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CHARACTERIZATION OF VOLATILE COMPOUNDS IN SELECTED CITRUS FRUITS FROM ASIA JORRY DHARMAWAN (B.Appl.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgments This project could not have been completed without the support of Food Science and Technology programme of the National University of Singapore and Firmenich Asia Pte. Ltd. for endorsing and financing the project. I am greatly indebted to the following supervisors and consultants who graciously lent me their technical expertise and encouragement throughout the project: • Associate Professor Stefan Kasapis for his supervision and guidance in journal publications, and for his advices, supports and encouragements. • Mr. Philip Curran for his supervision and guidance with his expertise and experiences in flavour industry. • Associate Professor Philip J Barlow and Associate Professor Conrad O Perera for their initial supervision in this research project. Gratitude is also expressed for the following people for their contribution to the project: • Dr. Martin J Lear and Ms. Praveena Sriramula from Department of Chemistry, NUS, for their assistance in the synthesis of (Z)-5-dodecenal. • Mr. Kiki Pramudya, Ms. Chionh Hwee Khim, Ms. Alison Tan, Ms. Yukiko, Ms. Susan Chua and Ms. Feng Peiwen from Firmenich Asia Pte. Ltd. for their advices and participation as panellists. • Ms. Mia Isabelle and Mr. Xu Jia for their contribution as panellists. i • Ms. Cynthia Lahey, Dr. Novalina Lingga and Mr. Mark Teo from Shimadzu Asia for their technical support in GC-MS. • Mdm. Lee Chooi Lan, Ms. Lew Huey Lee and Mr. Abdul Rahaman bin Mohd Noor for their continuous assistance whenever I need their lending hands. • Mdm. Frances Lim and Ms. Joanne Soong from HPLC Lab, NUS for their assistance in GC-FID. • Mr. Don Hendrix and staff at Firmenich Citrus Centre for their assistance and hospitality during my visit. • Mr. Gerald Uhde and staff at Firmenich Geneva for their assistance and hospitality during my visit. • Ms. Daisy Lam from Firmenich Asia Pte. Ltd. for her assistance in administrative matters Special thanks are owed to the following people: My parents, Mr. Hendy Dharmawan and Mdm. Phang Kim Jin, and my beloved family, together with my brothers and sisters from the Indonesian group of Hope of God Church, Singapore for their prayer support and encouragement. My gratitude is also for those whose names cannot be mentioned one by one here but have helped me in different ways throughout the duration of my postgraduate study and without them, this research will not be able to be completed. Finally and most importantly, I would like to acknowledge God’s grace and help, which has been critical to the success and completion of this project. ‘His grace is sufficient for me, for His power is made perfect in weakness.’ ii Table of Contents Page ACKNOWLEDGMENT i SUMMARY vi LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix LIST OF PUBLICATIONS AND PRESENTATION xi CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 3 2.1. Citrus Fruits 3 2.1.1. Fruit morphology 3 2.1.2. Chemical composition 5 2.1.3. Uses of citrus fruits 8 2.2. Citrus Variety 12 2.2.1. General classification 12 2.2.2. Selected citrus cultivars from Asia 14 2.3. Citrus Flavour 17 2.3.1. Important volatile compounds in citrus flavour 19 2.3.2. Factors affecting citrus flavour 21 2.4. Flavour Research 24 2.4.1. Challenges in flavour research 24 2.4.2. Systematic approach in flavour research 26 References 35 iii CHAPTER 3: CHARACTERIZATION OF VOLATILE COMPOUNDS IN HAND-SQUEEZED JUICES OF SELECTED CITRUS FRUITS FROM ASIA 55 3.1. Abstract 55 3.2. Introduction 56 3.3. Materials and Methods 57 3.3.1. Materials 57 3.3.2. Chemicals 57 3.3.3. pH, brix value and titratable acidity 58 3.3.4. SPME 59 3.3.5. Continuous liquid-liquid extraction 59 3.3.6. Gas Chromatograph-Flame Ionization Detector (GC-FID) 60 3.3.7. Gas Chromatograph/Mass Spectrometry (GC/MS) 60 3.3.8. Linear Retention Index 61 3.4. Results and Discussion 61 3.4.1. Chemical composition 61 3.4.2. Volatile compounds in citrus juices 63 References 75 CHAPTER 4: CHARACTERIZATION OF VOLATILE COMPOUNDS IN PEEL OIL OF SELECTED CITRUS FRUITS FROM ASIA 80 4.1. Abstract 80 4.2. Introduction 81 4.3. Materials and Methods 81 4.3.1. Materials 81 4.3.2. Chemicals 82 iv 4.3.3. Gas Chromatograph-Flame Ionization Detector (GC-FID) 82 4.3.4. Gas Chromatograph/Mass Spectrometry (GC/MS) 83 4.4. Results and Discussion 83 References 90 CHAPTER 5: EVALUATION OF AROMA ACTIVE COMPOUNDS IN PONTIANAK ORANGE PEEL OIL 92 5.1. Abstract 92 5.2. Introduction 92 5.3. Materials and Methods 94 5.3.1. Materials and chemicals 94 5.3.2. Gas Chromatograph/Olfactometry (GC-O) 95 5.3.2. Aroma Extract Dilution Analysis (AEDA), Relative Flavour Activity (RFA) and Odour Activity Value (OAV) 95 5.3.4 Aroma reconstitution and omission test 96 5.4. Results and Discussion 97 5.4.1. Aroma active compounds of Pontianak orange peel oil 97 5.4.2. Odour Activity Value (OAV) and Relative Flavour Activity (RFA) 104 5.4.3. Aroma reconstitution 110 5.4.4. Omission experiments 112 References 113 CHAPTER 6: CONCLUSION 118 CHAPTER 7: SUGGESTION FOR FUTURE WORK 120 APPENDIX 122 v Summary In this research, the characterization of volatile compounds in selected citrus fruits from Asia, namely Pontianak orange from Indonesia, Mosambi from India and Dalandan from the Philippines has been carried out for their juices and peel oils. Continuous liquid-liquid extraction with diethyl ether and Solid Phase Microextraction (SPME) were utilized to extract the volatiles from the juices prior to analysis with Gas Chromatography (GC), while direct injection to the GC was done for the hand-pressed peel oils. Flame Ionization Detector (FID) and Mass Spectrometer (MS) detector were used for quantitative and qualitative analysis respectively. There was a difference between juice and peel oil in the compounds characterized as the former contained more esters. Despite some differences, the profile of volatile compounds found in Mosambi was generally comparable to typical sweet orange whereas Dalandan’s profile resembled typical mandarin. On the other hand, Pontianak orange portrayed its unique citrus flavour profile. Consequently, further investigation has been explored to unveil the key compounds in Pontianak orange peel oil through a systematic approach. GC-Olfactometry (GC-O) was used to screen the potent odourants by using human nose as the detectors. Aroma Extract Dilution Analysis (AEDA) technique performed was effective in revealing 41 aroma active compounds, which were dominated by saturated and unsaturated aldehydes. Lastly, aroma reconstitution and omission test were carried out to verify the findings by sensory evaluation of aroma models. The outcome suggested that (Z)- 5-dodecenal and 1-phenyl ethyl mercaptan were the significant contributors to the flavour of Pontianak orange. vi List of Tables Table 3.1. Chemical composition of various orange juice cultivars 61 Table 3.2. Volatile compounds of freshly squeezed Pontianak orange, Mosambi and Dalandan juices 64 Table 4.1. Volatile compounds of the peel oil of Pontianak orange, Mosambi and Dalandan 84 Table 5.1. Aroma active compounds (FD ≥ 2) in Pontianak orange peel oil 99 Table 5.2. The Odour Activity Value (OAV) and Relative Flavour Activity (RFA) of aroma active compounds in Pontianak orange peel oil 105 Table 5.3. Potent odourants in Pontianak orange peel oil based on their Odour Activity Values (OAV>2000) 108 Table 5.4. Potent odourants in Pontianak orange peel oil based on their Relative Flavour Activity (RFA>6.5) 109 Table 5.5. Sensory evaluation for the aroma model of the Pontianak orange peel oil as affected by the omission of compounds 113 vii List of Figures Figure 2.1. Section of citrus fruit (Ranganna et al ., 1986) 4 Figure 2.2. Pontianak oranges 15 Figure 2.3. Mosambi 16 Figure 2.4. Dalandan 17 Figure 3.1. Diagram for the isolation of headspace flavour compounds of orange juice by SPME (Jia et al ., 1998) 59 Figure 5.1. Chromatogram (top) and aromagram (below) of aroma active compounds of Pontianak orange peel oil 103 Figure 5.2. Comparative flavour profile analysis of Pontianak orange peel oil and the reconstituted aroma model solutions based on all available compounds (Formula 1), Relative Flavour Activity (RFA; Formula 2) and Odour Activity Value (OAV; Formula 3) 111 viii List of Abbreviations AEDA Aroma Extract Dilution Analysis DVB Divinyl benzene ECD Electron Capture Detector EI Electron Ionization FD Flavour Dilution FID Flame Ionization Detector FPD Flame Photometric Detector GC Gas Chromatograph GC-FID Gas Chromatograph-Flame Ionization Detector GC/MS Gas Chromatograph-Mass Spectrometry GC-O Gas Chromatograph-Olfactometry LRI Linear Retention Index MNMA Methyl-N-methyl anthranilate MS Mass Spectrometry NIST National Institute of Standards and Technology NPD Nitrogen-Phosphorus Detector OAV Odour Activity Value PDMS Polydimethylsiloxane PLOT Porous-Layer Open Tubular RFA Relative Flavour Activity SAFE Solvent-Assisted Flavour Evaporation SBSE Stir Bar Sorptive Extraction SCOT Support Coated Open Tubular ix SDE Simultaneous Distillation/Extraction SPME Solid Phase Microextraction WCOT Wall-Coated Open Tubular x List of Publications and Presentation 1. Dharmawan J, Barlow PJ and Curran P. 2006. Characterization of Volatile Compounds in Selected Citrus Fruits from Asia. In: Bredie WLP and Petersen MA (eds). Flavour Science: Recent Advances and Trends. Proceedings of the 11 th Weurman Flavour Research Symposium held in Roskilde, Denmark on 21-24 June 2005. Amsterdam: Elsevier. p 319-322. 2. Dharmawan J, Kasapis S, Curran P and Johnson JR.

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