3.2 Pomegranate and α-Tocopherol 181 5.3.3 Borage Oil Blended with 1% Pomegranate Oil 181 5.3.4 Radical Scavenging Properties of Punicic acid 182 9 5.4 Conclusions 186 Chapter 6 Screening Pomegranate Oil for Oxidation Stabilising Potential 187 6.1 Introduction 187 6.2 Results 188 6.3 Results and Discussion 195 6.3.1 Oleic Rich Oils 195 6.3.2 Linoleic Rich Oils 197 6.3.3 Linolenic Rich Oils 199 6.4 Conclusions 200 Chapter 7 Processing of Oils 201 7.1 Introduction 201 7.2 Results 203 7.3 Results and Discussion 206 7.3.1 Colour Removal from Cotton Seed Oil 206 7.3.2 Cotton Seed Oil 206 7.3.3 Moringa Oil 207 7.3.4. Tocopherol Removal 208 7.4 Conclusions 210 Chapter 8 General Conclusions 211 References 214 Appendix 237 10 List of Figures Figure 1. Saturated and Unsaturated Fatty Acids Figure 2. Triacylglycerol Figure 3. Formation of Acetyl-CoA Figure 4. Formation of Malonyl-CoA Figure 5. Biosynthesis of Fatty Acids Figure 6. Desaturation of C18:0-ACP Figure 7. Cyclic Fatty Acids Cylcopropne and Cyclopropene Figure 8. Biosynthesis of Cyclopropene Figure 9. Cyclic Endoperoxides Figure 10. The Biosynthesis of Jasmonic Acid Figure 11. Fatty Acid Derivatives, Divinyl Ethers Figure 12. Furan Fatty Acids Figure 13. Allenic Acid Figure 14. Acetylenic Acid Figure 15. Branched Chain Fatty Acids Figure 16. Biosynthesis of Triacylglycerols Figure 17. Formation of Oil Bodies, Oleosome Figure 18. Oleic Acid Hydroperoxide Formation Figure 19. Linoleic acid Hydroperoxide Formation Figure 20. Linolenic Acid Hydroperoxide Formation Figure 21. The Kinetics of Oil Oxidation 11 Figure 22. Reaction Scheme for Peroxide Value Figure 23. Proposed Reaction Scheme for p-Anisidine Reaction with Oxidised Lipids Figure 24.