INACTIVATION OF PEROXIDASE AND LIPOXYGENASE IN GREEN BEANS, PEAS AND CARROTS BY A COMBINATION OF HIGH HYDROSTATIC PRESSURE AND MILD HEAT TREATMENT A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF THE MIDDLE EAST TECHNICAL UNIVERSITY BY ÇADA AKYOL IN PARTIAL FULFILLMENT ON THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN FOOD ENGINEERING NOVEMBER 2004 Approval of the Graduate School of Natural and Applied Sciences. Prof. Dr. Canan Özgen Director I certify that this thesis satisfies all the requirements as a thesis for the degree of Master of Science. Prof. Dr. Levent Bayındırlı Head of Department This is to certify that we have read this thesis and that in our opinion it is fully adequate, in scope and quality, as a thesis for the degree of Master of Science. Assist. Prof. Dr. Hami Alpas Prof. Dr. Alev Bayındırlı Co-Supervisor Supervisor Examining Comitee Members Prof. Dr. Ferhunde ahbaz (HU, FdE) Prof. Dr. Alev Bayındırlı (METU, FdE) Assoc. Prof. Dr. Esra Yener (METU, FdE) Assoc. Prof. Dr. Gülüm umnu (METU, FdE) Assist. Prof. Dr. Hami Alpas (METU, FdE) I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name: Çada AKYOL Signature : iii ABSTRACT INACTIVATION OF PEROXIDASE AND LIPOXYGENASE IN GREEN BEANS, PEAS AND CARROTS BY A COMBINATION OF HIGH HYDROSTATIC PRESSURE AND MILD HEAT TREATMENT. AKYOL, Çada M.Sc., Department of Food Engineering Supervisor: Prof. Dr. Alev BAYINDIRLI Co-supervisor: Asisst. Prof. Dr. Hami ALPAS November 2004, 66 pages In this study, the efficiency of high-pressure treatment (HHP, 250 - 450 MPa) with the combination of heat treatment (20 - 70oC) on peroxidase (POD) and lipoxygenase (LOX) inactivation in green beans, green peas and carrots was investigated for blanching purposes. Two steps treatments were also performed by pressurization at 250 MPa at 20°C for 15- 60 min and then by water blanching at 40- 70°C. For green beans, 25 % residual activity was obtained by water blanching at 50°C for 15 min after the pressurization at 250 MPa for 60 min. The enzyme inactivation in green peas was 78 % with water blanching at 50oC for 30 min after holding at 250 MPa for 60 min. When the carrots were water blanched at 50°C for 30 iv min after HHP treatment at 250 MPa for 15min, 13 % residual activity was obtained. During the experiments, the stability gain or the activation of latent form of the enzymes were observed prior to inactivation. For carrots, LOX activity could not be measured. For green beans, 22 % LOX inactivation was obtained by holding at 250 MPa for 15 min and then by water blanching at 40°C for 5 min. For green peas, the multiple treatment of 250 MPa for 30 min and water blanching at 50°C for 30 min provided 70% inactivation. To obtain the enzyme inactivation higher than 90 % for blanching purposes, the pressure applied must be increased. Key words: high hydrostatic pressure, green bean, green pea, carrot, blanching, peroxidase, lipoxygenase v ÖZ YÜKSEK HDROSTATK BASINÇ VE DÜÜK ISIL LEM KOMBNASYONU LE YEL FASÜLYE, BEZELYE VE HAVUÇTA PEROKSDAZ VE LPOKSGENAZIN NAKTVASYONU AKYOL, Çada Yüksek Lisans, Gıda Mühendislii Bölümü Tez Yöneticisi: Prof. Dr. Alev BAYINDIRLI Yardımcı Tez Yöneticisi: Yard. Doç. Dr. Hami ALPAS Kasım 2004, 66 sayfa Bu çalımada, Yüksek hidrostatik basınç (YHB, 250-450 MPa) ve düük ısıl ilem (20 - 70oC) kombinasyonunun yeil fasülye, bezelye ve havuçta bulunan peroksidaz (POD) ve lipoksigenaz’ın (LOX) inaktivasyonu üzerindeki verimlilii, farklı uygulama süreleri için aratırılmıtır. YHB ve su ile halamanın iki basamaklı uygulaması da yapılmıtır. Two steps treatments were also performed by pressurization at 250 MPa at 20°C for 15- 60 min and then by water blanching at 40- 70°C. Bu uygulamalar için, sebze numuneleri 20°C’de 15-60 dakika boyunca 250 MPa basınca tabi tutulmu ve daha sonra 40-70°C arasında su ile halanmıtır. Yeil fasülyeler için, 20°C’de 60 dakika boyunca 250 MPa basınca tabi tutulduktan sonra 15 dakika boyunca 50°C’de halama sonucunda enzim aktivasyonu yaklaık olarak 25%’e dümütür. Oda sıcaklıında 250 MPa (60 dakika) basınca tabi tutulduktan sonra 50°C’de 30 dakika boyunca su ile halanmı bezelyelerde 78% vi enzim inaktivasyonu görülmütür. 20°C’de 15 dakika boyunca 250 MPa basınca tabi tutulduktan sonra 50°C’de 30 dakika boyunca halanan havuçlarda 13% enzim aktivasyonu elde edilmitir. Deneyler süresince, inaktivasyondan önce, sebze tipi ve basınç-sıcaklık kombinasyonuna balı olarak, kararlılık kazancı veya enzimlerin gizli formlarının aktivasyonu gözlemlenmitir. Havuçlarda LOX aktivitesi ölçülememitir. Yeil fasülyeler için, 15 dakika boyunca 250 MPa basınç ve daha sonra 40°C’de 5 dakika boyunca halama sonucunda 22% LOX inaktivasyonu elde edilmitir Bezelyeler için, 30 dakika boyunca 250 MPa basınç ve 50°C’de 30 dakika halama uygulaması 70% inaktivasyon yaratmıtır. Daha etkili inaktivasyon elde etmek için uygulanan basıncın arttırılması gerekmektedir. Anahtar kelimeler: yüksek hidrostatik basınç, yeil fasülye, bezelye, havuç, halama, peroksidaz, lipoksigenaz vii ACKNOWLEDGEMENTS Completion of this thesis cannot be accomplished without many people’s contributions, help and encouragement. I would like to mention those who deserve my sincere acknowledgement. I extend my sincere appreciation to my family for their patience, moral support, and sacrifices not only throughout my graduate years, but my whole life. My special thanks go to my sister, Ceyda Akyol for her support in all my life. I can never forget the motivations and support of my very valuable, dear Cenk Özcan. Without his presence even for just being beside me during my boring times, this study would not finish on time. Thanks also to my colleagues Sencer Buzrul and Iraz Göksu for helping me during my graduate study, and being perfect friends during my M.S. life. I would like to thank my dear friends Muhsin Öcal, Gülen Algan and Pınar Pembeciolu whom I met during my time at METU for their help, suggestions and encouragement. I also thank to S. Firdevs Doan, Aslı çi and Pınar Demirekler for their presence during my experiments. I am also grateful to Prof. Dr. Mirzahan Hızal and Assist. Prof. Dr. Hami Alpas for their valuable guidance, professional help, understanding, support and encouragement throughout this study. Their solutions and suggestions for all hard times of my study are greatly acknowledged. And, most importantly, I would like to express my deepest appreciation to my advisor, Prof. Dr. Alev Bayındırlı for her academic support that made this thesis possible, for her generosity and patience in correcting my scientific errors, and for keeping me focused. It has been an honor and a pleasure for me to work under her guidance. viii TABLE OF CONTENTS Page PLAGIARISM ....................................................................................................... iii ABSTRACT ........................................................................................................... iv ÖZ .......................................................................................................................... vi ACKNOWLEDGEMENTS .................................................................................. viii TABLE OF CONTENTS........................................................................................ ix LIST OF TABLES.................................................................................................. xi LIST OF FIGURES ............................................................................................... xii CHAPTER 1. INTRODUCTION ...............................................................................................1 1.1Blanching.........................................................................................................2 1.2 Peroxidase ......................................................................................................5 1.3 Lipoxygenase..................................................................................................7 1.4 High Pressure Processing ................................................................................9 1.5 Effect of High Pressure Processing on Enzymes............................................12 1.6 Effect of high pressure processing on quality related enzymes in fruits and vegetables .....................................................................................................14 1.7 Effect of high pressure processing on the quality of fruits and vegetables......20 1.8 Aim of the study............................................................................................22 2. MATERIALS AND METHODS ........................................................................24 2.1 Vegetables ....................................................................................................24 2.2 High Pressure Treatment ...............................................................................24 2.3 Thermal Treatment (Water Blanching) ..........................................................25 2.4 Enzyme Assays .............................................................................................27 2.4.1 Preperation of Crude Enzyme Extracts.................................................27 2.4.2 Peroxidase Assay.................................................................................27