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Istanbul Technical University Institute of Science and Technology ISTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY CHANGES IN ANTIOXIDANT PROFILES, METABOLITES AND ENZYMES DURING DEVELOPMENT OF TOMATO FRUIT AND TOMATO PASTE PROCESSING Ph.D. Thesis by Esra ÇAPANOLU, M.Sc. 506022130 Department : Food Engineering Programme : Food Engineering Supervisor : Prof. Dr. Dilek BOYACIOLU MAY 2008 FOREWORD The health protective effects of tomato created a great interest in the effect of processing since its industrially processed products are highly consumed worldwide. The purpose of this study was to evaluate the effects of tomato processing on its antioxidant compounds, metabolites, enzymes and to investigate the effects of development stages on the metabolites which are present in the different tissues of tomatoe. I hope this study will be enhancing the current literature on the health characteristics of a processed tomato product, the paste. First of all, I would like to express my special thanks and gratitude to my supervisor and mentor, Prof. Dr. Dilek BOYACIOLU with whom I worked for many years and who helped me in countless ways to bring me up in the scientific and academic area, and supervised me to carry out this study. I also would like to thank Prof. Dr. Artemis KARAALI and Prof. Dr. Reat APAK with whom it was a pleasure to work with, and I do appreciate for their valuable contributions. This thesis was a product of long and intensive study which was partly realized in PRI (Plant Research International), The Netherlands and IPK (The Leibniz Institute of Plant Genetics and Crop Plant Research), Germany. This study was sponsored by the TINCEL Proficient (3 months) and DAAD (German Academic Exchange Programme) (5 months), and it was a part of the EU 6th Frame work FLORA Project. It is my privilege to express my special thanks and sincere appreciations to Dr. Jules BEEKWILDER, Dr. Ric DE VOS, Dr. Robert HALL, Dr. Sofia MOCO, Mr. Harry JONKER, and Mr. Bert SCHIPPER from PRI who not only helped me to shape my dissertation, but also contributed to it in many ways. I also would like to thank to Dr. Hans-Peter MOCK, Dr. Andrea MATROS, and Dr. Silke PETEREK in IPK, with whom I had the chance to work on enzymes and to learn many new techniques. I also would like to express my gratitude to Tamek Co, Inc., Karacabey and especially to Mrs. Özlem HALL for providing the samples from paste production. I would like to thank to all my instructors in the university who helped me during my education. I express my special thanks to Aslı CAN KARAÇA, and acknowledge to Assoc. Prof. Dr. Beraat ÖZÇELK, Baak AKMAN, Dilara NLÜFER ERDL, Nee AHN YELÇUBUK, Nalan DEMR, and all my friends who helped me in various ways to realize this study. Finally, a very special note of appreciation and thanks to my mother Vedia, my father Bilgin, my brother Tolga, and my sister Gizem for their love, help and support throughout my education and life. I would like to express my special thanks to my fiancé, Aybars, for his patience, encouragement, support, and help during these years. I would like to give this thesis as a gift to my family. April, 2008 Esra ÇAPANOLU, M.Sc. ii CONTENTS Page Number ABBREVIATIONS ................................................................................................... vi LIST of TABLES ....................................................................................................viii LIST of FIGURES ..................................................................................................... x LIST of SYMBOLS.................................................................................................xiii ÖZET........................................................................................................................ xiv SUMMARY ............................................................................................................. xix 1. INTRODUCTION.................................................................................................. 1 2. LITERATURE ....................................................................................................... 4 2.1. Tomatoes and Tomato Paste.............................................................................. 4 2.2. Healthy Compounds of Tomato and Tomato Products ..................................... 7 2.2.1. Flavonoids ................................................................................................. 10 2.2.2. Carotenoids................................................................................................ 14 2.2.3. Tocopherols ............................................................................................... 17 2.2.4. Ascorbic acid............................................................................................. 18 2.3. Changes in Antioxidants During Development of Tomato Fruit .................... 19 2.4. Effect of Industrial Food Processing on Tomato Antioxidants ....................... 23 2.4.1. Thermal treatments.................................................................................... 25 2.4.2. Non-thermal treatments............................................................................. 32 3. MATERIALS AND METHODS ........................................................................ 34 3.1. Materials .......................................................................................................... 34 3.1.1. Chemicals .................................................................................................. 34 3.1.2. Tomatoes and tomato products.................................................................. 35 3.1.3. Tissues of tomato at different developmental stages................................. 36 3.1.4. Tomato samples at different developmental dtages for measurement of enzyme activity ..................................................................................... 37 3.2. Methods ........................................................................................................... 38 3.2.1. Moisture analysis....................................................................................... 38 3.2.2. Determinations of optimum solvent for the analysis of phenolic content and antioxidant capacity ............................................................... 39 3.2.3. Analysis of total phenolics ........................................................................ 39 3.2.4. Analysis of total flavonoids....................................................................... 40 3.2.5. Total antioxidant capacity assays .............................................................. 40 3.2.6. On-line antioxidant capacity ..................................................................... 41 3.2.7. Flavonoid composition.............................................................................. 42 3.2.8. Carotenoids................................................................................................ 43 iii 3.2.8.1. Selection of extraction method............................................................ 43 3.2.8.2. Carotenoid composition ...................................................................... 43 3.2.9. Tocopherol composition............................................................................ 44 3.2.10. Analysis of vitamin C.............................................................................. 44 3.2.11. High Performance Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry based metabolomics ......... 45 3.2.11.1. Data analysis and alignment.............................................................. 45 3.2.11.2. Annotation of metabolites................................................................. 46 3.2.11.3. Multivariate analyses of LC-MS data ............................................... 46 3.2.12. Enzyme activity measurements ............................................................... 47 3.2.12.1. 3-O-Glucosyl transferase assay......................................................... 47 3.2.12.2. Protein determination........................................................................ 49 3.2.12.3. Phenylalanine ammonia lyase assay ................................................. 49 3.2.13. Statistical analysis ................................................................................... 51 4. RESULTS AND DISCUSSION .......................................................................... 52 4.1. Effect of Tomato Processing on Antioxidative Compounds ........................... 52 4.1.1. Moisture contents ...................................................................................... 52 4.1.2. Solvent choice for spectrophotometric methods ....................................... 53 4.1.3. Total phenolics .......................................................................................... 54 4.1.4. Total flavonoids......................................................................................... 56 4.1.5. Antioxidant capacity.................................................................................. 57 4.1.6. On-line antioxidant detection .................................................................... 61 4.1.7. Variation between batches......................................................................... 64 4.1.8. Flavonoid profile and chlorogenic acid..................................................... 67 4.1.9. Carotenoids................................................................................................ 70 4.1.9.1. Optimum extraction
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