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Isolation and Characterization of Ovomucin – a Bioactive Agent of Egg White

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Isolation and Characterization of Ovomucin – a Bioactive Agent of Egg White DOCTORAL THESES IN FOOD SCIENCES AT THE UNIVERSITY OF TURKU /ƐŽůĂƟŽŶĂŶĚ ŚĂƌĂĐƚĞƌŝnjĂƟŽŶŽĨKǀŽŵƵĐŝŶ ͲĂŝŽĂĐƟǀĞŐĞŶƚŽĨŐŐtŚŝƚĞ JAAKKO HIIDENHOVI Food Chemistry and Food Development Department of Biochemistry DOCTORAL THESES IN FOOD SCIENCES AT THE UNIVERSITY OF TURKU Food Chemistry Isolation and Characterization of Ovomucin - a Bioactive Agent of Egg White JAAKKO HIIDENHOVI Food Chemistry and Food Development Department of Biochemistry TURKU, FINLAND – 2015 Food Chemistry and Food Development Department of Biochemistry University of Turku, Finland Supervised by Director Eeva-Liisa Ryhänen, Ph.D. Green Technology Natural Resources Institute Finland (Luke) Jokioinen, Finland Professor Rainer Huopalahti, Ph.D. Department of Biochemistry University of Turku Turku, Finland Reviewed by Professor Francoise Nau, Ph.D. Agrocampus Ouest UMR1253 Dairy and Egg Science and Technology Rennes, France Professor Olli Lassila, MD, Ph.D. Department of Biomedicine University of Turku Turku, Finland Opponent Professor Markku Kulomaa, Ph.D. BMT / Molecular Biotechnology University of Tampere Tampere, Finland Research director Professor Heikki Kallio, Ph.D. Department of Biochemistry University of Turku Turku, Finland The originality of this dissertation has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service ISBN 978-951-29-6114-6 (print) ISBN 978-951-29-6115-3 (pdf) ISSN 2323-9395 (print) ISSN 2323-9409 (pdf) Painosalama Oy – Turku, Finland 2015 In memory of my parents Table of Contents TABLE OF CONTENTS ABSTRACT ........................................................................................................ iv SUOMENKIELINEN ABSTRAKTI ................................................................... v LIST OF ABBREVIATIONS ............................................................................. vi LIST OF ORIGINAL PUBLICATIONS .......................................................... viii 1 INTRODUCTION ........................................................................................... 1 2 REVIEW OF THE LITERATURE ................................................................. 3 2.1 Chemical and structural characterization of ovomucin .......................... 3 2.1.1 Ovomucin subunits ......................................................................... 3 2.1.1.1 α-Ovomucin – a proteinaceous subunit ................................ 4 2.1.1.2 β-Ovomucin – a carbohydrate-rich subunit .......................... 7 2.1.2 Ovomucin complex ........................................................................ 8 2.2 Isolation and purification of ovomucin ................................................. 12 2.2.1 Isoelectric precipitation method ................................................... 12 2.2.2 Chromatographic methods ........................................................... 17 2.2.3 Other methods .............................................................................. 21 2.3 Properties of ovomucin ......................................................................... 21 2.3.1 Biological role .............................................................................. 21 2.3.2 Role in egg white thinning ........................................................... 22 2.3.2.1 Enzyme action .................................................................... 23 2.3.2.2 Reactions of disulphide bonds of ovomucin ...................... 23 2.3.2.3 Ovomucin-lysozyme interaction ........................................ 24 2.3.2.4 Changes in the ovomucin subunit structure ....................... 24 2.3.2.5 Ovomucin and divalent cations .......................................... 25 2.3.3 Techno-functional properties ....................................................... 26 2.3.4 Bioactive properties ..................................................................... 27 2.3.4.1 Antiviral properties ............................................................. 27 2.3.4.2 Antitumour activity ............................................................ 28 2.3.4.3 Antiadhesive and antibacterial activity .............................. 29 2.3.4.4 Immunomodulating activity ............................................... 30 2.3.4.5 Antioxidant activity ............................................................ 30 2.3.4.6 Hypocholesterolemic activity ............................................. 30 2.3.4.7 Dipeptyl dipeptidase-4 (DPP4) inhibitory activity ............. 31 2.4.4.8 Antihypertensive activity .................................................. 31 2.4 Summary of the literature review ......................................................... 31 Table of Contents 3 AIMS OF THE STUDY ................................................................................ 34 4 MATERIALS AND METHODS .................................................................. 35 4.1 Egg samples .......................................................................................... 35 4.2 Separation of egg white fractions ......................................................... 35 4.3 Isolation of ovomucin ........................................................................... 36 4.3.1 Isoelectric precipitation method (I˗IV) ....................................... 36 4.3.2 Gel filtration chromatography (GFC) method (I, III) .................. 36 4.3.2.1 Preparative Sephacryl S-400 GFC with reducing agent (I) 36 4.3.2.2 Preparative Superose 6 GFC with reducing agent (III) ..... 36 4.3.2.3 Preparative Superose 6 GFC without reducing agent (III) 37 4.4 Characterization of ovomucin .............................................................. 37 4.4.1 Analytical GFC (II˗IV) ................................................................ 37 4.4.2 SDS-PAGE (I˗III) ........................................................................ 37 4.4.3 Amino acid analysis (I) ................................................................ 37 4.4.4 Carbohydrate analysis (II) ............................................................ 38 4.5 Enzymatic hydrolysis of ovomucin (III, IV) ........................................ 38 4.6 Characterization of ovomucin hydrolysates (III, IV) ............................ 38 4.7 Solubilisation of ovomucin (IV) ........................................................... 39 4.8 Solubilisation rate of ovomucin (IV) .................................................... 40 4.9 Yield determination of various ovomucin aggregates .......................... 41 4.9.1 Crude ovomucin (II˗IV) ............................................................... 41 4.9.2 Ovomucin-G (II˗IV) ..................................................................... 41 4.9.3 Ovomucin-C (III, IV) ................................................................... 42 4.10 Statistical analysis (III, IV) ................................................................... 42 5 RESULTS AND DISCUSSION .................................................................... 43 5.1 Isolation of ovomucin by using modified IEP method (I˗III) ............... 43 5.2 Characterization of ovomucin ............................................................... 48 5.2.1 Subunit structure of ovomucin (I-III) ........................................... 48 5.2.2 Subunit structure of ovomucin-G (III) ......................................... 51 5.3 Eggs as source of ovomucin (II, III) ..................................................... 52 5.3.1 Protein composition of crude ovomucin ...................................... 53 5.3.2 Various complex forms of ovomucin ........................................... 54 5.3.3 Ovomucin subunits ....................................................................... 58 5.4 Dissolving of ovomucin (IV) ................................................................ 58 5.4.1 Dissolving of ovomucin by using physical methods ................... 58 5.4.2 Dissolving of ovomucin by using enzymatic hydrolysis ............. 61 5.5 Antiviral properties of ovomucin .......................................................... 62 5.5.1 Antiviral properties of crude ovomucins obtained from various EW layers (III) ................................................. 62 Table of Contents 5.5.2 Effects of various dissolving methods on antiviral properties of ovomucin (IV) .......................................................... 64 6 SUMMARY AND CONCLUSION .............................................................. 67 ACKNOWLEDGEMENTS ............................................................................... 68 REFERENCES ................................................................................................... 69 APPENDIX: ORIGINAL PUBLICATIONS .................................................... 79 iv List of Abbreviations ABSTRACT The hen’s egg is a source of new life. Therefore, it contains many biologically active compounds. In addition to being a very nutritious food and also commonly used in the food industry due to its many techno-functional properties, the egg can serve as a source of compounds used as nutra-, pharma- and cosmeceuticals. One such interesting compound is ovomucin, an egg white protein responsible for the gel-like properties of thick egg white. Previous studies have indicated that ovomucin and ovomucin-derived peptides have several different bioactive properties. The objectives of the present study were to develop isolation methods for ovomucin, to characterize the structure of ovomucin, to compare various egg fractions
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