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Authentication of Edible Bird's Nest Using AUTHENTICATION OF EDIBLE BIRD’S NEST USING ADVANCED ANALYTICAL TECHNIQUES AND MULTIVARIATE DATA ANALYSIS CHUA YONG GUAN PETER (B.Sc.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Professor Li Fong Yau Sam, (in the laboratory S5-02-05), Chemistry Department, National University of Singapore between 03-08-2013 and 10-03-2014. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has not been submitted for any degree in any university previously. The content of the thesis has been partly published in: 1) Metabolite profiling of edible bird nest using GCMS and LCMS Chua, Y.G., Bloodworth, B.C., Leong, L.P. and Li, F.Y.S, Journal of Mass Spectrometry, 2014, 28,1-14. Chua Yong Guan Peter 10/03/2014 Name Signature Date ACKNOWLEDGEMENT ACKNOWLEDGEMENT First, I would like to express my grateful appreciation to my Ph.D supervisor Professor Li Fong Yau Sam and co-supervisor Dr Leong Lai Peng for their support in my Ph.D program in the last 5 years. Prof Li has taught me skills of designing and performing scientific research work that are of the highest quality. In addition, he has provided me with encouragement and sound advice whenever I needed them. I am also grateful to Dr Leong who often took time off from her busy schedule to discuss with me on my research work. Through these discussions, I was able to obtain useful scientific advice and knowledge which greatly aids in my understanding on the field of food authentication. I would also like to express my gratitude to National University of Singapore (NUS) and Singapore-Peking-Oxford Research Enterprise for Water Eco- efficiency (SPORE) for providing me with the research scholarship for my PH.D studies and all the staff from the Chemistry Department in NUS for the administrative support. Next, I would like to thank Applied Sciences Group at the Health Science Authority (HSA) and Shimadzu (Asia Pacific) Pte Ltd for providing me with the chance of conducting my research work in their laboratories and Eu Yan Seng (Singapore) for agreeing to sponsor the edible bird’s nest for the project. Throughout the course of my PH.D , it has been a great pleasure to work with the post-graduates from Prof Li’s laboratory - Dr Fang Guihua, Dr Ji kaili, Dr I ACKNOWLEDGEMENT Jon Ashely, Mr Ang Jin Qiang, Ms Anna Karen Carrasco, Mr Chen Baisheng, Ms Lee Si Ni, Mr Lin Xuanhao, Mr Peh En Kai Alister and Ms Zhang Wenlin. Without them, I am certain that that my research work would not have proceeded so smoothly. I am grateful to all their assistance and wish them the best of luck in the future. Also, I would like to thank Professor Bosco Chen Bloodworth and Ms Joanne Chan from HSA for their assistance in obtaining the edible bird’s nest from Malaysia and their advice on the research work. In addition, I wish to express my thanks to Dr Zhan Zhaoqi, Ms Hui-Loo Lai Chin, Ms Cynthia Lahey, Mr Ling Gee Siang and Ms Zeng Peiting from Shimadzu for their valuable technical advice and training in the analytical instruments. Special thanks are given to Bay Lianjie and Kee Jiahui for proofreading my thesis. Last but not least, I am always grateful to my family members and friends for their continuous support and understanding throughout the journey of my Ph.D . Chua Yong Guan Peter National University of Singapore March 2014 II TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGEMENT ............................................................................... I TABLE OF CONTENTS ............................................................................. III SUMMARY ................................................................................................ VIII LIST OF TABLES ........................................................................................ IX LIST OF FIGURES ...................................................................................... XI LIST OF ABBREVIATIONS .................................................................. XVII LIST OF SYMBOLS ................................................................................... XX CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ............ 1 1.1. Overview on food authentication ............................................................ 3 1.1.1. Identification of food items .............................................................. 5 1.1.2. Classification of food items .............................................................. 6 1.1.3. Discrimination of genuine food items from its spiked form ............ 8 1.2. Background information on EBN ......................................................... 10 1.2.1. Origin of EBN ................................................................................ 10 1.2.2. Production sites of EBN ................................................................. 13 1.2.3. Methods utilized to process the EBN ............................................. 16 1.2.4. Economic importance of EBN ........................................................ 18 1.2.5. Health effects of the consumption of EBN ..................................... 18 1.3. Analytical techniques applied for food authentication .......................... 21 1.3.1. Deoxyribonucleic acid based techniques ........................................ 21 III TABLE OF CONTENTS 1.3.2. Spectroscopic techniques ................................................................ 23 1.3.3. Chromatographic techniques .......................................................... 24 1.4. Multivariate data analysis...................................................................... 28 1.4.1. Scaling ............................................................................................ 30 1.4.2. Unsupervised model ....................................................................... 32 1.4.3. Supervised model ........................................................................... 34 1.5. Scope of thesis ....................................................................................... 40 CHAPTER 2. IDENTIFICATION OF EDIBLE BIRD’S NEST WITH AMINO ACIDS AND MONOSACCHARIDES ......................................... 42 2.1. Introduction ........................................................................................... 42 2.2. Materials and methods .......................................................................... 46 2.2.1. Information on the samples ............................................................ 46 2.2.2. Chemicals and materials ................................................................. 47 2.2.3. Amino acid analysis........................................................................ 48 2.2.4. Monosaccharide analysis ................................................................ 50 2.2.5. Statistical analysis........................................................................... 52 2.2.5. Hotelling T2 range plot................................................................... 52 2.3. Results and discussion ........................................................................... 54 2.3.1. Development and validation of an analytical method for the monosaccharide analysis of EBNs ........................................................... 54 2.3.2. Establishing the Hotelling T2 range plot to identify EBN ............. 62 2.3.3. Evaluation of Hotelling T2 range plot with non-EBN ................... 67 IV TABLE OF CONTENTS 2.3.4. Assessment of amino acid and monosaccharide contents of EBN . 72 2.3.5. Quality control of EBN with OPLS-DA......................................... 78 2.4. Conclusion ............................................................................................. 79 CHAPTER 3. CLASSIFICATION OF EDIBLE BIRD’S NEST WITH METABOLITE FINGERPRINTING .......................................................... 81 3.1. Introduction ........................................................................................... 81 3.2. Materials and methods .......................................................................... 84 3.2.1. Sample information ........................................................................ 84 3.2.1. Chemicals and materials ................................................................. 85 3.2.2. Analysis with GC/MS ..................................................................... 85 3.2.3. Analysis with LC/MS ..................................................................... 86 3.2.4. Pre-processing of GC/MS data ....................................................... 88 3.2.5. Pre-processing of LC/MS data ....................................................... 88 3.2.7. Statistical analysis........................................................................... 90 3.3. Results and discussion ........................................................................... 90 3.3.1. Profiling of metabolites using GC/MS ........................................... 90 3.3.2. Profiling of metabolites using LC/MS............................................ 95 3.3.3. Classification of EBNs with PCA ................................................ 103 3.3.4. Classification of EBN based on color ........................................... 106 3.3.5. Classification of EBN based on country ....................................... 110 3.3.6. Classification of EBN based
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