Functionality of Jerusalem Artichoke Derived Inulin in Vietnamese Coffee and Developing a Novel Food Grade Microemulsion System by Xuhao Zhao A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Carleton University Ottawa, Ontario © 2017, Xuhao Zhao Abstract Jerusalem artichoke (JA) is an important source of inulin, a dietary fiber with many health benefits. To investigate the potential of JA on antioxidant activity of coffee, JA coffee was made by adding roasted JA tuber to Vietnamese coffee at a ratio 1:1 (w/w). The TPC (total phenolic content) increased by 13.6% in JA-coffee effecting higher antioxidant activity by 48.0% and 26.9% in ORAC and DPPH assays, respectively. Additionally, JA improved such coffee characteristics as flavor and foam. Mass spectrometric analysis showed that the majority of inulin contents were DP 3 and DP 4. Furthermore, to utilize coffee waste, phenolic compounds were extracted from spent coffee. A food-grade microemulsion system was composed of grape seed oil, soya lecithin, ethanol, and water. The feasibility of this novel microemulsion system for the delivery of phenolic acids was studied by applying it to spent coffee phenolic extracts. Finally, the uniformity of microemulsions was improved by the LiposoFast which confirmed by Cryo-SEM. ii Acknowledgements My deepest gratitude is given to my supervisors Dr. Farah Hosseinian and Dr. Edward Lai for their patiently and professional guidance and encouragement in the past 2 years. I would not successfully complete my project without their support. They always share new ideas and their professional knowledge and abundant research experience. I would also like to express my appreciation to all of our lab members. Thanks to Dr. Rania Agil and Dr. Aynur Gunenc for their great advice and assistance. Thanks to Melanie and Tatiana who taught me operations of equipment in our lab. Dorsa and Natasha, I am so pleasant to be friends with you, and I wish you a bright future. Thanks to Diba, Allison and Paloma for all their assistance and companion. I could not express more thanks to our lovely group. A special thanks to Shari Levac who is so knowledgeable and helped me a lot at laboratory instruments. I also appreciate Dr. JJ Wang and Karl for their support for SEM and MS operations. Additionally, I would like to appreciate Chantelle Gravelle for warmly encouraging and helping me all the time. Thanks to Peter Mosher and Jim Logan who helped me a lot with technical problems. Thanks to Sijing and Alex for all the happy and tough time we had over two years. It is truly a pleasure for not being alone in science. Last but not least, I would like to thank my parents for supporting me all the time even we are far away from each other. Although they do not understand my work, they always share their wisdom of life which inspires me all the time. I would not be here all the way to the other side of the planet without the emotional and financial support from them. iii Table of Contents Abstract.......................................................................................................... ii Acknowledgements ...................................................................................... iii List of Tables ............................................................................................... vii List of Figures ............................................................................................ viii List of Abbreviations ..................................................................................... x 1 Chapter: Literature Review .................................................................................................... 1 1.1 Botanical fact, chemistry, and application of Jerusalem artichoke ....................... 1 1.2 Inulin and health benefits .......................................................................................... 3 1.3 Vietnamese coffee and health benefits ...................................................................... 5 1.4 Phenolic compounds as antioxidants ........................................................................ 6 1.4.1 Chemical structure of phenolic compounds ........................................................... 6 1.4.2 Phenolic compounds in coffee and biosynthesis ................................................... 8 1.5 Free radicals and antioxidant activity assays......................................................... 10 1.5.1 Oxygen radical absorbance capacity (ORAC) assay ........................................... 10 1.5.2 DPPH radical scavenging assay ........................................................................... 12 1.5.3 Relationship between total phenolic content (TPC) and antioxidant capacity .... 12 1.6 Microemulsions and applications ............................................................................ 13 1.6.1 Microemulsion systems, definition, characteristics and formations .................... 13 1.6.2 Applications of microemulsions .......................................................................... 15 2 Chapter: The potential of inulin in Jerusalem Artichoke to affect the bioactivity of Vietnamese coffee ......................................................................................................................... 17 2.1 Abstract ..................................................................................................................... 17 iv 2.2 Introduction .............................................................................................................. 17 2.3 Materials and methods ............................................................................................. 18 2.3.1 Sample preparation .............................................................................................. 18 2.3.2 Materials and instruments .................................................................................... 19 2.3.3 Extraction of phenolic compounds ...................................................................... 20 2.3.4 Characterization and determination of phenolic compounds ............................... 20 2.3.5 Oxygen radical antioxidant capacity (ORAC) assay ........................................... 22 2.3.6 DPPH (2-diphenyl-1-picryhydrazyl) radical scavenging assay ........................... 22 2.3.7 Total Phenolic Content ........................................................................................ 23 2.3.8 Inulin extraction ................................................................................................... 24 2.3.9 Mass Spectrometry (MS) sugar component analysis ........................................... 24 2.3.10 Statistical analysis ................................................................................................ 24 2.4 Results........................................................................................................................ 25 2.4.1 Determination and characterization of phenolic compounds in coffee and JA samples ................................................................................................................ 25 2.4.2 Antioxidant activity analysis ............................................................................... 26 2.4.3 MS analysis .......................................................................................................... 30 2.5 Discussion .................................................................................................................. 33 2.6 Conclusions ............................................................................................................... 36 2.7 Connecting statement to Chapter 3 ........................................................................ 37 3 Chapter: Development of a novel food grade microemulsion system ............................... 38 3.1 Abstract ..................................................................................................................... 38 3.2 Introduction .............................................................................................................. 38 3.3 Materials and methods ............................................................................................. 40 3.3.1 Materials and instruments .................................................................................... 40 3.3.2 Optimization of surfactant: co-surfactant ratios .................................................. 40 v 3.3.3 Formation of microemulsions and the pseudoternary phase diagram .................. 41 3.3.4 Preparation of spent coffee ground and phenolic compounds extraction ............ 42 3.3.5 Determination of phenolic compounds in extracts from spent coffee ground ..... 42 3.3.6 Application of the microemulsion system on phenolic compounds extracts ....... 43 3.3.7 Mini-extruder extrusion technique by the LiposoFast device .............................. 43 3.3.8 Cryo-SEM (cryogenic scanning electron microscope) ........................................ 44 3.4 Results........................................................................................................................ 44 3.4.1 Formations of microemulsions ............................................................................ 44 3.4.2 Determination of phenolic compounds in extracts from spent coffee ground ..... 46 3.4.3 Application of the microemulsion system on phenolic compounds extracts from spent coffee ground .............................................................................................. 46 3.4.4 Mini-extruder extrusion technique and Cryo-SEM ............................................. 47 3.5 Discussion .................................................................................................................. 49 3.6 Conclusions ............................................................................................................... 51 4 Chapter: General conclusions and future directions .......................................................... 52 References .....................................................................................................54 vi List of Tables Table 1-1 Comparisons of physicochemical characteristics of chicory and artichoke inulin ..............................................................................................................................................2 Table 1-2 Nutrients of Jerusalem artichoke .........................................................................4 Table 1-3 Classes of phenolic compounds in plants ............................................................7 Table 1-4 Comparison between microemulsions and emulsions .......................................14 Table 2-1 Phenolic acid and flavonoid standards and retention times ...............................21 Table 2-2 Phenolic composition in samples, mg/g ............................................................26 Table 2-3 Summary of results from multiple antioxidant activity assays .........................27 Table 3-1 Ratios of L/E to grape seed oil in Group A .......................................................42 Table 3-2 Ratios of L/E to grape seed oil in Group B ......................................................44 Table 3-3 Compositions of microemulsions in Group A ...................................................46 vii List of Figures Figure 1-1 JA plant (A) and JA tuber (B) ...........................................................................2 Figure 1-2 Chemical structure of inulin, n=2~60 ................................................................4 Figure 1-3 Chemical tructure of phenolic acids ...................................................................6 Figure 1-4 Chemical structure of flavonoids ......................................................................7 Figure 1-5 Chemical structure of CGA ...............................................................................8 Figure 1-6 Biosynthesis pathway of hydroxycinnamic acids and quinic acid. ....................9 Figure 1-7 Fluorescence decay curve ................................................................................11 Figure 1-8 Reactions of DPPH radical and an antioxidant (AH) or radical species (R●) .12 Figure 1-9 Hypothetical phase regions of microemulsion systems ...................................15 Figure 2-1 Roasted JA tuber sample ..................................................................................18 Figure 2-2 Total phenolic compounds content in samples determined in HPLC analysis, mg/g of sample ..................................................................................................25 Figure 2-3 Antioxidant activity of phenolic extracts from PC, INC, JAC, RST and FD ..28 Figure 2-4 The linear correlation of results of (A) ORAC vs. TPC, (B) DPPH vs. TPC…………………………………………………………………………… …………29 Figure 2-5 MS spectra of JA coffee inulin extracts. ..........................................................30 Figure 2-6 MS spectrum of single sugar standards, negative mode, singly charged. Peak a) D-glucose, D-fructose, peak b) sucrose. ......................................................31 Figure 2-7 MS spectra of plain coffee inulin extracts .......................................................32 viii Figure 2-8 Antioxidant activity of 1g coffee, 1g coffee with 0.11g and 1g coffee with 1g JA. .....................................................................................................................36 Figure 3-1 Comparison of L/E 75:25 (left) and L/E 80:20 (right) .....................................45 Figure 3-2 Pseudoternary phase diagram of lecithin: ethanol (80:20)/grape seed oil/water microemulsions .................................................................................................45 Figure 3-3 Pseudoternary phase diagram of lecithin: ethanol (80:20)/grape seed oil/water with phenolic extracts microemulsions .............................................................47 Figure 3-4 Structures of microemulsions under Cryo-SEM. ............................................48 ix List of Abbreviations AAPH 2, 2’-azobis(2-methylpropionamidine) dihydrochloride CGA Chlorogenic acid DP Degree of polymerization DPPH 2,2-diphenyl-1-picryhydrazyl FD Freeze-dried fresh Jerusalem artichoke tuber GAE Gallic acid equivalent GSO Grape seed oil HLB Hydrophile-lipophile balance HPLC High performance liquid chromatography IN Inulin INC Inulin coffee JA Jerusalem artichoke JAC Jerusalem artichoke coffee L/E Lecithin/ethanol ratio MW Molecular weight O/W Oil in water ORAC Oxygen radical absorbance capacity PC Plain coffee RNS Reactive nitrogen species ROS Reactive oxygen species RST Roasted Jerusalem artichoke tuber SEM Scanning electron microscopy x