EXTRACTION OF OMEGA-3 FATTY ACIDS FROM ATLANTIC HERRING (Clupea harengus) by Akila Bura Mohanarangan Submitted in partial fulfilment of the requirements for the degree of Master of Applied Science at Dalhousie University Halifax, Nova Scotia November 2012 © Copyright by Akila Bura Mohanarangan, 2012 i DALHOUSIE UNIVERSITY DEPARTMENT OF PROCESS ENGINEERING AND APPLIED SCIENCE The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled “EXTRACTION OF OMEGA-3 FATTY ACIDS FROM ATLANTIC HERRING (Clupea harengus)” by Akila Bura Mohanarangan in partial fulfilment of the requirements for the degree of Master of Applied Science. Dated: November 22, 2012 Supervisors: _________________________________ _________________________________ Reader: _________________________________ ii DALHOUSIE UNIVERSITY DATE: November 22, 2012 AUTHOR: Akila Bura Mohanarangan TITLE: EXTRACTION OF OMEGA-3 FATTY ACIDS FROM ATLANTIC HERRING (Clupea harengus) DEPARTMENT OR SCHOOL: Department of Process Engineering and Applied Science DEGREE: MASc CONVOCATION: May YEAR: 2013 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author iii DEDICATION TO: My parents Mohanarangan. B.V and Maduram. N iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................... ix LIST OF FIGURES ............................................................................................................. xii ABSTRACT ....................................................................................................................... xiv LIST OF ABBREVIATIONS AND SYMBOLS USED .................................................... xv ACKNOWLEDGEMENTS .............................................................................................. xvii CHAPTER 1. INTRODUCTION ......................................................................................... 1 CHAPTER 2. OBJECTIVES ................................................................................................ 4 CHAPTER 3. LITERATURE REVIEW .............................................................................. 5 3.1. Canadian Fish Production ........................................................................................ 5 3.1.1. Atlantic region ............................................................................................... 5 3.1.2. Pacific region ................................................................................................. 5 3.2. Fish Processing Waste .............................................................................................. 8 3.3. Utilization of Fish Processing Waste ..................................................................... 10 3.3.1. Animal feed .................................................................................................. 10 3.3.2. Fish meal ...................................................................................................... 11 3.3.3. Chitin/chitosan ............................................................................................. 11 3.3.4. Enzymes ....................................................................................................... 13 3.3.5. Biodiesel ...................................................................................................... 14 3.4. Fish Lipids .............................................................................................................. 15 3.5. Omega-3 Fatty Acids ............................................................................................. 17 3.5.1. Alpha-linolenic acid (ALA) ......................................................................... 17 3.5.2. Eicosapentaenoic acid (EPA) ....................................................................... 20 3.5.3. Docosahexaenoic acid (DHA) ..................................................................... 20 3.6. Dietary Sources of Polyunsaturated Fatty Acids.................................................... 22 3.6.1. Marine Sources ............................................................................................ 22 3.6.2. Plant sources ................................................................................................ 26 3.7. Medical uses of Omega-3 Oil................................................................................. 27 3.8. Extraction of Fish Oil ............................................................................................. 27 3.8.1. Cooking and pressing ................................................................................... 27 3.8.2. Solvent extraction ........................................................................................ 28 v 3.8.3. Enzymatic extraction ................................................................................... 30 3.9. Refining Edible Oils ............................................................................................... 31 3.9.1. Degumming ................................................................................................. 32 3.9.2. Neutralization ............................................................................................... 32 3.9.3. Bleaching ..................................................................................................... 32 3.9.4. Deodorization ............................................................................................... 33 3.10. Lipid Oxidation .................................................................................................... 33 3.10.1. Auto oxidation ........................................................................................... 33 3.10.2. Photo oxidation .......................................................................................... 35 3.10.3. Enzymatic oxidation .................................................................................. 36 3.11. Use of Antioxidants .............................................................................................. 36 3.11.1. Natural antioxidants ................................................................................... 37 3.11.2. Synthetic antioxidants ................................................................................ 38 3.12. Quality Parameters of Fish Oil ............................................................................. 38 3.12.1. Acid value .................................................................................................. 40 3.12.2. p-Anisidine value ....................................................................................... 41 3.12.3. Peroxide value ............................................................................................ 42 3.13. Concentration and Composition of Omega-3 Fatty Acids ................................... 42 3.13.1. Enzymatic hydrolysis ................................................................................. 43 3.13.1.1. Enzyme load. ............................................................................... 46 3.13.1.2. Temperature. ................................................................................ 46 3.13.1.3. pH. ............................................................................................... 47 3.13.1.4. Reaction time. .............................................................................. 47 3.13.2. Urea complexation ..................................................................................... 47 3.13.2.1. Crystallisation temperature. ......................................................... 51 3.13.2.2. Crystallisation time. ..................................................................... 52 3.13.2.3. Urea:fatty acid ratio. .................................................................... 52 3.13.3. Supercritical fluids ..................................................................................... 52 3.13.4. Molecular distillation ................................................................................. 53 3.13.5. Comparison of extraction methods ............................................................ 55 CHAPTER 4. MATERIALS AND METHODS ................................................................. 59 4.1. Experimental Materials .......................................................................................... 59 4.1.1. Fish ............................................................................................................... 59 4.1.2. Glassware ..................................................................................................... 59 vi 4.1.3. Chemicals and enzymes ............................................................................... 59 4.1.4. Reagents ......................................................................................................